- Volume 4, Issue 5, 2022
Volume 4, Issue 5, 2022
- Reviews
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Saliva as an alternative specimen to nasopharyngeal swabs for COVID-19 diagnosis: Review
More LessAlmost 2 years ago, the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was discovered to be the causative agent of the disease COVID-19. Subsequently, SARS-CoV-2 has spread across the world infecting millions of people, resulting in the ongoing COVID-19 pandemic. The current ‘gold standard’ for COVID-19 diagnosis involves obtaining a nasopharyngeal swab (NPS) from the patient and testing for the presence of SARS-CoV-2 RNA in the specimen using real-time reverse transcription PCR (RT-qPCR). However, obtaining a NPS specimen is an uncomfortable and invasive procedure for the patient and is limited in its applicability to mass testing. Interest in saliva as an alternative diagnostic specimen is of increasing global research interest due to its malleability to mass testing, greater patient acceptability and overall ease of specimen collection. However, the current literature surrounding the sensitivity of saliva compared to NPS is conflicting. The aim of this review was to analyse the recent literature to assess the viability of saliva in COVID-19 diagnosis. We hypothesize that the discrepancies in the current literature are likely due to the variations in the saliva collection and processing protocols used between studies. The universal adaptation of an optimised protocol could alleviate these discrepancies and see saliva specimens be as sensitive, if not more, than NPS for COVID-19 diagnosis. Whilst saliva specimens are more complimentary to mass-testing, with the possibility of samples being collected from home, the RT-qPCR diagnostic process remains to be the rate-limiting step and therefore interest in salivary rapid antigen tests, which negate the wait-times of RT-qPCR with results available within 15–30 min, may be an answer to this.
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- Research Articles
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Co-isolates of Acinetobacter baumannii complex in polymicrobial infections: a meta-analysis
More LessBackground. Acinetobacter baumannii complex (ABC) infections are commonly polymicrobial. Examining which pathogens are most commonly co-isolated with ABC is an important first step for assessing disease potential due to pathogen-pathogen interactions.
Methods. Based on a systematic search of PubMed, Scopus and CENTRAL, we estimated percent proportions of co-isolates in polymicrobial pulmonary and bloodstream ABC infections using random-effects meta-analysis.
Results. Twenty-eight eligible studies were analysed reporting 575 polymicrobial bloodstream and 290 polymicrobial pulmonary infections. Common co-isolates in pulmonary infections were P. aeruginosa (36%, 95% CI 24–49%, I2 71%), S. aureus (28%, 95% CI 19–38%, I2 44%) and Klebsiella spp. (11%, 95% CI 6–20 %, I2 56%), while the prevalence of other co-pathogens did not exceed 5%. Most common co-isolates in bloodstream infections were coagulase-negative Staphylococci (21%, 95% CI 12–34 %, I2 84%), Enterococci (15%, 95% CI 9–26%, I2 73%), P. aeruginosa (12%, 95% CI 6–22%, I2 74%), Klebsiella spp. (10%, 95% CI 6–16%, I2 42%), Enterobacter spp. (10%, 95% CI 6–16 %, I2 38%) and S. aureus (8%, 95% CI 4–15%, I2 58%).
Conclusion. The common co-isolation of certain pathogens (especially P. aeruginosa ) with ABC suggests potential beneficial between-pathogen interactions, which may have treatment implications for polymicrobial infections and requires further study.
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Human Bocavirus infection among children with respiratory tract infection in Ibadan, Nigeria
More LessBackground. Human Bocavirus (HBoV), which is an ssDNA virus of the family Parvoviridae, is responsible for 21.5 % of childhood respiratory tract infections (RTIs) annually. Among the four genotypes currently known, HBoV-1 has been associated with acute RTI. Although there have been studies on HBoV in some countries, there is limited information on this virus in sub-Saharan Africa where there is the highest burden of RTI. This study aimed to characterize the circulating strains of HBoV in Ibadan, Nigeria.
Methods. Nasopharyngeal and oropharyngeal swab samples were collected from 333 children ≤5 years old presenting with RTI attending hospitals in Ibadan, whose parents assented, from 2014 to 2015. Twenty-three HBoV isolates were sequenced after a nested PCR and phylogenetic analysis was carried out using mega 6 software.
Results. A total of 27 children tested positive for the HBoV-1 genotype by PCR and 23 of the 27 isolates were successfully sequenced. The 23 HBoV-1 isolates from this study have been assigned GenBank accession numbers KY701984–KY702006. Phylogram analysis indicated that the isolates belong to the same clades. Six isolates aligned closely to the reference strains ST1 and ST2, while 17 isolates showed a high level of divergence to the reference isolates.
Conclusion. This study highlights the contribution of HBoV to RTIs in Nigeria and that HBoV-1 strains are associated with the infection.
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Genome sequence of the aurodox-producing bacterium Streptomyces goldiniensis ATCC 21386
More LessWe report the genome sequence of Streptomyces goldiniensis ATCC 21386, a strain which produces the anti-bacterial and anti-virulence polyketide, aurodox. The genome of S. goldiniensis ATCC 21386 was sequenced using a multiplatform hybrid approach, revealing a linear genome of ~10 Mbp with a G+C content of 71%. The genome sequence revealed 36 putative biosynthetic gene clusters (BGCs), including a large region of 271 Kbp that was rich in biosynthetic capability. The genome sequence is deposited in DDBJ/EMBL/GenBank with the accession number PRJNA602141.
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Utilization and accumulation of compatible solutes in Halomonas pacifica: a species of moderately halophilic bacteria isolated from a saline lake in South Libya
More LessWhen grown in high salt concentrations, halophilic bacteria often accumulate compatible solutes, which have major applications in biotechnology because they stabilize cells and proteins. Four Gram-negative bacterial strains, belonging to the family Halomonadaceae, were isolated from Qaberoun and Um-Alma lakes in South Libya using high-salinity medium. The strains were identified using 16S rRNA gene sequencing as belonging to Halomonas pacifica (strain ABQ1), Halomonas venusta (ABQ2), Halomonas elongata (ABU1) and Halomonas salifodinae (ABU2). H. pacifica ABQ1 is a moderate halophile (salinity range 0.05 to 2.5 M NaCl), with a broad tolerance to pH (7 to 9) and temperature (25–37 °C). Addition of the compatible solutes glycine betaine (betaine) and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) to the medium had a positive effect on growth of H. pacifica at 2 M NaCl. In rich LB medium, betaine was the major compatible solute accumulated, with ectoine only being accumulated at salinities in excess of 1 M NaCl. In minimal M9 medium, betaine was not produced, but increasing amounts of ectoine were synthesized with increasing salinity, and hydroxyectoine [(4S,5S)−5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was also synthesized when the cells were grown in very high salt. We have thus identified H. pacifica as a producer of ectoine and hydroxyectoine, with more being produced at higher salinities. As industrial demand for these compatible solutes continues to increase, this system has biotechnological potential.
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- Short Communications
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RNA-based stable isotope probing provides no indication for rapid α-synuclein assimilation by murine gut bacteria
In Parkinson’s disease (PD), α-synuclein is a key protein in the process of neurodegeneration. Besides motor symptoms, most PD patients additionally suffer from gastrointestinal tract (GIT) dysfunctions, even several years before the onset of motor disabilities. Studies have reported a dysbiosis of gut bacteria in PD patients compared to healthy controls and have suggested that the enteric nervous system (ENS) can be involved in the development of the disease. As α-synuclein was found to be secreted by neurons of the ENS, we used RNA-based stable isotope probing (RNA-SIP) to identify gut bacteria that might be able to assimilate this protein. The gut contents of 24 mice were pooled and incubated with isotopically labelled (13C) and unlabelled (12C) α-synuclein. After incubation for 0, 4 and 24 h, RNA was extracted from the incubations and separated by density gradient centrifugation. However, RNA quantification of density-resolved fractions revealed no incorporation of the 13C isotope into the extracted RNA, suggesting that α-synuclein was not assimilated by the murine gut bacteria. Potential reasons and consequences for follow-up-studies are discussed.
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Use of whole-genome sequencing to detect transmission of group A Streptococcus in Houston, TX
More LessWe used a combination of local, comprehensive strain surveillance and bacterial whole-genome sequencing to identify potential transmission events of group A streptococcus (GAS) in Houston, TX, USA. We identified pharyngeal and skin and soft tissue sources of infection as having important roles in community GAS transmission, including invasive diseases.
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A comparison of SARS-CoV-2 RNA extraction with the QuickGene-810 Nucleic Acid Isolation System compared to the EZ1 Advanced DSP Virus Kit
More LessThe QuickGene-810 Nucleic Acid Isolation System is a semi-automated extraction platform which may be used for RNA extraction. New methods were required to support the rapid increase in respiratory virus testing during the SARS-CoV-2 pandemic. The aim of this study was to assess SARS-CoV-2 RNA extraction using the QuickGene-810 kit compared to the EZ1 Advanced Extraction Platform for use on the AusDiagnostics SARS-CoV-2, Influenza and RSV 8-well RT-PCR assay. Qualitative results from all clinical samples were concordant between the QuickGene-810 and the EZ1 extraction methods, demonstrating that the QuickGene-810 kit is suitable for use in pathogen diagnostics. However, there was an average difference of approximately two cycles between the cycle threshold (Ct) values for both SARS-CoV-2 targets, suggesting that the EZ1 kit yields a higher concentration of nucleic acid extract, possibly related to its use of carrier RNA and/or smaller elution volume, which infers the possibility of false negative results for samples with very low viral loads.
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Comparative evaluation of chlorous acid and sodium hypochlorite activity against SARS-CoV-2
A novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suddenly emerged in China in 2019, spread globally and caused the present COVID-19 pandemic. Therefore, to mitigate SARS-CoV-2 infection effective measures are essential. Chlorous acid (HClO2) has been shown to be an effective antimicrobial agent. However, at present there is no experimental evidence showing that HClO2 can inactivate SARS-CoV-2. Therefore, in this study, we examined the potential of HClO2 to inactivate SARS-CoV-2 in presence or absence of organic matter and the results were compared with that of sodium hypochlorite (NaClO), another potent antimicrobial agent. When concentrated SARS-CoV-2 was incubated with 10 ppm HClO2 for 10 s, viral titre was decreased by 5 log of 50% tissue culture infective dose per mL (TCID50 ml−1). However, the same concentration of NaClO could not inactivate SARS-CoV-2 as effectively as HClO2 did even after incubation for 3 min. Furthermore, 10 ppm HClO2 also inactivated more than 4.0 log of TCID50 within 10 s in the presence of 5 % fetal bovine serum used as mixed organic matters. Our results obtained with HClO2 are more effective against SARS-CoV-2 as compared to NaClO that can be used for disinfectant against SARS-CoV-2 .
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Ralstonia mannitolilytica: an emerging multidrug-resistant opportunistic pathogen in a tertiary care hospital setting
More LessIntroduction. Ralstonia mannitolilytica is a rare opportunistic pathogen capable of causing a serious infection in immunocompromised patients. Our objective was to describe all cases of R. mannitolilytica bloodstream infection identified within 2 years at our tertiary care centre, focusing on clinical characteristics, risk factors, antibiotic sensitivity patterns, management and outcomes.
Case Series. We compiled a descriptive case series including 14 non-duplicate R. mannitolilytica isolates obtained from bloodstream infection samples from the microbiology laboratory of a tertiary care centre from June 2019 to June 2021. All isolates were initially identified based on their morphological properties and biochemical reactions, and then underwent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) examination for confirmation of identity. Antibiotic susceptibility testing was performed using the Kirby–Bauer disc diffusion method and Vitek 2. All 14 patients presented with symptoms of fever and/or chills, and a positive blood culture for R. mannitolilytica . After 48 h of incubation, no Ralstonia growth was reported from any of the current environmental or pharmaceutical water samples. Chemotherapy (9/14), mechanical ventilation (4/14), steroid use (2/14) and diabetes mellitus (1/14) were associated risk factors in our patients. The antibiotic sensitivity panel showed maximum resistance to aminoglycosides (64.3%) and no resistance to cefoperazone/sulbactum. Patients received treatment with cefoperazone/sulbactum and meropenem or ceftazidime. Thirteen patients recovered with antibiotic therapy and one patient succumbed to his illness.
Conclusion. R. mannitolilytica can cause bloodstream infections in immunocompromised patients. It is likely to be missed or underreported due to lack of clinical awareness. MALDI-TOF MS is helpful in rapid identification. R. mannitolilytica is resistant to many routinely used antibiotics, including carbapenems.
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- Case Reports
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Detection of Enterococcus avium in a case of urinary tract infection and haematuria
More LessEnterococci have been recognized as major pathogens causing nosocomial and community-acquired infections. The emergence of antimicrobial-resistant enterococci is one of the major public health challenges worldwide. While many enterococcal species have been identified, Enterococcus avium is rarely detected in humans. Here we present an interesting case of urinary tract infection and haematuria involving E. avium in a 72-year-old patient. The patient underwent antibiotic therapy and surgical procedures with excellent improvement. This case report highlights the important role of E. avium in clinical settings.
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Cellulitis and transient bacteremia by Capnocytophaga canis after a cat scratch in an immunocompetent patient
Capnocytophaga canis is still a rare cause of infection. We present a case of an immunocompetent patient admited in the hospital with functional impotence, pain and erythema in his left leg after suffering two scratches from his cat 48 h ago. After obtaining blood and wound cultures, broad-spectrum antimicrobial therapy with intravenous amoxicillin clavulanate was initiated. After 1 day and with a clear improvement of the symptoms the patient was discharged from the hospital with cellulitis and transient bacteremia as diagnosis and completing 1 week of antimicrobial therapy orally. After 80 and 92 h of incubation, both anaerobic flasks were positive. In the Gram-stain Gram-negative rod-shaped bacteria could be observed. Despite subculturing in brucella blood agar, tripticase soy agar with 5 % of sheep blood and chocolate agar, in both anaerobic and microaerophilic conditions, the strain could not be recovered. However, these Gram-negative rods could be identified as C. canis by 16S rRNA sequencing, Capsular typing was performed to study the strain, but none of the studied capsule-types tested positive. C. canis is still a rare cause of human infection, but it must be considered in the differential diagnosis of infections related to bites, scratches and licks from dogs or cats.
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Capnocytophaga tricuspid valve endocarditis: a case report and literature review
More LessCapnocytophaga canimorsus is a Gram-negative zoonotic pathogen capable of causing serious infection following dog or cat bite. Infections often manifest as sepsis, fatal septic shock, gangrene, bacteraemia, meningitis and endocarditis. Here we report a case of C. canimorsus bacteraemia complicated by tricuspid valve infective endocarditis and septic pulmonary emboli.
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Cerebral venous sinus thrombosis as a complication of cranial melioidosis – a rare case report
Cerebral venous sinus thrombosis is a rare complication of cranial melioidosis. We report a case of an adult male who presented with skull osteomyelitis, transverse sinus thrombosis and multiple brain abscesses. His blood cultures grew Burkholderia pseudomallei . The patient finally succumbed after multiple recurrences of the infection despite surgical excision of the osteomyelitic bone and the recommended antibiotic treatment. The management of cerebral venous sinus thrombosis in patients with cranial melioidosis is discussed along with a brief review of the literature.
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- Abstracts from Annual Conference 2021
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- Oral Abstracts
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Inactivation of antibiotic-resistant microorganisms by physical plasma
Wastewater treatment plants are “hotspots” for the dissemination of antibiotic-resistant microorganisms (ARM).1 General treatment methods only insuffiently reduce the load.2 Conversely, physical plasma methods have proven to be promising to inactivate microorganisms.3
Different plasma sources were tested according to their efficacy to inactivate ARM. Synthetic wastewater containing Escherichia coli GW-AmxH19 (isolate from hospital wastewater, Greifswald, Germany)4 was treated with the respective plasma source for 30min. The viable count was determined before and after plasma treatment.
In dependence of the source a reduction of the viable count of 1-7 log 10 was achieved.
Thus, physical plasma can be utilized as additional treatment stage in wastewater remediation and may also support the effort of “one health”.
1Rizzo, L.; Manaia, C.; Merlin, C.; Schwartz, T.; Dagot, C.; Ploy, M. C.; Michael, I.; Fatta-Kassinos, D. Science of the Total Environment 2013, 447, 345-360.
2Prado, T.; Pereira, W. C.; Silva, D. M.; Seki, L. M.; Carvalho, A. P. D.; Asensi, M. D. Letters in Applied Microbiology 2008, 46 (1), 136-141.
3Hahn, V.; Dikyol, C.; Altrock, B.; Schmidt, M.; Wende, K.; Ercan, U. K.; Weltmann, K. D.; von Woedtke, T. Plasma Processes and Polymers 2019, 16 (5).
4Schneider, D.; Zühlke, D.; Petscheleit, T.; Poehlein, A.; Riedel, K.; Daniel, R. Germany. Microbiology Resource Announcements 2020, 9 (21), e00279-20 (2pp).
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Recycling-derived Phosphorus Fertilizers as a Sustainable Alternative to Triple Superphosphate Fertilizers
More LessMicrobial activity is adamant for the nutrient cycling in soil. Generally, mineral phosphorus (P) fertilizer is applied to soil to improve plant growth, however, significant amounts are immobilized quickly. Mineral fertilizer can cause soil degradation, affecting the microbial community. Alternative, recycling-derived fertilizers (RDFs) need to be evaluated as suitable replacement for finite mineral P fertilizer. The impact of four RDFs (two ashes, two struvites) on the soil microbiome in comparison with a P-free control and triple superphosphate (TSP) as mineral fertilizer was investigated in a pot trial and a subsequent microcosm trial (subset of samples). For both experiments, perennial ryegrass was cultivated for 54 days. The pot trial was conducted at P fertilization rates of 20 and 60 kg P ha-1 in quadruplicates. After the pot harvest, the bulk soil was stored until the microcosm trial was conducted, using the control, TSP and the two ashes at 60 kg P ha-1 in six replicates. Struvites displayed highest P bioavailability at high P application rates in the pot trial, yielding higher biomass on average. Furthermore, P solubilization from tri-calcium phosphate was enhanced in the RDFs treatments, while the TSP treatments were negatively affected. For the microcosm trial, most probable number (MPN) analysis showed that phytate-utilizing bacterial abundance was significantly increased in one of the ashes. Non-metric multidimensional scaling (NMDS) analysis of phoD illumina sequencing data showed significant separation between all treatments of the microcosm trial. Understanding the impact of RDF application on the soil P cycle is vital to sustainable agriculture.
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Recombination, and Then What? How Zika Virus Hybrids Improve Their Fitness
More LessZika virus (ZIKV) is an emerging flavivirus primarily transmitted through the bite of Aedesmosquitoes. It spread explosively around the tropics in the 21st century, and still presents a threat to human health. There is a need to better understand the drivers of emergence of ZIKV and likely consequences of its future evolution. RNA recombination is a process of genetic exchange which occurs in positive-sense RNA viruses and contributes to virus evolution. We have developed a recombinant-specific PCR assay for detection of ZIKV recombinants in mammalian and insect cell co-infections. Using this system, we demonstrated the ability of distinct geographic and genetic isolates to recombine and tested their viability in tissue culture. Initial ZIKV recombinants produce plaques with a significantly smaller diameter than either parental strain. Recombinant large-plaque variants were isolated through serial passage and plaque purification, and the genome analysed for adaptive mutations. A single nucleotide coding mutation in NS1 was detected in several independent large-plaque variants. The role of this mutation in virus replication was investigated through reverse genetics to assess whether it gave the recombinant a replicative advantage. Understanding the genetic changes that arise during, and following recombination is important in the context of virus evolution. It is particularly relevant for ZIKV, as co-circulation of the African and Asian strains of ZIKV in nature has already occurred in parts of Latin America and Africa. Current and future studies will focus on the phenotype of recombinant ZIKV, their replication in mammalian and insect cell lines, and in mosquitoes.
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Uncovering the residues responsible for Dis3L2 specificity
More LessExoribonucleases from the RNB-family of enzymes are widely distributed in nature. DIS3 is the eukaryotic homolog of the bacterial exoribonuclease II and is the only catalytic subunit of the core exosome complex. In humans, there are three members of DIS3 family that can be distinguished according to the sequence conservation of the active site:
DIS3, DIS3L (DIS3L1) and DIS3L2. Unlike its family counterparts, DIS3L2 does not interact with the exosome since it lacks the PIN domain, which is essential for the interaction with this multiprotein complex. Dis3L2 is involved in several cellular mechanisms, such as apoptosis, cellular differentiation and proliferation and its mutations have been associated with Wilms tumor formation and Perlman syndrome in children. Distinct studies on Dis3L2 enzyme unraveled a novel eukaryotic RNA decay pathway that challenged the models already established. Dis3L2 activity is stimulated by the addition of untemplated uridine residues to mRNAs, tRNAs, microRNAs, snRNAs among other classes of RNA.
The first insight on the uridylation involvement in controlling the stability of poly(A)-containing mRNAs was reported in S. pombe. However, the precise mechanism of action of this enzyme is not yet fully understood. In this work, the activity of fission yeast Dis3L2 mutant proteins was analyzed over different RNA substrates. The aim was to characterize the amino acid residues that distinguish Dis3L2 substrate specificities regarding its family homologues, namely the preference for uracil residues. The results show that some of the mutant Dis3L2 ribonucleases lose or acquire activity regarding the degradation of different RNAs. Furthermore, this will enable us to understand the mechanism of action of Dis3L2 and its function in different eukaryotic cells.
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Development of a lentivirus-mediated gene therapy targeting HIV-1 RNA to eliminate HIV-1-infected cells
More LessOnly two patients have ever been cured of HIV-1. The latent HIV-1 reservoir is the major roadblock to this and necessitates lifelong therapy. In the ‘shock and kill’ approach to eliminate cells harbouring dormant virus the latency reversing agent (LRA) vorinostat increased HIV-1 RNA levels, but this failed to enable a reduction in reservoir size by immune- or virus-mediated cytotoxicity. To leverage LRAs’ ability to heighten HIV-1 transcription, we are developing a ‘kill’ strategy that hijacks the HIV-1 splicing process with a therapeutic trans-splicing RNA, encoding an incomplete Herpes simplex virus thymidine kinase (HSV-tk) that gains functionality by trans-splicing onto HIV-1 tat pre-mRNA. HSV-tk activates the exogenous prodrug ganciclovir for selective killing of HIV-1-infected cells. Following proof-of-principle transfection studies, therapeutic constructs were engineeredfor lentivirus-mediated delivery to HIV-infected cells in vitro. We optimized lentiviral production for high infectious titer (>106 TU/mL) and low vector plasmid carryover (<1 copy/cell). In a tissue culture model of HIV-1 infection we confirmed lentiviral delivery of therapeutic (and control) vectors. Successful trans-splicing of the HSV-tk RNA onto HIV-1 RNA in cells co-transduced with HIV-1 and therapeutic vector at an MOI as low as 4 was confirmed by sequencing. In MTT assays the most potent therapeutic vector killed approximately 80% of HIV-1-expressing cells. Next-generation vectors are being evaluated for enhanced therapeutic potential and improved HIV-1-targeting RNA expression. The lead candidate culminating from this work will be assessed for selective killing of HIV-1-infected cells both productively infected and with virus reactivated from latency.
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Automated pseudogene detection reveals insights into historical gene sharing dynamics in prokaryotes
More LessIn recent years it has become apparent that prokaryotic genomes contain large numbers of pseudogenised genes which may provide valuable insights into the recent functional history of an organism. However, pseudogenes are difficult to detectab initioand are not routinely reported by gene prediction tools.
We present StORF-R(Stop-ORF-Reporter), a tool that takes as input an annotated genome and returns putative missed genes (functional and/or pseudogenised) from the intergenic regions. We show that this methodology can recover gene-families that the state-of-the-art methods continue to misreport or completely omit.
We applied StORF-R to the intergenic regions of2,665E. coligenomes and found on average 244 previously missed pseudogenised genes (with in-frame stop codons) per genome, many of which had high scoring similarity to known Swiss-Prot proteins. Many of these pseudogenised genes form widespread gene families across E. coli strains.
To investigate if this phenomenon exists in other taxa we further applied the methodology to 44,048 bacterial genomes representing 8,244 species from Ensembl. This revealed manygene-families spanning multiple species with large (>10,000) numbers of copies of both intact and pseudogenised versions. Many of these families had only previously been reported in a single or few genomes, though we detected many hundred pseudogenised versions with StORF-R, changing our understanding of how widespread these genes truly are.
These pseudogenised genes represent a pangenomic ‘graveyard’ which may alter our understanding of the definition of core and accessory genes for many species.
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HCMV UL148 and UL148D regulate multiple immune pathways by impairing expression of ADAM17
Human cytomegalovirus (HCMV) is one of the most widespread, highly successful herpesviruses, establishing a life-long viral infection in humans. HCMV has been described as a paradigm of immune evasion able to manipulate many immune functions in the host. Here, we describe a novel, post-translational mechanism in which HCMV downregulates a disintegrin and metalloproteinase 17 (ADAM17), a ‘sheddase’ that cleaves and releases over 80 membrane-anchored cytokines, cell adhesion molecules and other receptors. A screen of HCMV deletion mutants identified UL148 and UL148D as the HCMV genes responsible for ADAM17 downregulation, working synergistically to alter ADAM17 levels in infected cells. We demonstrate that UL148/UL148D interfere with ADAM17 maturation, resulting in expression of only the intracellular immature precursor, and absence of mature ADAM17 on the surface of wildtype HCMV-infected cells. The consequences of ADAM17 downregulation by HCMV were analysed using proteomics and validated using biochemical and flow cytometric techniques, revealing impact on multiple cell surface and secreted host proteins. This included stabilisation of surface TNF Receptor 2, as well as Vasorin and Jagged1, which have recognised roles in Treg development. Other known ADAM17 targets were not stabilised, suggesting specific control by HCMV. Vaccinia virus, another paradigm of immune evasion, also impaired surface ADAM17 expression, suggesting that manipulation of ADAM17 may represent a novel immunoregulatory hub targeted by large DNA viruses.
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Identifying long-term colonization factors of Vibrio cholerae O1 El Tor in the zebrafish natural host model
The human disease cholera, marked by acute, voluminous watery diarrhea, is caused by the gram-negative, aquatic bacterium Vibrio cholerae. All seven cholera pandemics since 1817 were identified as being caused by just 2 of over 155 known V. choleraeserogroups: O1 and O139. The O1 serogroup is divided into two biotypes: classical and El Tor. Classical biotype is associated with pandemics 1 through 6, but the El Tor biotype has since displaced classical as the causative agent of the ongoing 7th cholera pandemic over the past 60 years. The El Tor genome resembles that of classical but has acquired two unique pathogenicity islands known as Vibrio Seventh Pandemic (VSP) -1 and -2. El Tor biotype has been associated with prolonged colonization, infection, and disease both in humans and in the zebrafish natural host model. The zebrafish model allows for complete observation of Vibrio cholerae infection in a system undisrupted by antibiotic use or immune suppression. El Tor strains colonize the zebrafish intestine for up to 10 days longer than classical strains. Preliminary studies demonstrate VSP-2 is required to observe this phenotype, but VSP-1 is not. By creating targeted regional knockouts of the VSP-2 island, the specific gene(s) essential for enabling prolonged colonization will be identified and applied to the understanding of how El Tor interacts with a natural host. By identifying the genes used by El Tor to colonize a natural host for prolonged periods, we gain insight into how this pathogen may persist in the environment and perpetuate disease.
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Mobile antimicrobial resistance in Neisseria gonorrhoeae
More LessNeisseria gonorrhoeae (the gonococcus) is the causative agent of the sexually-transmitted infection gonorrhoea, and has developed resistance to all classes of antimicrobials. In gonococci, plasmids can mediate high-level antimicrobial resistance (AMR) to tetracyclines and ß-lactams. Plasmids can spread through bacterial populations by transformation and conjugation, resulting in the rapid dissemination of traits. Characterisation of plasmids, including understanding their distribution in bacterial populations, is therefore key to understanding bacterial evolution, and in particular the spread of AMR. N. gonorrhoeae can harbour three plasmids, conjugative (pConj), ß-lactamase (pbla) and cryptic (pCryp). Using genomic and phylogenetic analyses, we show that plasmids are widespread in a large collection of gonococcal isolates from 56 countries. We found that variants of pConj (which can mediate tetracycline resistance) and pbla expressing TEM-135 ß-lactamase are associated with distinct gonococcal lineages. Furthermore, AMR plasmids are significantly more prevalent in gonococci from less wealthy countries. Over 94% of gonococci possess the cryptic plasmid (pCryp), and its absence can be correlated with the presence of a novel chromosomal Type IV secretion system. Our results reveal the extent of plasmid-mediated AMR in the gonococcus, particularly in less wealthy countries, where diagnostic and therapeutic options can be limited, and highlight the risk of their global spread.
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Host environment induces daptomycin tolerance in Staphylococcus aureus
More LessDaptomycin is a last-resort antibiotic for the treatment of invasive diseases caused by methicillin-resistant Staphylococcus aureus. However, despite potent activity in vitro,daptomycin fails to resolve up to 30% of cases of staphylococcal bacteraemia, suggesting that the host environment reduces bacterial susceptibility to the antibiotic. Using human serum as a model of bacteraemia, we demonstrated that the host environment induced daptomycin tolerance via activation of the GraRS two-component system. Testing of various antimicrobial peptides present in serum revealed that the human cathelicidin LL-37 was able to bind GraS, induce GraRS signalling and confer daptomycin tolerance. Activation of GraRS by serum led to daptomycin tolerance via changes in the staphylococcal cell envelope. For example, GraRS-dependent increases in positive surface charge and peptidoglycan content occurred in serum, leading to reduced daptomycin binding. Additionally, incubation in serum led to a Cls2-dependent increase in the cardiolipin content of the membrane which contributed to daptomycin tolerance. Finally, inhibition of both peptidoglycan and cardiolipin synthesis together completely abolished acquisition of daptomycin tolerance in serum, demonstrating that these processes fully explain the tolerance phenotype. In summary, host LL-37 activates staphylococcal GraRS signalling, causing changes in the cell surface which confer daptomycin tolerance. This demonstrates how host defences can compromise antibiotic efficacy, and also provides a rationale for combination therapies to prevent the development of daptomycin tolerance and reduce rates of treatment failure.
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Understanding the Role of Norovirus VP1 in Viral Infectivity and Persistence
More LessHuman noroviruses (HNV) are a prevalent cause of gastroenteritis that contribute to >200,000 deaths each year and cost >£40 billion worldwide per annum. There is currently no approved vaccine or therapy, and a greater understanding of the virus life-cycle could help develop new approaches towards disease control. Although HNV infection is usually self-limiting, persistent infections can establish in immunocompromised people - however the underlying mechanisms are poorly understood. Our studies use the murine norovirus (MNV) model system to investigate fundamental virus biology, and several strains of MNV can also persist in the murine host. The primary receptor for MNV, CD300lf, interacts with a network of amino acids (AAs) on the protruding domain of the virus major capsid protein (VP1). We hypothesised that genetic variations leading to changes within this network of AAs could influence the VP1-CD300lf interaction and viral persistence.
Bioinformatic analysis of the VP1-receptor interface highlighted variation in just a single AA that correlates with persistent MNV strains. To confirm this AA is important for receptor interactions we conducted in vitro evolution experiments on suspension or adherent grown cells. Passage through suspension cells resulted in the selection of hydrophobic residues at this position co-incidental with a 1.5-fold increase in viral titre. In contrast, small polar residues were maintained at this position during passage on adherent cells. Furthermore, infectivity assays with infectious clones suggest that hydrophobic residues favour infection of suspension cells over adherent cells. Work is ongoing to understand the importance of this AA on viral infectivity and persistence.
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Nitrofurantoin-resistant Escherichia coli in the UK: genetic determinants, diversity, and undetected occurrences
BackgroundAntimicrobial resistance in enteric or urinary E. coli might predispose invasive E. coli infection and bacteraemia. Nitrofurantoin resistance occurs in <6% of UK urinary E. coli isolates, however, 2018 national recommendations to prescribe nitrofurantoin for uncomplicated urinary tract infection (UTI) raised concerns for increased prevalence of nitrofurantoin-resistant E. coliin the future. Therefore, we investigated mechanisms of nitrofurantoin resistance in UK E. coli isolates and assessed their occurrences in a large dataset of E. coli genomes.
MethodsTo elucidate chromosomal and acquired genetic determinants of nitrofurantoin resistance in E. coli, we analysed whole-genome sequences of nine randomly selected nitrofurantoin-resistant UTI E. coli isolates from West London. We then performed targeted analysis of 12,412 E. coli genomes collected from across the UK and predicted nitrofurantoin susceptibility from identified genotypes.
ResultsUsing comparative genomics, we found known and novel point mutations or insertion sequences (ISs) in chromosomal genes encoding oxygen-insensitive nitroreductases NfsA and NfsB in the nine isolates. Most of these genetic alterations resulted in gene inactivation. We also identified the same kinds of mutations in NfsA, NfsB, and their associated enzyme RibE in a number of 12,412 E. coli genomes. We also observed homoplasic mutations in all these proteins. By contrast, multidrug efflux pump OqxAB, which confers resistance when horizontally transferred, was only encoded by one genome.
ConclusionsChromosomal de novo mutations and ISs are main causes of nitrofurantoin resistance in UK E. coli. Prevalence of nitrofurantoin resistance should be monitored among urine, blood, and enteric isolates as nitrofurantoin exposure increases.
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Biochemical and Biophysical Characterization of Exoribonucleases from the Human Pathogen Mycobacterium tuberculosis
More LessMycobacterium tuberculosis remains the leading cause of mortality from a single infectious organism, infecting nearly one-third of the global population. The current emergence of multidrug-resistant strains represents a serious health problem nowadays. Moreover, regulation of gene expression through RNA metabolism is a key mechanism for bacterial growth, division and rapid accommodation to environmental conditions. Ribonucleases are enzymes present in all living organisms that play an important role in RNA processing and degradation. Particularly, ribonucleases belonging to the RNB-family are often essential for viability of prokaryotes and are implicated in the establishment of virulence of several pathogens. In the present study we aim to structurally and functionally characterize two putative exoribonucleases from the RNB-family of enzymes inM. tuberculosis. Overexpression and purification of the proteins was performed and further in vitro activity, binding and helicase assays using synthetic RNA substrates were accomplished. In parallel, a biophysical characterization proceeded with several crystallization trials and protein stability tests. We have demonstrated that both RNases are 3’-5’ exoribonucleases with different degradation properties and unravelled the importance of highly conserved residues for catalysis. Moreover, we were able to identify improved buffer formulations that increase protein stability, possibly enhancing their propensity to crystallize. The information regarding RNA metabolism in M. tuberculosis is limited and RNB-family enzymes have not been previously characterized in this important human pathogen. Thus, a complete knowledge of these ribonucleases is an approach to recognize their influence in M. tuberculosis metabolism and to better understand the post-transcriptional control in this pathogen.
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Engineered anti-viral shRNAs are more effective than lhRNAs in transgenic Aedes aegypti
The Aedes aegypti mosquito is a major vector of chikungunya virus (CHIKV), which has no licensed vaccine. Engineered mosquitoes expressing long RNA hairpins (lhRNA) or small RNAs against selected arboviruses have been developed to limit virus replication, but their silencing efficiency has not been compared.
We developed lhRNA and short hairpin RNA (shRNA) arrays against CHIKV non-structural protein nsP2. We used a Tet-response element (TRE) and a tTA transactivator to control expression of lhRNAs (TRE-lhRNA) and shRNAs (TRE-shRNA). Constitutive expression in Aag2 cells was assessed with a PUb-tTA driverand midgut specific expression in transgenic mosquitoes was assessed using Carboxypeptidase A (AeCPA)-tTA as driver. In vitrointerference ability was determined with a CHIKV split replication system, and a synthetic luciferase reporter with mRNA containing the targeted CHIKV sequence (CHIK-FF). In vivo interference was tested by inserting the TRE-lhRNA and TRE-shRNA constructs into a AeCPA-tTA line so both constructs expressed from the same locus, and a TRE reporter line that expressed an AmCyan reporter with an N terminal CHIKV fusion (CHIK-AmC).
In Aag2 cells, shRNAs were more effective than lhRNA in silencing both the CHIKV split replication system (99.7%, S. D. ± 0.47 and 72.8%, S. D. ± 9.50, respectively, P<0.001) and CHIK-FF (98.8%, S. D. ± 0.71 and 50.9%, S. D. ± 7.92, respectively, P<0.05). Similar results were observed in transgenic mosquitoes when comparing AmC expression in the midgut.
This study demonstrates that, in mosquitoes, effectively chosen shRNAs can induce greater interference of the desired viral target than the corresponding lhRNA.
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Multi-omics analysis of the specialised metabolism of two novel Pseudonocardia spp. isolated from the deep Southern Ocean
More LessMultidrug-resistant pathogens have become a global threat. In this context, filamentous Actinobacteria has been proven to be an exceptional source of antimicrobial metabolites. In particular, rare actinomycetes isolated from marine environments have been proposed as a potential source of yet untapped specialised metabolites. In this study, two novel species, Pseudonocardia abyssalis sp. nov. and Pseudonocardia oceani sp. nov, isolated from deep Southern Ocean sediments are described, both in terms of their phenotypic and genomic characterization. Furthermore, the genomic architecture, with a focus on Biosynthetic Gene Cluster (BGC), across eight strains belonging to the two novel species were investigated. A total of 14 Gene Cluster Families (GCF) were identified, of which five GCFs comprise BGCs from both species, and nine were specific to each species. Moreover, a correlation of GCFs to phylogeny was observed. Following genome analysis, a comparative mass-spectrometry based metabolomics analysis was carried out with one strain from each new species, as well as Pseudonocardia ammonioxydans and Pseudonocardia sediminis, also of marine origin. The metabolomics profiles agreed with the GCF distribution, where a group of ubiquitous metabolites were produced by both new Pseudonocardia spp., while groups of species-specific metabolites were also detected. This metabolic-repertoire was found to be elicitated through the addition of N-acetyl glucosamine (GlcNAc), revealing chemically-inducible bioactivity against the fungi Candida albicas and multidrug-resistant Candida auris. These results showcase the power of a combined genomic-metabolomics approach to investigate rare-actinomycetes from understudied locations and have uncovered a wealth of both biosynthetic and chemical diversity for further investigation.
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Respiratory pathogens co-infection in patients with COVID-19 pneumonia in Kazakhstan
More LessBackgroundClinical evidence of the presence of bacterial co-infection in patients with SARS-CoV-2 pneumonia is important for an adequate treatment with antibacterial drugs. Objectives: to determine the secondary bacterial flora in patients with COVID pneumonia in patients in the Republic of Kazakhstan.
MethodsProspective, microbiological, multicenter study, which was conducted at the Medical University of Karaganda in the Shared Resource Laboratory. Sputum samples were collected from three cities of Kazakhstan with the worst SARS-CoV-2 epidemiological situation. Microbiological examination was carried out using classical methods. All investigated isolates were identified to species by MALDI-TOF mass spectrometry. Susceptibility to antibiotics was performed by the disk diffusion method in accordance with the CLSI 2019 recommendations.
Results133 patients were included with a mean age of 60.9±12.7 years old, 53/133 (39.8%) had confirmed SARS-CoV-2 PCR test. Microbiological examination showed the growth of normal microflora in 31.45%. Difference in secondary bacterial co-infection etiology in COVID-19 positive and negative patients was not found. The leading place in general etiological structure belonged to K.pneumoniae - 22.64%, E.coli - 11.95% and A.baumannii - 11.32%. A significant relationship (p = 0.003) between such parameters as the use of antibacterial drugs and the isolated microflora was found.
ConclusionsIsolated microorganisms are etiologically significant and are pathogens of the ESKAPE group. It is important to limit the risk of infection and spread of resistant microorganisms by closely monitoring nosocomial infections and drawing attention to secondary infections caused by resistant bacteria that can increase the mortality rate of patients with COVID-19.
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Different molecular routes to mat formation in environmental Pseudomonas isolates
More LessMany bacterial species secrete polymers and form mat-like structures. These mats can be useful (e.g., in bioremediation), or problematic (e.g., in hospital settings). The molecular bases of mat formation have been investigated in a number of species, including various pseudomonads. One well-characterized example is the plant symbiont Pseudomonas fluorescens SBW25. Laboratory populations of SBW25 readily acquire mutations in one of three regulatory loci (wsp, aws, mws), leading to the over-production of the secondary messenger cyclic-di-GMP. In turn, this activates the production of a cellulose-like polymer, the major structural component of the SBW25 mat. Here, we dissect and compare the molecular mechanisms of mat formation in two further plant-associated pseudomonads: P. simiae PICF7 and P. fluorescens A506. We find that both PICF7 and A506 are capable of mat formation in the laboratory, by distinct molecular routes. Mat formation in PICF7 involves mutations in wsp, aws, or mws that serve to activate the production of Pel (as opposed to cellulose in SBW25). Contrastingly, A506 mat formation does not require mutation of wsp, aws, or mws (despite their retention in the genome). Instead, our results are consistent with a readily reversible, non-mutational route to polymer production and mat formation in A506. Overall, our results demonstrate the presence of multiple molecular routes to mat formation among environmental, plant-associated pseudomonads.
The presented work has since been published: Mukherjee A, Dechow-Seligmann G, Gallie J. 2022. Molecular Microbiology 117(2): 394-410.
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Control Of Hepatitis E Virus Polyprotein Processing By Cellular Proteases
More LessHepatitis E Virus (HEV) is one of the leading causes of acute viral hepatitis, with ~20 million HEV infections worldwide per annum, and mortality rates up to 25% in pregnant women. However, many aspects of the biology of the virus are poorly understood. HEV has a positive-sense single-stranded RNA genome. ORF1 encodes the non-structural polyprotein required for viral RNA replication. This polyprotein (sometimes termed pORF1) is predicted to contain seven domains based on sequence homology to related viruses and it is hypothesised that this polyprotein must undergo proteolysis to generate functional protein units. However, it is unknown if the pORF1 polyprotein undergoes full proteolysis, the potential locations of any cleavage boundaries and whether a viral or host cell protease is responsible.
We have adapted our in vitro-based proteolysis assays to investigate cleavage of HEV pORF1. In comparison to related RNA viruses, our data suggest that pORF1 has no auto-catalytic activity. Previous studies have shown that the liver-produced protease, thrombin, is essential for replication. In the presence of thrombin, we have shown that the ORF1 polyprotein undergoes specific proteolysis to produce eight distinct protein products. Combining bioinformatics with pORF1 truncations/mutagenesis, we have located the position of the pORF1 thrombin cleavage sites. Interestingly, these cleavage sites correspond to the junctions between the predicted pORF1 protein domains. Our data suggests that thrombin is an important cellular protease for controlling pORF1 proteolysis. Work is ongoing to understand the importance of each thrombin cleavage site in viral replication.
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Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation
Paula Corsini, Sunjun Wang, Saima Rehman, Katherine Fenn, Amin Sagar, Slobodan Sirovica, Leanne Cleaver, Charlotte Edwards-Gayle, Giulia Mastroianni, Ben Dorgan, Lee Sewell, Steven Lynham, Dinu Iuga, Trent Franks, James Jarvis, Guy Carpenter, Michael Curtis, Pau Bernado, Vidya Darbari and James GarnettEscherichia coli is a Gram-negative bacterium that colonizes the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and some commensal E. colistrains. It can degrade mucins in the intestine, promotes biofilm maturation and in virulent strains, it is a major determinant of infection, although how it carries out these functions is not well understood. Here we examine SslE from the E. coli Waksman and H10407 strains and using electron microscopy (EM), small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy and biochemical analyses we show that SslE has a highly dynamic structure in solution. We also directly observe acidification within mature biofilms, describe a mechanism where SslE forms unique functional fibres under these conditions and determine that these SslE aggregates can bind cellulose, a major exopolysaccharide of many E. coli biofilms. Our data indicates that the spatial organization of SslE polymers and local pH are critical for biofilm maturation and SslE is a key factor that drives persistence of SslE-secreting bacteria during acidic stress.
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Bacterial diversity of two types of Wagashi, a traditional Beninese cheese, using High-Throughput Amplicon Sequencing
More LessWagashi, also called Gassiréin the local Fulfulde language, is a traditional cheese produced from cow milk in Benin. For its preparation, milk is heated and coagulated with Calotropis procera extract. After coagulation, the heated curd obtained is drained, moulded and stained, or not, using Sorghum vulgare or Sorghum codatum panicles. It is the most commonly consumed dairy product as surrogate for meat or fish. In order to get better insights into the bacterial diversity of Wagashi, Illumina MiSeq amplicon sequencing targeting the V1-V2 region of the bacterial 16S rRNA gene was performed on two different Wagashi types (stained and not stained) from Benin. The results showed that the Lactobacillaceae (60%) and Streptococcaceae (38%), were the most abundant bacteria in the unstained cheese, whereas Streptococcaceae (89%) and Bacillaceae (10%) were the most prevalent in stained cheese. Moreover, at the species level, the microbial community structures of stained and unstained cheeses were significantly different. The main differentiating species were Lactobacillus bulgaricus, Streptococcus thermophilus, and Lactobacillus fermentum in unstained cheese, compared to Streptococcus infantarius, Aeromonas sp., Bacillus cereus, Bacillus subtilis and S. thermophilus in stained cheese. This work provides valuable insights into microbiology of cheese Wagashi and shows that the staining of this dairy product affects its bacterial composition. In particular, the presence of potential useful Lactobacilli and Streptococci in unstained cheese is to be further investigated for selecting functional bacteria for potential biotechnology applications. On the contrary, the presence of Bacilli in stained cheese needs additional study to detect potential foodborne strains or pathotypes.
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Actin dynamics regulate proteasome homeostasis
More LessCells require thousands of unique proteins to be in the right place, at the right time, in the right amounts and with the right modifications. They do this through several processes collectively known as protein homeostasis. TORC1 is a principal regulator of protein homeostasis, coordinating protein synthesis and degradation. The proteasome, composed of a core particle and one or two regulatory particles, degrades unwanted protein. Diverse stresses cause a protein homeostasis imbalance: inhibiting TORC1 and the misfolded/damaged protein load. Following TORC1 inhibition, proteasome regulatory particle assembly chaperone (RPAC) translation is increased and thus cells assemble more proteasomes to degrade the damaged and misfolded proteins, thereby restoring protein homeostasis.
Using yeast, we identify an endocytic protein, Ede1, that interacts with and is critical for translation of RPAC mRNA following TORC1 inhibition. We find two further endocytic proteins important for RPAC translation regulation. Mutants of these proteins cause altered Arp2/3 activity, and hence altered formation of actin patches/endocytic sites. We show that RPAC mRNA is transported on actin cables and interacts with actin patches. TORC1 inhibition depolarises the actin cytoskeleton, causing RPAC mRNA accumulation on actin patches concurrent with translation. We demonstrate Ede1 is essential for RPAC mRNA localisation regulation following rapamycin treatment.
This work shows that, upon actin depolarisation, RPAC mRNA is recruited to actin patches, likely by Ede1, and translation occurs. Actin regulation is therefore a key element of proteasome (and therefore protein) homeostasis.
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Intraspecies-Resolution Metabarcoding: automated primer design and a plant pathology case study
We present results demonstrating species-specificity and sub-species resolution by novel, automatically-designed metabarcoding primers for environmental DNA analysis.
Conventional metabarcoding remains a cornerstone of rapid, high-throughput environmental DNA (eDNA) community analysis and biodiversity assessment. Standard barcodes such as 16S (prokaryotes) and ITS1 (fungi/oomycetes) have been instrumental in identifying the complex composition of communities using total eDNA. However, standard barcodes have limitations in terms of resolution and quantitation and, though genus-level identification can be reliable, species-level identification is often not possible.
To overcome the limitations of resolution, we implemented extensions to the diagnostic primer design tool pdp (https://github.com/widdowquinn/find_differential_primers [https://github.com/widdowquinn/find_differential_primers]) that enable automated design of metabarcoding markers and corresponding primers that are (i) specific to a prescribed taxon at species level and (ii) capable of discriminating between members of the same species. This allows for rapid, high-throughput measurement of diversity below species level for a target organism.
We aimed to survey geographical distribution and pathogen transfer of the widespread plant pathogenic bacterium Pectobacterium atrosepticum (Pba). This organism has considerable sub-species taxonomic structure identifiable using MLST and with whole-genome methods, but which is not accessible using standard barcodes. We designed metabarcoding primers (202bp) specific to Pba using `pdp`, and established that these have resolution comparable to eight-gene MLST, revealing sub species-level diversity within single fields, and on the same individual plant host.
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Defining the pknH Transcriptional Network in the Animal Tuberculosis bacillus, Mycobacterium bovis
More LessThe Mycobacterium tuberculosiscomplex (MTBC) is a group of bacteria that show more than 99% genetic identity, yet they diverge in their host preference and the severity of associated disease. Mycobacterium bovis causes the disease in cattle and poses an economic challenge, with the cost of Irelands TB Eradication Scheme predicted to exceed €90 million in 2020.
The aim was to focus on the RD900 and pknH gene network in MTBC members. ThepknH gene encodes a transmembrane serine threonine protein kinase, that plays a role in the regulation of signalling pathways in the mycobacterial cell and has been linked to virulence. The RD900 or “region of difference 900” contains the tbD2 and pknH2 genes. The part of the genome that contains this region varies substantially across MTBC members.
The results of RNA-seq analysis showed that there are statistically significant levels of differential gene expression in wildtype and knock-in M. bovis strains.
The RD900 analysis across the MTBC showed that there is variation in this important genomic region. Analysis carried out by Mata et al. 2020, showed that this RD900 region had been independently lost in different MTBC lineages and strains. M. bovis has retained the RD900 region and this is believed to be linked to its increased virulence.
The majority of the M . africanum strains were found to possess the standard pknH, tbD2, pknH2, embR gene arrangement as is found in M. africanum GM041182, the strain that is typically used as a reference.
Based on work of Mata et al., 2020.
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Localization dynamics of N-acetylglucosamine transporter, Ngt1 in Candida albicans
More LessThe amino sugar N-acetylglucosamine (GlcNAc), widely present in multiple cell-surfaces is efficiently utilized by human pathogen Candida albicans at the sites of infection as a survival strategy in different host niches. GlcNAc import inside the cell is mediated by GlcNAc transporter, Ngt1. In this present study, to investigate the Ngt1 dynamics, we have checked the sensitivity of Ngt1 for GlcNAc, expression kinetics and dynamics of Ngt1, stability of Ngt1 in the presence of unrelated carbon source like, glucose and endocytic trafficking of Ngt1. For this study we have used epi-fluorescence microscopy, Western blot and Northern blot analysis. We have observed that Ngt1 expression is prolific and highly sensitive to even minute amount (<2 μM) of GlcNAc. Ngt1 maintains its turnover in the plasma membrane by sugar stimulated endocytosis via UBI4 (polyubiquitin encoding locus) mediated ubiquitylation. Co-localization studies with different sub-cellular markers have revealed that Ngt1 follows a trafficking route via early endosome-late endosome-multi vesicular body (MVB)-Trans Golgi network for degradation. In conclusion, the study will provide a better understanding of endosomal trafficking for other membrane proteins in particular sugar transporters and also will open new therapeutic aspects in Candida albicans.
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Redundancy of AcrA and AcrE to support efflux of antibiotics through AcrD in Salmonella Typhimurium
More LessRND efflux pumps are important mediators of antibiotic resistance. RND efflux pumps including AcrB, are organized as tripartite systems, consisting of an inner membrane RND transporter, a periplasmic adaptor protein (PAP) and an outer membrane factor. We have previously identified the residues required for binding of the major PAP AcrA to the major RND pump AcrB and shown that there is promiscuity between the PAPs such that they can function with non-cognate pumps. AcrE is a PAP homologous to AcrA and AcrD and AcrF are RND pumps homologous to AcrB. This study aimed to determine whether the PAP AcrE can function with AcrD, which does not have its own PAP, to mediate efflux and whether the previously identified RND binding residues in AcrA and AcrE were also required for AcrD binding. The acrD and acrE genes were cloned into compatible vectors and co-transformed into a strain lacking acrAB, acrD and acrEF. When expressed together, acrDand acrE significantly decreased susceptibility of the efflux mutant strain to AcrD substrates including aztreonam, carbenicillin, cloxacillin, fusidic acid, nafcillin, novobiocin, oxacillin and ticarcillin, indicating that AcrE can also form a functional complex with AcrD. These experiments also highlighted that the substrate profile of AcrD in S. entericaand E. coli are different. Point mutations in the previously defined RND binding residues of AcrA and AcrE impaired AcrD-mediated efflux of substrate drugs which validates the interchangeability of AcrA and AcrE and highlights these residues as ideal drug targets for efflux inhibition to combat antimicrobial resistance.
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Transcriptional profiles of Streptococcus pneumoniae associated with adaptation to the nasopharynx environment
More LessStreptococcus pneumoniae is a major cause of global morbidity and mortality. It behaves as a commensal in the host nasopharynx, but can become pathogenic, invading the lungs, blood, and meninges. As such, identification of pneumococcal virulence and colonisation factors remains a major research objective. We previously described an experimental evolution approach for the identification of pneumococcal genes that make niche-specific contributions to fitness and virulence. Sequential passage of pneumococci through mouse models of nasopharyngeal-carriage and pneumonia was performed, generating bacterial lineages adapted to the nasopharynx and lungs, respectively.
Using RNA-Seq differential gene expression analysis, this study compared the transcriptomic profile of a nasopharynx-evolved pneumococcal lineage that showed evidence of enhanced nasopharyngeal colonisation potential, with a lab-adapted ancestor strain. Here, we describe how the genomic adaptations acquired by this lineage, and which we have demonstrated facilitate survival in the nasopharynx, can influence bacterial gene expression.
One key finding was the identification of five adjacent upregulated genes, representing a putative pneumococcal operon. These poorly characterised genes are predicted to encode a carbohydrate-scavenging pathway. Expression of the operon within the nasopharynx may facilitate sugar acquisition from host glycoproteins. Of note, the nasopharynx evolved pneumococcal lineage carries a single nucleotide insertion mutation immediately adjacent to the -10 element of the operon predicted promotor sequence. This may contribute to increased operon gene expression in the nasopharynx-adapted pneumococcus, thereby enhancing colonisation potential. Confirmatory mechanistic investigations are underway, which will aid the identification of pneumococcal virulence factors involved in the commensal to pathogen switch.
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Diversity Analysis and Genome Mining of Microbial 16S rRNA Gene and Metagenomic Data for the Discovery of Novel Antibiotics
More LessThere is an urgent need for new antimicrobials due to constantly advancing antimicrobial resistance. Here, we worked with environmental samples from diverse habitats including different savannah and forest soils, volcanic caves, and termite mounds and assessed their microbial communities for the potential of biosynthesis of secondary metabolites. We analysed and compared microbial composition by applying the QIIME2 pipeline to 16S rRNA gene data. We focused on the abundance of Actinobacteria and Streptomyces as they are important producers of antimicrobials. Out of the samples analysed, the highest abundance of Actinobacteria was found in termite mound and volcanic cave samples. Moreover, the termite mound samples also had the highest abundance of Streptomyces. When comparing microbial composition, soil samples and termite mound samples each formed their own clusters, but volcanic cave samples appeared more dispersed. We assessed the antimicrobial potential of a subset of samples by analysing metagenomic data to predict biosynthetic gene clusters (BGCs) using antiSMASH5.2.0, which resulted in over 800 hits per sample. This number was narrowed down by evaluating identified BGCs based on antimicrobial potential, completeness, size, presence/absence of regulatory and transport-related genes, and dissimilarity with known BGCs. This resulted in an average of 20 BGCs per sample. These BGCs will be subjected to further sequence-based analyses before attempting heterologous expression. Following successful expression, antimicrobial potential will be assessed by screening for growth inhibition of multidrug resistant E.coli strains and the ESKAPE pathogens.
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Investigation of the impact of food preservatives on avian pathogenicEscherichia coli(APEC) and their role in driving zoonotic disease
More LessThe excessive use of antibiotics in agriculture is routinely described as a major contributor to bacterial antimicrobial resistance. Globally, antibiotics are also widely used as growth supplements in livestock. This has led to concerns regarding use of human-use antibiotics in food and food-producing animals. In more recent times organic acids such as propionic acid (PA) and formic acid (FA) have been used as alternative antimicrobials or preservatives in place of antibiotics.
PA is a short chain fatty acid naturally abundant in the human and animal intestine as a breakdown product of non-digestible carbohydrates. In the human intestine it plays important roles in regulating the immune response in the human body. Recently, a study has shown that exposure of a Crohn’s Disease associated bacterial pathotype, adherent-invasive Escherichia coli(AIEC),to PA significantly altered its phenotype resulting in increased adhesion and invasion of epithelial cells and increased persistence through biofilm-formation.
AIEC are both evolutionarily and phylogenetically related to avian pathogenicEscherichia coli (APEC). PA and FA use is widespread in chickens a known source of zoonotic disease. Our results indicate that virulence of some APEC strains is increased by exposure to alternative antimicrobials such as PA and FA. This included increased adhesion of APEC strain E. coli 601 to human intestinal epithelial cells after exposure to FA which could be a potential risk of zoonotic disease. Further investigation of APEC strains is currently underway in a fermentation model of the poultry gut. This approach will improve our understanding of how commonly used.
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Diversity and dynamics of particle-associated and free-living bacteria in eelgrass (Zostera marina) bed along the coast of Japan
More LessAs the seagrass leaves remain underwater, the primary source of leaf microbes is considered to be seawater heterotrophic bacterioplankton, which possess the ability to degrade biopolymers and are known to attach to surface and form biofilms. In this study, 16S rRNA gene amplicon sequencing was used to assess bacterial diversity and dynamics of the particle associated (PA) and free-living (FL) fraction of the seagrass-covering seawater (inside) and bulk seawater (outside) among different seagrass bed around Japan. Samples were collected from the three Zostera marinabeds (Ikuno-shima Is., Hiroshima; Nanao Bay, Ishikawa; Mutsu Bay, Aomori Prefecture) around Japan during summer (June-August 2015; July 2016). Prokaryotic DNA was extracted from samples using a FastDNA spin kit according to the manufacturer’s protocol. After extracting DNA, 16S ribosomal RNA (16S rRNA) genes were sequenced by Illumina Miseq platform. The Results showed that PA bacterial communities had a higher (p <0.001) diversity than FL ones. Compared to the outside of the seagrass bed, the inside had lower diversity both in PA and FL fraction. Taxonomic analysis revealed a different community composition between lifestyle (PA vs FL) and sampling point (inside vs outside). Differential abundance analysis showed that PA were significantly enriched in a diversity of Cyanobacteria (Synechococcaceae), Saprospiraceae and Hyphomonadaceae. Conversely, FL were more abundant in Gammaproteobacteria (including Halomonadaceae, Alteromonadaceae), Microbacteriaceae, Campylobacteraceae, Pelagibacteraceae,Acidimicrobiia(OCS155). The present data provide a comprehensive description of the PA and FL microbial community in the seagrass bed and can be useful for better understanding the seagrass microbe interactions.
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Teaching Microbial Genomics in the COVID-19 age: principles, theory, and practice
More LessThe importance of genomics in the COVID-19 age cannot be overstated and genomics has a key place in the advanced undergraduate microbiology curriculum. The COVID-19 pandemic, and its attendant lockdowns, have necessitated a change in the delivery of our microbial genomics module. The key challenges for delivering a remote computer-based genomics module, are ensuring student engagement and learning; and supporting the technical aspects of remote computer work.
In the absence of in-person classes, we have adapted our material covering the principles and theory of microbial genomics, to asynchronous videos and synchronous online workshops. Practical training for all undergraduate microbiologists in the UK has been severely reduced, which has forced us to focus skills training towards computational aspects of genome assembly, analysis, and interpretation. The set up and implementation of a robust and scalable Linux-based genome analysis pipeline for students presents many challenges: from organising remote access to computer clusters; software support; and managing hardware.
Assessment for the module is based on the demonstrating learning and skills development through the analysis of SARS-CoV2 genomes to identify spike protein variation and mapping this to published structures. Terminal assessment is through the analysis of newly sequenced bacterial genomes and the preparation of a genome report suitable for publication.
We will outline the implementation and management of our Microbial Genomics module as a model for computer-based skills training for undergraduate microbiologists. Furthermore, we will discuss the impact of the COVID-19 pandemic on the module and the opportunities it has presented.
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Improving phage genome annotation to understand phage biology: the case of Pseudomonas aeruginosa LES prophages
More LessPseudomonas aeruginosais an important opportunistic pathogen, causing nosocomial infections. The Liverpool Epidemic Strain (LES), a major cause of mortality and morbidity in cystic fibrosis patients, harbours five prophages associated with increased fitness and survival in models of infection. However, ~76.5% of the LES prophage genes are hypothetical proteins. Also, little is known about the LES prophage interactions with the lysogen and other prophages. In this study, we used the VIral Genome Annotation (VIGA) pipeline to re-annotate the LES prophage genomes and improve the prediction of gene function. The RefSeq viral proteins database was used for homology-based gene prediction and the Prokaryotic Virus Orthologous Genes (PVOGs) and Reference Viral DataBase (RVDB) were used for HMM-based protein function prediction. InterProScan 5.47-5.82 and Infernal 1.1.3 (with Rfam 14.3) were applied to enrich the gene function prediction for all genes and ncRNA elements, respectively. The number of putative coding sequences had increased by 1.17-1.43 times. Multiple genes related to DNA recombination and host cell lysis were identified in this reannotation. Also, we have identified new ncRNA elements in these prophages, such astRNAs in prophages 2 and 5 and a putative regulatory ncRNA, a Hammerhead-II ribozyme, in prophage 4. All this new information will be combined with data from future RNAseq experiments to map the expression profiles of each LES prophage under inducing and non-inducing conditions to characterise interactions between the prophages and their lysogen host.
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Development of CRISPR/Cas9-based Novel Vaccines against Poultry Viruses
More LessVaccines remain the primary means of disease prevention throughimmunisationschemes in the poultry sector. Novel approaches in vaccine development, such as reverse genetic systems and genome editing technologies (i.e. CRISPR/Cas9), are currently being utilized to overcome challenges in establishing an immunogenic platform that is safe and capable of inducing long-term immunity. The CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats associated protein nuclease 9) technology offers an effective, fast and simple novel approach to edit genomes for the development of viral vectors against poultry diseases such as Newcastle disease (ND), avian influenza (AI) and infectious bronchitis (IB). In this study, we demonstrate the application of CRISPR/Cas9-based genome editing to generate recombinant viral vectors that express the F gene of NDV. Validation of gene integration, protein expression, and insert stability was carried out by PCR, immunostaining and Western blot analysis, respectively. This approach offers an efficient platform for the generation of multivalent recombinant vaccines that can provide simultaneous protection against major poultry diseases.
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How do bacteria change their coats? Structural analysis of acyltransferases involved in O-antigen modification
More LessAcyltransferase-3 (AT3) domain-containing proteins are involved in acylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, antimicrobial resistance, and antibiotic biosynthesis. Despite this, the mechanism of action is largely unknown. AT3 proteins fromSalmonellaspp. are responsible for acetylation of lipopolysaccharides which can generate a specific immune response upon infection. Here we analysed two AT3 proteins fromSalmonellaspp., some differences exist but both contain an integral membrane AT3 domain fused to a periplasmic SGNH domain. Identification of essential residues from each domain suggests both domains are required for acylation.
The crystal structure of the SGNH domain and periplasmic linking region was determined. Novel structural features are seen in comparison to other SGNH domains. In particular, the periplasmic linking region is structured and forms an extension of the SGNH domain (SGNH-extension). Removal of the SGNH-extension suggests that this region is important for stability of the SGNH domain. The structure of the SGNH-extension suggests the SGNH domain is in close proximity to the acyltransferase domain and the domains may interact. In silicoco-evolution analysis, used to make predictions about the structure of both domains, suggests likely inter-domain interactions. This analysis also predicted which transmembrane helices in the acyltransferase domain interact giving an insight into the overall structure.
Combining these data we propose a refined model of AT3-SGNH proteins which enhances our understanding of the mechanism and function of AT3 proteins required for modification of cell-surface carbohydrates.
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genomeRxiv: a microbial whole-genome database and diagnostic marker design resource for classification, identification, and data sharing
genomeRxiv is a newly-funded US-UK collaboration to provide a public, web-accessible database of public genome sequences, accurately catalogued and classified by whole-genome similarity independent of their taxonomic affiliation. Our goal is to supply the basic and applied research community with rapid, precise and accurate identification of unknown isolates based on genome sequence alone, and with molecular tools for environmental analysis.
The DNA sequencing revolution enabled the use of cultured and uncultured microorganism genomes for fast and precise identification. However, precise identification is impossible without
1. reference databases that precisely circumscribe classes of microorganisms, and label these with their uniquely-shared characteristics
2. fast algorithms that can handle the volumes of genome data
Our approach integrates the highly-resolved classification framework of Life Identification Numbers (LINs) with the speed and computational efficiency of sourmash and k-mer hashing algorithms, and the precision and filtering of average nucleotide identity (ANI). We aim to construct a single genome-based indexing scheme that extends from phylum to strain, enabling the unique and consistent placement of any sequenced prokaryote genome.
genomeRxiv includes protocols for confidentiality, allowing groups to identify and announce the identities of newly-sequenced organisms without sharing genome data directly. This protects communities working with commercially- and ethically-sensitive organisms (e.g. production engineering strains, potential bioweapons, and to enable benefit sharing with indigenous communities).
genomeRxiv will also provide online capability to design molecular diagnostic tools for metabarcoding and qPCR, to enable tracking of specific groupings of bacteria directly in the environment.
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Regulatory cascade in SaPI activation
Staphylococcal pathogenicity islands (SaPIs) are mobile genetic elements encoding superantigens and other toxinsand are induced for excision, replication, packaging and intercell transfer by phage-encoded anti-repressors that counter the SaPI master repressor. Though SaPI induction has heretofore been assumed to be the exclusive province of helper phages, we report here the remarkable discovery that one of the SaPIs, SaPI3, can instead be induced only by a second, co-resident SaPI, which must first be induced by a phage. This induction cascade thus represents intricate regulatory triad; SaPI3, the beneficiary of this intracellular largess, is the prototype of a hitherto uniquely immobile SaPI lineage. We report that members of this lineage are controlled by a novel regulatory module and are induced by a highly conserved but previously uncharacterised SaPI protein. SaPI3 and its cousins are thus SaPI satellites, just as most other SaPIs are phage satellites.
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Amoeba & Biofilms in UK Chlorinated Drinking Water Distributions Systems: Impact on Water Safety
More LessAmoeba-related diseases have been related with the presence of certain amoebas in domestic water, including drinking water. Biofilms in drinking water distribution systems are able to support amoeba growth by providing a food source and protecting them against disinfectants. Additionally, amoeba growth can be favoured by warm temperatures and climate change appears to contribute to its geographic spread.
The presence of amoeba and its association with potential pathogenic bacteria was studied in a real-scale chlorinated DWDS. The test facility comprised three independent pipe loops fed with water from the local water supply and for this study a varied flow hydraulic profile was applied based on daily patterns observed in real UK distribution networks. The daily regime was repeated for a biofilm growth phase of 30 days. Amoeba viability was tested by a culture-based method, non-nutrient agar (NNA)-E. coli plates, and then confirm by qPCR using specific primers to detect species of amoeba including Naegleria and Acanthamoeba. Amplicon sequencing of the 16s rRNA gene was used to characterise the biofilm and planktonic bacterial communities.
Several amoeba species belonging to the genera Acanthamoeba, Vermamoeba and Naegleria were identified in 30-day old biofilm samples. While the bacterial communities in biofilms were dominated by Variovorax, Pseudomonas and Aquabacterium. This study yielded new insights on the dynamics of amoeba and bacterial communities in DWDS. However, more research is required to accurately establish the impact of these inter-kingdom associations on human health.
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Antimicrobial resistance: revisiting the mechanisms of resistance
Resistance to antibiotics persists as a critical challenge in public health. Currently, the emergence of multi-drug resistant (MDR) bacteria is a primary concern globally, resulting in a dramatic increase in epidemiological relevance and importance of nosocomial and chronic infections. Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae has recently been classified as critical in the World Health Organization (WHO) priority pathogens. Among these bacterial pathogens, resistance seems to be a natural trait. The acquisition and development of resistance by bacteria is through several mechanisms. The genetic background and intrinsic resistance mechanisms largely contribute to competitive advantage and resistance in a highly resistant pool. The acquisition of resistance genes driven by mobile genetic elements (MGE) and several biochemical mechanisms also plays a central role in resistance development among pathogenic bacteria. This review discussed the recent underlying multiple resistance mechanisms among the priority pathogens. This review also provides an up-to-date regional epidemiological data and implications of antimicrobial resistance. Given the severity of infections caused by these bacteria, their less susceptibility to the available antimicrobials, and the limited antimicrobial arsenal to treat these pathogens, current insight on resistance mechanisms becomes timely and highly relevant. This information will help develop better therapeutic strategies against resistance microbes, especially those of urgent priority.
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Beyond fluorescein: Use of fluorescent protein calibrants for direct and absolute quantification of protein production in synthetic biology
More LessWhile inter-lab calibration standards are approaching mainstream usage in synthetic biology, such calibrations are not in fact sufficient for absolute protein quantification required for modelling synthetic circuits. Fluorescein-based calibration of plate reader and flow cytometry instruments allows the measurement of green fluorescent protein (GFP) in synthetic cells to graduate from arbitrary units to calibrated units, but retains important caveats. Fluorescein is only a good calibrant for green FPs, leaving other FPs uncalibrated, and only provides conversions to units of brightness, not to molecule numbers.
Ideal assay calibrants in molecular biology consist of the same molecule as the one to be measured – in this case, a purified preparation of the appropriate fluorescent protein. Here we show that by using purified FP calibrants, all protein species in a synthetic circuit can be quantified in absolute terms using no advanced instrumentation. We develop a SEVA (Standardised European Vector Architecture)-based expression vector that allows the high-level production of soluble protein and describe a straightforward 2-day protocol for the purification of micrograms of FP, followed by a calibration that relates fluorescence activity to protein mass.
We validate this protocol by calculating conversion factors for a panel of commonly-used FPs including superfolder GFP, mCherry, mScarlet-I and mTagBFP2 on multiple laboratory instruments, and use these calibrations to debug synthetic circuits. We also demonstrate that the suspected bias of the presence of mCherry on OD600 measurements is real, but in practice requires extreme overexpression (~100,000 proteins per cell) to have a meaningful impact on cell density estimates.
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Investigation of the Localisation of FtsZ in Pathogenic E. coli Upon Bacteriophage Addition in a Human Cell Model as a Biomarker for Antibacterial Agents
More LessAntimicrobial resistance is a growing problem worldwide and has created a need for novel antibacterial agents and strategies. Escherichia coli is one of the most common Gram-negative pathogens and is responsible for infection leading to neonatal meningitis and sepsis. The FtsZ protein is a bacterial tubulin homolog required for cell division in most species, including E. coli. Agents that block cell division have been shown to mis-localise FtsZ, including the bacteriophage λ encoded Kil peptide, resulting in defective cell division and a filamentous phenotype, and therefore FtsZ may be an attractive target for new antimicrobials.
In this project, we are interested in studying the localisation of FtsZ in pathogenic E. coli in the presence and absence of human cell cultures, in order to establish how and if this localisation changes upon infection. We are also interested in whether bacteriophages specifically attacking pathogenic E. coli have an effect on the localisation of FtsZ in a human cell environment and want to study the mechanism of this process. We have observed E. coliFtsZ localising to the cell midbody as a ring in both a K12 strain and in the K1 strain EV36 using confocal microscopy. These strains were used to infect human cerebral microvascular endothelial cells (hCMEC/D3) to create a meningitis model. We will present our results showing the effect of the Kil peptide and bacteriophages on this localisation within the model system, in an effort to validate FtsZ as a potential biomarker for antibacterial agents.
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The ecology and antimicrobial resistance of Staphylococci colonising neonates
Coagulase Negative Staphylococci (CoNS) are common commensals of human skin, account for nearly 20% of the microbiota in infants and are thought to promote early immune responses in healthy babies. However, CoNS are opportunistic pathogens and in the UK between 2005 and 2014 were responsible for 57% of episodes of Late Onset Sepsis (LOS). In neonatal intensive care units (NICUs) this is a major concern and antiseptics are used to prevent vascular catheter infections. Chlorhexidine (CHX) and Octenidine (OCT) are the most common agents used for skin antisepsis, but evidence is emerging of antiseptic tolerance amongst CoNS.
We undertook a longitudinal survey of CoNS from skin and rectal swabs isolated from babies in two NICUs from countries with different antiseptic regimens (UK and Germany). Over 1000 isolates were characterised for antimicrobial susceptibility and sequenced. The most frequent species isolated were S. epidermidis and S. haemolyticus with similar strain types present in both units. Reduced susceptibility to CHX and OCT was observed in UK isolates (where CHX is used), compared to German isolates (where OCT is used).
Analysis of genome data using GWAS and clustering techniques has identified loci associated with antimicrobial susceptibility. Comparison of isolates taken on admission and thereafter, demonstrated that babies acquired isolates with decreased antiseptic tolerance after admission. This data provides new information about the phylogeny of CoNS in NICUs and suggest different potentials for selection of resistance between antiseptics commonly used in neonatal care.
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Vegetables and rhizospheres as the reservoir for extensively drug-resistant (XDR) Pseudomonas aeruginosa
More LessPseudomonas aeruginosais an urgent threat pathogen due to its evolving resistance to multiple antibiotics. Agricultural soil and plants are the vast reservoirs of this much-dreaded opportunistic bacterium. A few human isolates of P. aeruginosa are known to infect plants and insects. However, there is no report on the occurrence of multi-drug resistant P. aeruginosa in edible vegetable crops. This study compared 18 P. aeruginosa isolates from the rhizosphere and endophytic niches of four different vegetable crops (cucumber, tomato, eggplant, and chili) with three known clinical strains. All the isolates were tested for virulence traits such as resistance to various antibiotics classes, motility, biofilm, and production of virulence factors (rhamnolipid, pyocyanin, hemolysin, proteases, and lipases). Hierarchical clustering based on Ward minimum variance with Manhattan distance matrix grouped the strains into three clusters based on their phenotypic traits. Strains were exhibiting the highest virulence co-clustered with the human pathogenic isolates. These strains were resistant to cephalosporins, aminoglycosides, macrolides, nitrofurans, tetracyclines, and sulfonamides. These extensively drug-resistant (XDR) strains were only susceptible to polymyxin (colistin) and quinolone (cephalosporin). This study shows that the virulence traits are shared between plant- and human-isolates of P. aeruginosa. More importantly, the occurrence of XDR strains in vegetable crops is a serious global threat.
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Exploring bacterial diversity via a curated and searchable snapshot of archived DNA
The open sharing of genomic data provides an incredibly rich resource for the study of bacterial evolution and function, and even anthropogenic perturbations such as the widespread use of antimicrobials. Whilst these archives are rich in data, considerable processing is required before a biological question can be addressed. Here, we have assembled, quality controlled and characterised 661,405 bacterial genomes that were in the European Nucleotide Archive (ENA) at the end of November of 2018, using a uniform standardised approach. A searchable index has been produced, facilitating the easy interrogation of the entire dataset for a specific gene or mutation. Our analysis shows how uneven the species composition is within this database, with just 20 of the total 2,336 species making up 90% of the high-quality genomes. The over-represented species tend to be acute/common human pathogens, often aligning with research priorities at different levels from individuals with targeted but focussed research questions, areas of focus for the funding bodies or national public health agencies, to those identified globally as priority pathogens by the WHO for their resistance to front- and last-line antimicrobials. Whilst this is a rich resource which often forms the context or references for multi-‘omic’ studies and supports discovery research in many domains, understanding the actual and potential biases in bacterial diversity depicted in this snapshot, and hence within the data being submitted to the public sequencing archives, is essential if we are to target and fill gaps in our understanding of the bacterial kingdom.
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A study on the antibacterial efficacy of Halohydantoin-containing foams
More LessHeterocyclic N-halamine compounds such as 1-monochloro-5,5-dimethylhydantoin (MCDMH) and 1,3-dichlor-5,5-dimethylhydantoin (DCDMH) show antimicrobial activity and odour control. Both MCDMH and DCDMH stabilise and display oxidative chlorine (Cl+1) and, in aqueous environments, generate hypochlorous acid (HOCl). It is well known that HOCl, produced by neutrophils as part of the respiratory immune response, is used as a defence mechanism against foreign bodies. This study aimed to investigate the antibacterial efficacy of proprietary foaming formulations containing mixtures of MCDMH and DCDMH provided by MedeSol Inc., as a potential delivery mechanism for HOCl. The efficacy of the foams containing active chlorine concentrations of 350 ppm and 1050 ppm was tested against single and multi-species bacterial mixtures of S. aureus, P. aeruginosa, E. coli, E. faecalisand K. pneumoniae. Cellulose filters were contaminated with single/multispecies bacterial suspensions and left to air-dry; the two foam concentrations were applied for 15, 30 and 60 seconds at a time. Samples were taken and the total viable count method was employed to determine antibacterial efficacy of the foams. Following the use of both foam concentrations, no colonies were recovered at any time-points in both single and multi-species experiments. In contrast, the negative controls where the foam application was omitted, resulted in high numbers of bacterial colonies. MCDMH-containing foams have very strong antibacterial potential as a result of active chlorine release. Further studies are needed to determine concentration dependence and use in other applications including hand sanitation.
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Investigation of the influence of plant tissue damage on the attachment of Listeria to fresh salad produce
More LessVegetables and fruits are an important for a healthy life due to them containing nutritionally important minerals and vitamins. Green salads (spinach, lettuce) are popular convenience consumer products, however evidence is increasing that these ready to eat salads have been identified as a source of foodborne illness. There are many pathogenic bacteria which are transmitted by food that can cause serious disease. One of the most important is Listeria monocytogenesbecause it can cause invasive infections and especially because of its ability to grow at low (food refrigeration) temperatures. The aim of this study is to investigate whether compounds released from damaged fresh salad leave have an effect on Listeria growth and virulence. The results in this report show that Listeria was highly responsive to the salad extracts and that growth and biofilm formation were both increased compared to un-supplemented control cultures. Plant derived chemicals also stimulated Listeria growth in serum-SAPI medium. The results also showed that Listeria treated with salad extract was more virulent in a Galleria mellonella infection model.
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How to sequence 10,000 bacterial genomes and retain your sanity: an accessible, efficient and global approach
Blanca Perez-Sepulveda, Darren Heavens, Caisey Pulford, Alexander Predeus, Ross Low, Hermione Webster, Gregory Dykes, Christian Schudoma, Will Rowe, James Lipscombe, Chris Watkins, Benjamin Kumwenda, Neil Shearer, Karl Costigan, Kate Baker, Nicholas Feasey, Jay Hinton, Neil Hall and The 10KSG ConsortiumNon-typhoidal Salmonella(NTS)are typically associated with enterocolitis and linked to the industrialisation of food production. In recent years, NTS has been associated with invasive disease (iNTS disease) causing an estimated 77,000 deaths each year worldwide; 80% of mortality occurs in sub-Saharan Africa. New clades of S. Typhimurium and S. Enteritidis have been identified, which are characterised by genomic degradation, altered prophage repertoires and novel multidrug resistant plasmids. To understand how these clades are contributing to the burden and severity of iNTS disease, it is crucial to expand genome-based surveillance to cover more countries, and incorporate historical isolates to generate an evolutionary timeline of the development of iNTS.
We developedand validateda robust and inexpensive method for large-scale collection and sequencing of bacterial genomes. The “10,000 Salmonella genomes” project established a worldwide research collaboration to generate information relevant to the epidemiology, drug resistance and virulence factors of Salmonellae using a whole-genome sequencing approach. By streamlining collection of isolates and developing an efficient logistics pipeline, we gathered 10,419 clinical and environmental isolates from collections in low and middle-income countries within six months. Genome sequences are now available for isolates from 51 countries/territories dating from 1949 to 2017, with ~80 % representing African and Latin-American datasets. Our method can be applied to other large sample collections that require maximisation of resources within a limited timeframe. Detailed genome analyses are in progress and it is hoped that the resulting data will contribute to public health control strategies in low and middle-income countries.
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Clostridioides difficile surveillance at the University Hospital in Košice reveals high prevalence of the ribotype 176
BackgroundClostridioides difficile infection (CDI) has become a serious health problem worldwide in recent years, the severity of which lies in the ability to spread epidemically in hospitals and in frequent diseases relapses. CDI Surveillance was performed at the Louis Pasteur University Hospital in Košice from January to February 2020 to analyse the molecular characteristics ofC. difficile(CD) isolates from local patients with CDI.
MethodsCDI was initially diagnosed using the C. difficile rapid test (for enzyme GDH and toxin A/B). A total of 36 stool samples (29 GDH and toxin positive, 7 GDH positive and toxin negative) were cultured anaerobically on selective media (Brazier’s agar). Culture was positive for CD in 31 samples. Bacterial DNA was extracted from all CD isolates. Genes tcdA, tcdB, cdtA and cdtB encoding toxin A, toxin B and binary toxin were detected by multiplex PCR and ribotypes of CD were analysed by capillary electrophoresis-based PCR.
ResultsMolecular typing showed that toxin A as well as toxin B was confirmed in 30 of 31 isolates, binary toxin in 18 isolates. Ribotype 176, characterized by production of all 3 toxins, was the most prevalent and was detected in 18 isolates (58%). Toxin A and toxin B producing ribotypes 001, 014 and 020 were also confirmed.
ConclusionThe high incidence of epidemic ribotype 176 with higher capacity to spread in a hospital setting emphasises the need to implement strict epidemic measures and the importance of implementing continuous surveillance programmes for CDI in Slovakia.
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Insights into Otitis Media: Dissecting the interaction of C-Reactive Protein with Non-Typeable Haemophilus influenzae
More LessOtitis Media (OM) is the inflammation of the middle ear (ME). Non-typeable Haemophilus influenzae (NTHi) is one of the leading otopathogens in causing OM. Phosphocholine (PCho) on the NTHi lipopolysaccharide influences host-pathogen interaction. C-Reactive Protein (CRP), an acute phase protein recognizes PCho, and can mediate bacterial killing. However, some strains of NTHi survive even in the presence of CRP. We aim to study the interaction of CRP with NTHi to understand its role in bacterial survival and OM.
NTHi can efficiently infect the Junbo mouse, a characterised model of chronic and acute OM. CRP levels were highest 1 day post-intranasal inoculation in the ME fluid (MEF) and nasal passage (NP) washes. We show CRP is a localized response to NTHi as serum CRP levels were unaffected in NTHi inoculated and non-inoculated mice at 1, 3 and 7-day post intranasal inoculation. Further, we confirm the presence of NTHi influences CRP levels in the MEF and NP washes. We show CRP binding is influenced by the position and expression of PCho on the NTHi surface. Serum bactericidal assays indicate that the expression and position of PCho affects NTHi survival. The removal of CRP from the serum restores NTHi survival. The expression of PCho also influences opsonophagocytosis activity in macrophages, thereby confirming the importance of PCho in NTHi survival.
The CRP-NTHi interaction is currently under investigation to advance our understanding of its role in the complex biological processes that influence bacterial killing and the onset, progression and resolution of OM caused by NTHi.
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Identification and characterisation of a Novel SXT/R391 ICE mobile genetic element isolated from an Irish wastewater environment
More LessHuman and animal pathogenic bacteria are constantly being released into the environment through human activity. Many of these organisms can harbour genes such as virulence genes, antibiotic resistance and heavy metal resistance genes that are inserted into plasmids, transposons and Integrating Conjugating elements (ICEs), leading to potential spread. Such spread can be detected among water and soil communities and in particular in wastewater treatment plants. This makes wastewater and treatment plants a potential reservoir for mobile genetic elements including SXT/R391 ICEs, commonly detected amongst enterobacterial genera. Many plasmid and ICE genomes have been detected serendipitously from clinical sources but few have been identified without selection. Here we examined a domestic wastewater treatment plant to identify, isolate and characterize SXT/R391 ICE’s without selection. Standard microbial replica plating in conjunction with ICE specific (conserved integrase gene) PCR techniques were employed to identify an SXT/R391 ICE MGE using a range of enterobactial selective media. A Novel SXT/R391 ICE MGE was identified from a wastewater Proteus mirabilisstrain. Whole genome sequencing using Ilumina sequencing technology revealed a novel 81 kb element which on annotation contained 75 open reading frames. The “hotspot regions”, which contain adaptive genes, encoded a novel bacteriophage defence mechanisms but lacked other selectable determinants. With the continuous arms race between bacteria and phage, bacteria have developed novel resistance mechanism systems that protect the bacteria from phage. Such systems may be key adaptive mechanisms harboured by ICEs particularly in wastewater systems which will contain large phage populations.
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The Rtc RNA repair system is linked to virulence in Escherichia coli
More LessAntibiotic resistance is one of the biggest public health challenges of the 21st century. The Rtc RNA repair system is present in many pathogenic bacterial species, including the model organism and putative pathogen Escherichia coli, and may play a role in antibiotic resistance. Here we explore its physiological role during infection. Pathogenicity assays are being performed using the infection model Galleria mellonella, to study the phenotypes and survival rates following larvae infection with E. coli K12 and variants lacking rtc genes. Larvae infected with wild-type bacteria survive for up to 10 days as opposed to those lacking any of the rtc genes, which survive for up to 14 days on average. Complementation of the rtc gene deletions with wild-type Rtc proteins, but not functionally catalytic mutants, reverses the infection phenotype to that of wild-type. Similarly, viable larvae infected with wild-type or complemented bacteria score much lower in a health index scoring system than those infected with strains either lacking any of rtc genes or expressing catalytically inactive Rtc proteins. To further explore the importance of Rtc in infection, bacterial burden assays will be conducted to measure the bacterial load. The results so far suggest that bacteria with a fully functional Rtc system are more aggressive in the G. mellonellainfection model. This outcome supports the notion that the Rtc RNA repair system is involved in bacterial virulence and opens the window for further research to understand the ways in which Rtc may contribute to resistance, with potential for broader health benefit.
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Evolutionary histories and antimicrobial resistance inShigella flexneri and Shigella sonnei in Southeast Asia
Conventional disease surveillance for shigellosis in developing country settingsrelies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s-1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia.
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Failing to control Maedi-Visna
More LessMaedi-Visna is a lentivirus of sheep that causes lung disease and chronic wasting. It has been designated an “Iceberg disease” by the UK sheep industry levy board with a very large burden of subclinical disease that is often not apparent until losses in an individual flock become catastrophic. Disease prevalence in the UK is thought to have doubled in the last 10 years, however farmer and veterinary awareness of the disease is poor. There is no vaccine and treatment is not cost effective, meaning that the only realistic control option is culling of affected animals.
Current testing protocols use MV gag protein ELISAs. A long lag time between infection and antibody production means that many animals are missed on flock screening and repeated rounds of testing over a period of years are necessary to remove all infected animals. Preliminary testing of flocks that have attempted eradication indicates that those that do not keep testing until all animals are negative fail to eliminate the disease and that prevalence rates can even increase substantially in these flocks. The viruses extreme variability confounded attempts to develop a qPCR capable of detecting all variants, indeed deep sequencing was required to establish which strains of virus are currently present in UK sheep as there has been substantial genetic drift since the last sequencing studies (performed more than 20 years ago). More promisingly virus was detectable in nasal swabs of experimental animals at least offering sampling methods that can be done by farmers themselves.
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Synergistic biodegradation of a compostable film by a marine microbial community
More LessBackgroundThe need for replacing conventional plastics has led to an increase of the use biodegradable plastics. Most biodegradable plastic materials are certified for compostability, and their degradation mechanisms by marine bacterial communities, are still largely unknown.
MethodsBacterial communities that degrade a poly (butylene adipate-co-terephthalate)-based biodegradable film (PF) were enriched from marine samples. DNA, RNA and proteins were extracted simultaneously from the biofilm and free-living bacteria. Genes of hydrolases similar to the ones involved in polyethylene terephthalate (PET) and monoester mono-2-hydroxyethyl terephthalate (MHET) degradation (PETase and MHETases, respectively) were detected. A MHETase-like gene (Mle046) was then recombinantly expressed. The activity of Mle046 was tested against the end product of PET and PF degradation: MHET and 4-(4-hydroxybutoxycarbonyl) benzoic acid (Bte), respectively. The optimal incubation temperature and pH of Mle046 activity was determined.
ResultsPETase-like (Ples) and MHETase-like (Mles) hydrolases and other enzymes needed for PF degradation were expressed within the microbial community. Within the biofilm, Ples were abundant and upregulated while Mles and terephthalate dioxygenases were abundant in the free-living fraction. Mle046 was the only Mle produced in this fraction and it was highly expressed. The purified Mle046 could degrade MHET and Bte. The optimum temperature of Mle046 activity was 20°C.
ConclusionPF degradation is achieved synergistically by labour division among film-attached and free-living bacteria. Understanding the biodegradability of these plastics will facilitate the development of more degradable materials. In addition, the discovery of new PETases- and MHETases-like enzymes will enable their future use in plastic recycling.
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Isolation and characterization of bacteriophage Ib_pec2 against Shigatoxigenic E.coli
The current study aimed to isolate and characterize bacteriophage against drug resistant, Shiga toxigenic E.coli, one of the zoonotic, food-borne organisms associated with ruminants, mainly cattle. STEC were isolated (n=35) from neonatal calves, dairy workers and the surrounding environment and their antimicrobial resistance pattern was studied. Out of the 35 isolates tested, 17 isolates were found to be multi-drug resistant to important antibiotics like as ampicillin, amoxicillin-clavulanate, ciprofloxacin, streptomycin and tetracycline. Bacteriophage namely Ib_pec2 was isolated against NM—18-040 isolate and its morphology, genetic and proteomic characterization was done. Morphological analysis by TEM revealed bacteriophage belonging to myoviridae family. The genetic characterization of g23 gene revealed that the bacteriophage belonged to Tequatrovirus of myoviridae family. SDS-PAGE analysis of structural proteins followed by LC-MS/MS analysis could able to identify five proteins identical to Tequatrovirus of myoviridae family. One step growth curve experiments revealed a latency period of 40 mins and a burst size of 893 PFU/ bacteria. Temperature and pH ranging from 40-50°C, pH 7-8, respectively were ideal for phage survival and multiplication. Phage was able to lyse 22 out of 35 STEC isolates. Thus, the study could able to characterize Ib_pec2 which could be used in control of STEC in the near future. STEC is a commensal organism in the gastrointestinal tract of ruminants but pathogenic in humans. Bacteriophages can be used as alternatives to antibiotics to control its growth in ruminants and prevent its further spillage in the environment.
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Sessile Clostridioides difficilecontribute towards recurrent C. difficileinfection
C. difficile, an anaerobic spore-forming intestinal pathogen, produces up to three toxins that cause host cell damage resulting in disease, C. difficileinfection (CDI). Therapies include antibiotic treatment; however, up to 30% of cases fail primary therapy, resulting in recurrent disease, which increases patient morbidity and places a burden on worldwide healthcare systems. We have little understanding of why these therapies fail. Using a clinically validated in vitro gut model, we assess the contribution of biofilms towards recurrent disease and to investigate biofilm microbiota-C. difficile interactions. During induction of simulated CDI, C. difficile spores and vegetative cells became associated with the colonic biofilm microbiota. Vancomycin treatment did not effectively remove the biofilm C. difficile cells and recurrent infection was observed. Additionally, vancomycin therapy followed by faecal microbiota transplant did resolve the recurrent infection, but the biofilm C. difficile cells remained unaffected. In a biofilm transfer experiment, we showed that transferring biofilm encased C. difficile cells into a C. difficile naïve (but CDI susceptible model) induced CDI. Furthermore, we show that members of the biofilm community can impact C. difficile biofilm formation either acting in an antagonistic or synergistic manner. We highlight the importance of biofilms as a reservoir for C. difficile, which can be a cause for recurrent infections.
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Use of Equine Herpesvirus 1 glycoprotein pseudotyped lentiviral particles for the development of serological tests and assessment of lyophilisation for transport and storage
Equine herpesviruses (EHVs) are enveloped DNA viruses predominantly infecting members of the Equidae family. EHVs primarily cause respiratory disease, however EHV-1 can produce cases of a neurological disease, abortion and neonatal death. Thus, these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 exhibits a complex array of 12 glycoproteins on its surface envelope, but it is unclear precisely which are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a useful study tool. We have successfully generated functional EHV-1 pseudotyped lentiviruses bearing four glycoproteins, gB, gD, gH and gL (sequences derived from an aborted foetus during a large EHV1 outbreak strain in Normandy, France). PVs were employed in a pseudotype virus neutralisation test (PVNT) to measure levels of specific neutralising antibodies serum samples (n=52) taken longitudinally from experimentally infected ponies, compared with uninfected controls.
PVs routinely require -80oC for long term storage and a dry ice cold-chain during transport which can impede dissemination and utilisation in other laboratories. Consequently, we further investigated whether freeze-drying (lyophilisation) of EHV-1 PV could address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions, sampling at different timepoints. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests.
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Disruption of the mce operon from Streptomyces affects spore resistance and results in precocious germination
More LessStreptomyces coelicoloris a non-pathogenic soil saprophytic bacterium and is a model organism for antibiotic production. This species contains a single copy of a nine gene cluster known as the mammalian cell entry (mce) operon. This operon was originally characterised in Mycobacterium tuberculosisas an important virulence factor acting in invasion and survival within macrophages and encodes an ABC transporter for cholesterol import. As the function of the mceoperon in S. coelicoloris currently unknown, this study aims to characterise the operon through deletion of the mcelocus and resulting impact on bacterial morphology and survival. SEM images demonstrate that spores of a mcedeletion mutant (Δmce) display a wrinkled, and ‘fragile’ phenotype, with spores appearing to germinate whilst on the spore chain. Germination assays show that spores of Δmcegerminate earlier than WT S. coelicolor, and impression mounts indicate spore chains emerge earlier in the Δmcestrain. As spores of Δmce possess an altered spore coat, it was hypothesised they might show reduced resistance to stressors. Heat kill assays show that deletion of the mce operon results in S. coelicolor spores which are less tolerant to temperatures of 60, 70, 80, 90 and 100°C compared to WT S. coelicolor spores. Heat activation of Δmce spores was also consistently absent at all temperatures tested. Spores of the Δmcestrain are also less resistant to triclosan, and sulfobetaines. Preliminary results indicate that the mce operon of S. coelicolor may be a cholesterol importer, as Δmce shows reduced growth in the presence of cholesterol, compared to WT S. coelicolor.
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Embedding 21st century employability into assessment and feedback practice through a student-staff partnership
More LessAlthough commonly thought to be only a measure of academic performance, assessments can also provide students with an opportunity to apply skills and knowledge acquired at university into written literacies that prepare them for the transition into prospective careers. Within a Level 2 Microbiology and Immunology course, students struggled to engage with standalone timetabled careers-related sessions, yet they showed enthusiasm when employability was embedded into assessments. A staff-student partnership project explored these issues, with the overall aim of understanding how to effectively embed employability skills into assessment and feedback, which supports the theme of supporting students to position themselves for the future.
Through student-run focus groups, this project investigated the reasons for low student engagement with the timetabled employability session and used student views to develop digital initiatives that could be applied to assessment and feedback practice with an emphasis on developing 21st-century competencies. These initiatives were then implemented as pre-session self-directed activities, which helped students link course feedback with employment skills and future career planning, followed by a newly developed in-class reflective feedback session that allowed students time to consider what skills they have developed and make links with future careers. Project evaluation was conducted using a quantitative survey of students involved.
This project aims to make feedback more meaningful and better valued by students. The approaches were designed in partnership with former students from the course, this project possesses the unique ability to deliver a genuine student voice both on employability concerns and the role of assessment.
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Epigenetic Modification Impacts on Gene Regulation in Streptococcus pneumoniae
More LessMethylation by the type I restriction modification system (RMS) SpnIII in Streptococcus pneumoniae is hypothesised to regulate gene expression via epigenetic changes (Manos). The phase variable SpnIII generates six host specificity determinants (hsdS) alleles through site-specific recombination (DeSteCroix), each allele correspond with different methylation pattern.
In addition to known functions of the RMS in restricting phage infection (Furi) and transformation (Kwun), we have now tested impact on gene expression. RNAseq was used to analyse S. pneumoniae strains expressing a single spnIII allele (spnIIIA, spnIIIB or spnIIIE) to determine differences in gene expression profiles. The data have identified six genes which show differential expression and have a methylation site mapping to their predicted promoter region. Three synthetic promoters with the wild type and altered methylation target site were cloned in front of a luciferase gene in strains expressing a single spnIII alleles. For the first three sets of constructs analysed, data indicate that the methylated promoters show a three- to twenty-fold higher activity compared to non-methylated promoters. Results obtained were further confirmed by qPCR analysis. Preliminary data using drugs targeting DNA topoisomerases indicate that the mechanism by which methylation impact gene expression could be related to DNA topology. These data demonstrate for the first-time RMS dependent variations in gene expression and propose an alternative mechanism for this epigenetic regulation.
References:
* Manso, Nature Communication, 2014; 5:5055.
* De Ste Croix, J. Bacteriol. 2019; 201(15). pii: e00233-19.
* Furi, J. Bacteriol. 2019; 201(19). pii: e00370-19.
* Kwun, Nucleic Acids Res. 2018; 46(21):11438-11453.
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Immuno-proteomics of sera from gonorrhoea patients identified potential vaccine candidates
More LessGonorrhea is a sexually transmitted disease caused by Neisseria gonorrhoeae and is one of the leading reportable STDs in adults, with ~87 million cases annually and globally. Gonorrhoea remains a major global public health concern not only because of rising incidence each year, but also because of rising antimicrobial resistance. There is an urgent need for long-term solutions to prevent gonorrhoea such as vaccines, but none currently existand research is focused on identifying potential antigens for inclusion in new vaccines. In our study, we used an immuno-proteomics approach to try and identify potential vaccine candidates.
A heterologous N. gonorrhoeae strain P9-17 was grown under iron-limiting conditions and whole cell lysates prepared. These were separated by isoelectric focusing (pH 3-10 range) and fractions were separated by SDS-PAGE. Western blots were prepared and reacted with sera from 20 patients with uncomplicated gonorrhoea and with sera from 5 controls with no history of gonorrhoea. Immuno-reactive bands were excised from the corresponding gels and subjected to mass spectrometry, which provided a profile of gonococcal proteins. After comparing patterns of reactivity, we identified 180 bands in sera from gonorrhoea patients. Using a bio-informatics approach we refined the list to 18 bands of interest and identified 13 novel proteins associated with an increase in immuno-reactivity with gonococci.
In summary, we have identified a unique set of gonococcal proteins that have not yet been investigated as potential vaccine antigens and that are the focus of current studies to develop a gonococcal vaccine.
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Antimicrobial potentials of Lantana camara montevidensis leaf extract on wounds infected with Candida isolates using animal models
More LessBackgroundThere is an increased focus in the development of antimicrobials of herbs origin because they possess active chemotherapeutic effect on different kinds of infectious diseases. Many communities in Nigeria and other African countries use plants to treat various infections, including wounds. New antimicrobial agents are being developed in response to the emergence of resistance to existing antibiotics and antifungal agents. Lantana camara leaves are easily accessible at low or no cost in resource poor communities. The aim of this study was to determine the antimicrobial activity of Lantana camaraleaf extract against selected Candida isolates from infected wounds invitro and in vivo using animal models.
MethodsAqueous and methanol leaf extraction was done using Soxhlet apparatus. TenCandidaisolates associated with wound infectionswere re-identified and used for the study. Antifungal susceptibility testing was done using the E-Test strips. Fifteen healthy male Wistar rats were used for the study. The rats were anesthetized before making incision wounds on the neck region. The rats were treated with 100mg/ml, 50mg/ml and 25mg/ml concentration of extract topically. The skin tissues of the sacrificed rats were obtained for histological examination using Heamatoxylin and eosin technique.
ResultsThe susceptibility rate of the Candida isolates ranged from (0.0% - 40.0%). Fluconazole was the most effective antifungal. Isolates were most susceptible(40.0%)to 100mg/ml concentration of extract. Rats treated with 100mg/ml methanol extract had significant mean wound contractions of 70.33±0.58 withdamaged tissue repair.
ConclusionMethanol extract of Lantana camara leafhas antimicrobial activity on Candidaisolates and topical healing effect on Candida.
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Probing mycobacterial metabolism in tuberculosis and leprosy to identify vulnerable metabolic nodes for drug development
Metabolism of pathogens in infectious diseases is important for their survival, virulence and pathogenesis. Mycobacterial pathogens successfully scavenge multiple host nutrient sources in the intracellular niche. It is therefore important to identify the intracellular nutrient sources and their metabolic fates in these pathogens. Metabolic phenotype of an organism is defined by metabolic fluxes. We quantified in vivo fluxes of the pathogens and probed host-bacterial metabolic cross talks in tuberculosis (TB) and leprosy using systems-based strategies and techniques of isotopic labelling, metabolic modelling and metabolic flux analysis (MFA). We show that the TB pathogen metabolizes a number of carbon and nitrogen sources in human macrophages and identified vulnerable nodes such as glutamine and serine biosynthesis as potential drug targets. Mycobacterium leprae, the leprosy causing pathogen, uses host cell glucose in infected schwann cells and the enzyme, phoenolpyruvate carboxylase is a potential drug target. Our research provides an understanding of the intracellular diets and metabolism of these important human pathogens and identified vulnerable metabolic nodes that can be used for developing innovative chemotherapies in TB and leprosy.
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In silico Approaches for Targeting an Essential Mycobacterial Protein, PrsA
More LessMycobacterium tuberculosis is a leading cause of death worldwide and with increasing cases of drug-resistance, new treatments options are required. PrsA is an essential protein in mycobacteria and is necessary for arabinogalactan production and nucleotide biosynthesis. This protein is not currently targeted by any drugs in clinical development and represents a potential drug development avenue.
Currently, no crystal structure of M. tuberculosis PrsA (MtPrsA) is available, hence the SWISS-MODEL structure, based on M. smegmatis (93% similarity) was utilised. Molecular Dynamic simulations were performed on the modelled structure, to gain information on the protein’s predicted flexibility. The output trajectory was clustered, and representatives of each major cluster were used in downstream analysis. Ensemble docking, molecular docking on the ensemble of MtPrsA structures, of the GSK-177 prioritised compounds was undertaken. The docking predictions were also re-evaluated using machine learning based programmes, including NNScore.
This approach yielded several compounds which could be taken forward for further computational analysis. The compound rankings were also retroactively compared to experimental data studying the GSK-177 compounds’ interactions with mycobacteria.
This research represents a potential workflow for future in silico drug development efforts against M. tuberculosis and further work may allow the identified compounds to be used for the treatment of TB.
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Searching for new microbial enzymes for plastic polymer degradation: Polyurethanes and Lycra
Julia Linke and John WardNew enzymes and biocatalysis could provide routes to help limit global plastic pollution, as suggested by the example of PETases for the PET degradation. Experiments were conducted to screen soil microorganisms for enzymes able to break down the plastic polymer Lycra, which contains polyurethane linkages and could be utilized as a carbon and nitrogen source.
The methodology is based on a plate assay, which compared the growth of bacteria at 50°C and 65°C on minimal media and minimal media with a top layer, that contained DMF-dissolved Lycra. The medium provided the main nutrients required for bacterial growth, with Lycra being the only substantial carbon source. Promising candidates were identified as bacterial colonies with visibly enhanced growth on the Lycra-enriched minimal medium plate. The selected soil microorganisms were then tested with a liquid culture assay, where the strain was cultivated at 50°C or 65°C in liquid broth medium, that contained dissolved Lycra. The liquid culture samples were collected at 4 time points between 0-48h, and the aliquots tested on HPLC for detection of released Lycra monomers.
Around 50 different bacterial strains from 6 various soil and compost sources were tested with the described plate screening method and 7 bacterial thermophilic species were detected, that showed visibly enhanced growth on the Lycra-enriched plates. These results could support the development of Lycra enzymatic degradation. New enzymes could be engineered, optimized and yield a sustainable solution for the textile industry, which would not require toxic chemicals or contribute to landfill.
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Understanding the bug that make our drugs: Cellular responses to therapeutic protein secretion in E. coli
More LessProduction of therapeutic proteins is a $200 billion industry with E. coli being one of the main expression systems used and proteins often being secreted to the periplasm to produce disulphide bonds. However, little is known about how E. coliresponds to secretion of these therapeutic proteins into the periplasm under industrial conditions.
Proteomics and transcriptomics were conducted on cultures grown at high cell density in industrial production conditions to look at the effect of secreting a model therapeutic protein (scFv) to the periplasm compared to expressing the same protein in the cytoplasm.
82 proteins and 1653 transcripts were differentially expressed when overexpressing the scFv in different cellular compartments. ScFv secretion had a significant effect on expression of genes involved in the envelope stress response. However, while certain envelope stress pathways where upregulated upon secretion others were downregulated (including expression of some periplasmic chaperones). Scfv secretion also led to an increase in expression of genes involved in degradation of membrane proteins and the SEC secretion system, as well as iron transport, Outer membrane porins and flagella operons.
In future and current experiments, we are testing whether knocking out and overexpressing a selection of these genes improves secretion and folding of scFvs and other recombinant proteins.
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Identification of Residues within Flavivirus Non-Structural Protein Associated with Mosquito Transmission
More LessFlavivirus non-structural protein 1 (NS1) is present at high levels in patient blood and has been previously implicated in increasing virus acquisition by mosquito vectors, which may facilitate increased transmission. Therefore, flaviviruses transmitted by Aedes aegypti mosquitoes were hypothesised to have similarities within NS1, and potentially other NS proteins, that may correlate with mosquito-borne (rather than tick-borne or no vector) transmission. Representative flavivirus amino acid sequences were downloaded from the NCBI Protein database, aligned using viprbrc.org and then manually analysed to identify residues correlating with arthropod vector tropism. All available sequences for individual clinically important flaviviruses were then analysed on viprbrc.org to determine sequence conservation within viral species for those residues correlating with vector tropism. Overall, we found five residues in NS1 that were highly correlated with transmission by mosquitoes, had functionally relevant differences in amino acid side chain properties between mosquito- and tick-borne viruses and had conservation within viral species. Additionally, a further nine residues in NS3 and four residues in NS4A were identified using the same methodology. Importantly, for NS1, for which more data is available regarding its role in influencing transmission, the residues we identified have not been implicated in increased transmission thus far. Hence, future work would aim to test the impact of mutating these NS1 residues to elucidate whether they modulate NS1 secretion into the blood stream, virus replication and/or transmission. Gaining an understanding of the molecular determinants underpinning vector tropism could be useful in the creation of transmission-incompetent vectors.
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Microbial community of MX80 bentonite and their interaction with iron
More LessMX80 bentonite has been selected as the buffer and backfill in a proposed method of long-term deep geological storage of nuclear waste. Extensive studies have been carried out on the geomechanical properties of MX80; however, it is not clear what effect microbes will have on its ability to function as an effective barrier. Specifically, in the UK, as carbon steel waste canisters will contribute iron oxides and rust products to the immediate environment, iron-reducing bacteria are of interest. Iron-reducing bacteria can reduce structural or external Fe (III) to Fe (II) and some species are adapted to high temperatures and low water availability, in keeping with conditions within the waste repository. Indigenous iron-interacting bacteria have been identified in compacted MX80 and microbially-influenced iron-reduction was observed in groundwater salinity up to 0.45M NaCl. Experiments investigating gas production, and silica-solubilising abilities of this community were carried out. Further experiments in pressurised test cells investigated microbial activities at the clay / steel interface. Significant increases in hydrogen production were observed when microbes were present, and biogenically influenced changes in structure and appearance of MX80 were seen in all experiments. Additionally, silica release occurred, likely coupled to metal / microbe interactions. Corrosion products differed depending on microbial presence following incubation in test cells. Biogenic transformation of clay minerals through iron reduction or release of silica to groundwater could significantly impact the geomechanical properties of MX80, as indicated by observed changes in clay plasticity, and ultimately this could affect the behavior of the material as a barrier.
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Comparison of the Antimicrobial Efficacy and Germicidal Efficiency of 405-nm Light for Surface Decontamination
More LessBackgroundThe persistence of infectious organisms on hospital surfaces presents a significant challenge to healthcare environments. Low irradiance visible 405-nm light has recently been developed as a method for environmental decontamination, with studies demonstrating successful reductions of environmental bacteria in wards and operating theatres. This study investigates the antimicrobial efficacy of 405-nm light for decontamination of surfaces, and how the dose-response kinetics are affected by use of differing light irradiances.
MethodsSurface-seeded Staphylococcus aureus and Pseudomonas aeruginosa (selected as model Gram-positive and Gram-negative species) were exposed to increasing doses of 405-nm light (≤ 90 Jcm-2) at three discrete irradiances (0.5, 5 and 50 mWcm-2). For both species, inactivation kinetics at each respective irradiance was established and susceptibility at equivalent light doses compared.
ResultsResults demonstrate increased bacterial susceptibility to 405-nm light inactivation when exposed at lower irradiance treatments. For both species, 3 Jcm-2 was required when exposed using 0.5 mWcm-2 irradiance to achieve significant bacterial inactivation (P < 0.05; 26.7-73.7% reduction). When exposed at 5 mWcm-2, double the energy (6 Jcm-2) was required to achieve similar reductions. Exposure at the highest irradiance (50 mWcm-2) required 3-5 times greater dose (9-15 Jcm-2) to achieve similar reductions to the lowest irradiance tested (0.5 mWcm-2).
ConclusionThis study provides evidence of the enhanced germicidal efficiency of low irradiance 405-nm light, highlighting its efficacy for continuous environmental decontamination applications. Further investigation into the photo-chemical inactivation mechanisms will be crucial for its optimisation for a range of infection control applications.
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A Legionella’s outbreak in a COVID-19 pandemic setting
More LessIn this report, the author intends to reinforce the need to test for another potencial causes of pneumonia which present with similar symptoms of SARS-CoV-2 infection and can also be responsible for outbreaks and sometimes fatal, particularly Legionnaires’ disease.
Legionellabacteria are aerobic, gram-negative, ubiquitous freshwater and soil inhabitants. Pneumonia is the most commonly described manifestation of Legionella infection (Legionnaires’ disease), acquired through inhalation of aerosolized water droplets containing the bacteria.
Legionella pneumophilais the most consistently reported species, being an important cause of severe community-acquired pneumonia that often requires hospitalization and is fatal in approximately 10% of cases. Most cases present sporadically, however, outbreaks can occur when there are appropriate conditions for bacteria to grow and spread, like stagnating water in piping systems in large facilities. Furthermore, the pathogenesis of Legionella pneumonia is complex and it is clinically and radiographically similar to other forms of pneumonia.
In the context of pandemic COVID-19, the use of Legionella urinary antigen testing continues to be crucial and strongly recommended. Thus, in the outbreak faced in the author’s hospital, this screening method proved to be effective and all cases of Legionellapneumonia were promptly recognized and notified, allowing public health measures to be taken to prevent the development of other potencial clusters.
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K1 Escherichia coli form foci in the brain at 24h post infection
More LessK1 capsule type Escherichia coli is the predominant cause of neonatal meningitis. In an intravenous (IV) infection model with a clinical K1 isolate IHE 3034 adult mice die of sepsis within 24 hours. However, by utilising cefazolin, a first-generation cephalosporin known for its inability to pass through the blood brain barrier, it is possible to prevent bacteraemia and study the early phases of E. coli meningitis in the brain. Mice were infected IV with E. coli IHE 3034 and treated with cefazolin at 12 and 24h intraperitoneal to ensure the survival of animals to the determined end points. The results of the experiment show IHE 3034 is present in the brain as early as 12h and it remains in the tissue 72h post infection even if blood is clear. Despite this, at the cellular level, clear foci formation can be observed in the meninges and the choroid plexus around the vascular endothelial cells at 48h, showing IHE 3034’s ability for intracellular replication inside the meningeal region The foci do not appear to be present at 24 or 72h, indicating for a secondary replication site around the major vascular endothelia sites of the brain. This further promotes the bacterial load increase through cell bursting in the tissue with or without a sustained bacterial influx from the blood. These findings show that K1 E. coli can form foci in the brain which may lead to escalated disease progression and severe morbidity following an initially successful clearance of bloodborne bacteria during sepsis.
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Bioinformatic Analysis for Iron-Uptake System of Campylobacter spp. in humans and chickens
More LessAsymptomatic Campylobacter jejuni colonisation is highly prevalent in chickens, however, it’s a major cause of bacterial foodborne disease in humans. Iron is an essential co-factor in many physiological processes and Campylobacter strains employ several non-redundant iron acquisition systems for survival and colonization. The outer membrane receptors CfrA and CfrB are involved in iron acquisition fromcatecholamine siderophores and are required for colonisation of the chicken GI tract. In contrast, ChuAB are part of an uptake system, enabling the utilization of iron in haem and is not required for colonisation. This study aims to compare differences in the iron-uptake systems present in Campylobacter strains isolated from chickens or humans.
Analysis of Campylobacter jejuni/coli genome sequences in PubMLST (https://pubmlst.org/organisms/campylobacter-jejunicoli [https://pubmlst.org/organisms/campylobacter-jejunicoli]) added from the UK in 2018 reveals differences in sequence type (ST) and chuA, chuB, cfrA and cfrB between isolates from chicken (CI) or human (HI) sources. ST828 was the most common ST for the C. coliCI (45%) and ST827 (32%) for the HI. C. jejuniST5136 was the most common of the HI (10%) and CI (13%), ST50 (8%) for the HI and ST21 (7%) for the CI. >95% of isolates contained chuAand chuB, we also found the Chu system to be highly conserved. For cfrA81% of HI and 74% of CI contained the gene, and 92% of HI and 96% of CI harboured cfrB.<3% of all isolates had neither cfrAor cfrB, cfrBmore associated with CI than HI. This data analysis reveals potential gene targets towards vaccine development against Campylobacter strains.
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Detection of Extended Spectrum Beta-lactamase Gene (CTX-M) among Representative Multidrug-Resistant Gram Negative Bacterial Isolates from Patients with Urinary Tract Infection in Ekiti State, Nigeria
More LessUrinary tract infection is huge public health burden and the emergence of extended spectrum beta lactamase producing bacterial pathogens increases the burden of infectious diseases in Nigeria. This study determined the current prevalence of cephalosporin resistance among Gram-negative bacteria isolated from patients with urinary tract infections between February 2018 and June 2018. Non-repetitive Gram–negative bacteria were recovered from 106 individuals with urinary tract infections who reported at two tertiary healthcare centers in Ekiti-State, Nigeria. A total of 106 bacterial isolates were obtained which included: Klebsiella pneumoniae 34 (29.1%), Klebsiella oxytoca 17 (16.0%), Proteus vulgaris 10 (9.4%), E. coli 24 (22.6%), Proteus mirabilis 18 (16.9%) and Pseudomonas aeruginosa 3 (2.8%). Sixty five of these organisms showed resistance to ceftazidime while 76 organisms showed resistance to ceftriaxone. Forty representative organisms were selected and tested for presence of extended spectrum beta-lactamase (ESBL) genes using primers specific for different ESBL genes. A total of eight (20.0%) organisms carried the blaCTX-M gene and other variants of the ESBL genes were not detected. The organisms carrying the blaCTX-M gene included E. coli 3 (37.5%), K. pneumoniae 1(12.5%), P. mirabilis 1(12.5%),) and K. oxytoca 3(37.5%). The high prevalence of cephalosporin resistant Gram-negative bacteria among patients with UTI is a serious threat to public health and efforts must be intensified to regulate the clinical use of the cephalosporins.
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Profiling the effects of acne therapeutics, including the novel antibiotic sarecycline, on the human microbiota
Many factors shape the human intestinal microbiota, some of which can confer a deleterious effect on the microbiota, e.g. antibiotic therapy. Disruption to the microbiota has been implicated in the progression of C. difficile infection (CDI), multiplication of multi-drug resistant organisms and many extra-intestinal diseases. Thus, determining the off-target effects of antibiotics is essential to determine a patient’s risk of these diseases, particularly for new therapies. Here we characterise the effect of sarecycline, a novel tetracycline antibiotic for the treatment of moderate to severe acne vulgaris; and compared its effect to the gut microbiota with other acne treatments. Using four independent in vitro gut models, we exposed the human microbiota to either sarecycline, minocycline, doxycycline, or clindamycin, and monitored the changes to the bacterial populations, and whether these changes were sufficient to induce CDI.
Sarecycline or doxycycline exposure caused a temporary reduction in the bacterial diversity upon initial exposure. Sarecycline exposure was characterised by a transient increase in Enterococcus spp. and Enterobacteriaceae, and a decrease in Bifidobacterium spp. Doxycycline exposure caused longer-term changes to the Lactobacillaceae and Ruminococcaceae populations. Minocycline exposure resulted in a dramatic reduction to the bacterial diversity, with extensive expansions to the Enterococcus spp. and Enterobacteriaceae populations, whilst the Lactobacillaceae and Ruminococcaceae populations contracted. Whilst clindamycin did induce simulated CDI, neither sarecycline, minocycline, nor doxycycline created a niche conducive for CDI.
These data show that long-term sarecycline use has a lower potential for disruption of the colonic microbiota, compared with the current treatments for acne vulgaris.
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Cell-to-cell ATP differences can modulate cellular decision-making
More LessCells generate phenotypic diversity both during development and in response to stressful and changing environments, aiding survival. Functionally vital cell fate decisions from a range of phenotypic choices are made by regulatory networks, the dynamics of which rely on gene expression and hence depend on the cellular energy budget (and particularly ATP levels). However, despite pronounced cell-to-cell ATP differences observed across biological systems, the influence of energy availability on regulatory network dynamics is often overlooked as a cellular decision-making modulator, limiting our knowledge of how energy budgets affect cell behaviour. Here, we consider a mathematical model of a highly generalisable, ATP-dependent, decision-making regulatory network, and show that cell-to-cell ATP variability changes the sets of decisions a cell can make. Our model shows that increasing intracellular energy levels can increase the number of supported stable phenotypes, corresponding to increased decision-making capacity. Model cells with sub-threshold intracellular energy are limited to a singular phenotype, forcing the adoption of a specific cell fate. We suggest that energetic differences between cells may be an important consideration to help explain observed variability in cellular decision-making across a broad range of biological systems, including bacteria and the blood stem cell system.
*NOTE: Our work is highly interdisciplinary and if you believe it would be better suited to another topic area, then I would be delighted to discuss that further.
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Shiga toxin-producing E. coli (STEC) isolated from wild mammals in Portugal
More LessBackgroundZoonoses are diseases common to humans and animals (livestock, wildlife, and pets). In 2018 about 360 000 zoonoses were reported in European Union. Shiga toxin-producing Escherichia coli (STEC) infections were among the most reported causes of these zoonotic diseases.
MethodsFaecal samples of mammal species (n=286) with distinct phenology (wild boar, red deer, otter, and red fox) were collected in Portugal. After the initial processing, the presence of STEC was screened by PCR, and suspicious samples were platted on CHROMagar STEC. STEC positive isolates were tested for antibiotic susceptibility. Thephylogenetic relationship of STEC strains was evaluated by PFGE. Of these, 20 representative strains were selected for whole genome sequencing with the Illumina NovaSeq 6000 system. For the assembly, annotation and genome characterization, multiple web-based bioinformatic tools were employed.
ResultsCultivable STEC (n=52) were recovered from 17% (n=49) of the samples collected from the four mammals. All the isolates were non-O157:H7 STEC encoding stx1 (n=2; 4%) and/or stx2 genes (n=51; 98%). Only one strain (2%) of red fox was resistant to ceftazidime, aztreonam and nalidixic acid. The 20 strains that were sequenced belong mainly to serotype O27:H30 (n=15), followed by O146:H28 (n=2), O146:H21 (n=1), O178:H19 (n=1) and O103:H2 (n=1). In addition to stx, all strains encode several virulence factors, mainly toxins, adhesins, fimbrae, secretion systems, among others. Additionally, several pathogenicity islands have been predicted for these strains.
ConclusionsOur results show that wild animals are reservoirs of STEC, potentially pathogenic to humans.
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Ruminococcus gnavus GH98 substrate specificity to blood group A antigen contributes to mucin glycan foraging
The human gut symbiont Ruminococcus gnavus displays a strain-specific repertoire of glycoside hydrolases (GHs) contributing to its spatial location in the gut. Sequence similarity networks showed that R. gnavus GH98 (RgGH98) sequence fell in a cluster different from sequences of GH98 enzymes functionally characterised to date. We heterologously expressed and purified RgGH98, and determined its substrate and linkage specificity. We showed that RgGH98 is specific for blood group A antigen (BgA), as also confirmed by isothermal titration calorimetry (ITC) and saturation transfer difference (STD) NMR, revealing affinity for blood group A over blood group B and H antigens. The molecular basis of RgGH98 specificity was further investigated using a combination of site-directed mutagenesis and X-ray crystallography. The crystal structure of the complex between RgGH98 and BgA trisaccharide and RgGH98 inactive mutant with BgA tetrasaccharide identified residues involved in RgGH98 unique specificity. RNAseq and qPCR analysis showed that the gene encoding RgGH98 is part of an operon that is overexpresssed in vitro when R. gnavusis grown on mucin as sole carbon source. We showed that RgGH98 releases BgA trisaccharide from mucin and that pretreatment of mucin with RgGH98 conferred other R. gnavusstrains lacking this enzyme the ability to grow through BgA metabolism and access to the underlying mucin glycan chain. These data further support that the GH repertoire of R. gnavus strains enables them to colonise different nutritional niches in the gut and provide a source of enzymes with unique specificities for potential applications in diagnostic or therapeutics.
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Dissecting meningococcal disease and carriage traits using high throughput phenotypic testing
Despite on-going vaccination programmes, Neisseria meningitidisis a majorcause of septicaemia and meningitis. In 2017-18, the MenW and MenY capsular groups caused 38% of all UK invasive meningococcal disease cases. Current policy is to generate genome sequences of all meningococcal disease isolates. Using this resource, we aim to determine how genetic variation contributes to phenotypic differences between carriage and disease isolates. We have adapted assays, mimicking carriage and disease behaviours, for high-throughput phenotypic testing of 335 MenW cc11 and MenY cc23 isolates. We are currently testing MenW cc11 disease and carriage isolates for cytotoxicity in a human lung epithelial cell line, growth in media and biofilm formation. Phenotypic differences are utilised as inputs for Genome Wide Association Studies enabling linkage of specific genomic variants, or variant combinations, with phenotypic variation. Genomic data include whole genome sequences and repeat-mediated phase variation states.
The MenW cc11 isolates span two known phylogenetic clusters, original and 2013. Our preliminary data from high-throughput growth and biofilm assays showed no significant differences between these groups or sources (disease versus carriage); however, variations were observed within groups with, for example, distinctive cytotoxicity or biofilm differences between isolates. These variations may reflect physiological divergence due to minor genetic modifications between highly phylogenetically-related strains. Our assay systems are robust, reproducible, and easily scalable for efficient high-throughput genotypic and phenotypic testing. Thus, large-scale screening of phenotypic variation for infectious diseases is achievable and harnessable for cost-effective, direct evolutionary and epidemiological studies.
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Controlling the lytic switch; can m6A-modified RNA be used as an Anti-Viral Target?
KSHV has a biphasic life cycle encompassing a latent state and lytic replication. The KSHV replication and transcription activator viral protein, encoded from open reading frame 50 (ORF50), is the key viral protein which drives the switch between the latent and lytic phases (Guito and Lukac, 2012). We have recently demonstrated that KSHV manipulates the host cell N6-methyl adenosine (m6A) RNA modification pathway to enhance viral gene expression. Specifically, we have shown that the KSHV ORF50 transcript is m6A methylated, allowing the recruitment of the m6A reader protein, Staphylococcal nuclease domain-containing protein 1 (SND1), resulting in the stabilisation of the ORF50 transcript and efficient KSHV lytic replication (Baquero-Perez et al. 2019).
Further analysis of the m6A modified site with the ORF50 transcript has identified an RNA stem-loop, termed ORF50-1, which is a m6A-modified 43-mer, essential for SND-1 binding, thought to occur in a secondary structure/ sequence-dependent manner.
Generating in silico 3D structures of the ORF50-1 RNA in its native ‘N’ and m6A-modified ‘M’ form confirms that the presence of the m6A-modification has repercussions in the lower part of the stem-loop and predicts a different secondary structure(collab. Pasquale and Roeder).
Due to the obvious structural differences anticipated, RNA-binding ligands have been identified using Small Molecule Microarrays (SMMS) (collab. Fullenkamp and Schneekloth). By investigating these ligands in biophysicalexperiments, we have verified RNA-binding and optimised cell-based assays to assess anti-viral properties. It is hoped these experiments will highlight the importance of A versus m6A within the lytic phase of KSHV’s lifecycle.
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Genetic manipulation of Lactobacillus to produce optically pure lactic acid following fermentation using hydrolysed organic fraction of municipal solid waste as a feedstock
More LessBackgroundLactic acid for polymer applications must be optically pure to allow the final plastic properties to be controlled. We have isolated a strain of Lactobacillus plantarum capable of utilising a range of carbohydrates and tolerant to high sugar and organic acid concentrations. However, like most Lactobacillus plantarum, this strain produces D- and L-lactic acid. To improve the commercial application of this strain gene editing has been used to ensure only one isoform is produced.
MethodsUsing two-step homologous recombination, ldhL was replaced with a truncated version interrupted by a chloramphenicol acetyltransferase resistance marker, then with an unmarked truncated version.
The optical purity shift was quantified biochemically by enzymatic assay and HPLC and fermentation performance compared against the unmodified strain using enzymatically hydrolysed municipal solid waste pulp (MSW sugars) as a feedstock.
ResultsThe ldhL gene was successfully deleted, leading to a dramatic shift in the optical purity of lactate produced in both synthetic media and MSW sugars. The unmodified strain produced D-lactic acid with an optical purity of 40%, while the mutant achieved 94%. In MSW sugars, the modified strain performed as well as the unmodified control, consuming all available glucose in less than 48 hours with a volumetric productivity of 1.02 g/L/h.
ConclusionsDeletion of ldhL from our strain of Lactobacillus plantarum allowed exclusive production of D-lactic acid from MSW sugars. This provides a laboratory scale demonstration of the production of optically pure lactic acid from renewable sugars for use in the production of biodegradable plastics.
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Palmatine and berberine chloride synergistically Inhibit NanH sialidase of Tannerella forsythia
More LessThe periodontal pathogen Tannerella forsythia is associated with severe periodontitis, and expresses NanH sialidases that cleave sialic acids by hydrolyzing the glycosidic bonds to underlying sugars. Palmatine and berberine chloride are plant-derived alkaloids with pharmacological effects, including anti-inflammatory and anti-bacterial properties. Recombinant NanH sialidase was purified using HisTag affinity chromatography while sialidase activity was determined using 4-methylumbelliferyl N-acetyl-α-D-neuraminic acid (MUNANA) as a substrate. The individual and synergistic effects of palmatine and berberine chloride on NanH sialidase inhibition was determined as well as their antimicrobial effects. The IC50 values of palmatine and berberine chloride were found to be 0.143 and 0.474 mM respectively. A significant synergistic effect was observed when a 0.20 mM:0.50 mM Palmatine:Berberine chloride mixture was used, inhibiting NanH sialidase by almost 100%, as compared to 0.2 mM palmatine and 0.5 mM berberine chloride invidually, which inhibit sialidase activityby 60.33 and 55.94%, respectively. Additionally, an antimicrobial viability assay was conducted and, 0.5 mM palmatine and 0.45 mM berberine showed a significant antimicrobial activity against Tannerella forsythia. Lastly, to examine potential toxicity to host cells, thecytotoxic effects of palmatine (0.15 mM) on H357 oral squamous carcinoma cells was investigated using a trypan blue assay and palmatine was found not to be toxic. In summary, a combination of palmatine and berberine display significant synergistic inhibitory effects on NanH with minimal cytotoxic effects as well as potential antimicrobialeffects on the oral pathogen T. forsythia. Suggesting thatthese compounds may have potential for future development.
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Covariation analysis of the master bacterial DNA replication initiator DnaA
More LessBacterial DNA replication initiation is governed by DnaA: an oligomeric protein belonging to the ATPases associated with diverse cellular activities (AAA+) superfamily. The master replication initiator is present in all known bacterial pathogens; thus, it is an attractive target for drug discovery. Here, an in-silico evolutionary covariation analysis of DnaA amino acid residues was conducted using EVcouplings, a Python framework, to investigate connections between key protein residues. Several co-evolving amino acid pairs were identified and characterized using PyMol to visualize the residues on DnaA crystal structures. I hypothesize that some co-evolving pairs, particularly those spatially connected, are likely important for structural integrity of the protein. Interestingly, one co-evolving pair of residues (A. aeolicus Val121 and Ser229) was found to be separated by ~27 angstroms, suggesting a more complex relationship connects these two sites. Thus, analysing co-evolution of DnaA residues supports known structural information and may predict either allosteric connections within DnaA or contacts between DnaA and other binding partners.
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Global diversity and potential functioning of prophages in plant pathogenic Ralstonia solanacearum bacterium
More LessRalstonia solanacearum is a destructive plant pathogenic bacterium which harbours a wide variety of virulence genes allowing it to infect over 200 plant species worldwide. Its virulence is also affected by the presence of integrated bacteriophages, termed prophages. While several such prophages have been identified, the global distribution and diversity of R. solanacearum prophages is unknown.
To study this, we first identified prophages present in a diverse collection of 192 assembled R. solanacearum genomes. Prophage diversity was explored by calculating prophage genetic distances and clustering with characterised prophages in a neighbour-joining tree. Prophage clusters were further verified by assessing gene content, GC content, and prophage length. Prophage identities were determined using the NCBI Virus database, and prophage-encoded virulence genes identified by analysing pangenome content.
343 intact prophages were identified, forming ten prophage clusters with distinct gene content, GC content, and length profiles. Five prophage clusters, containing 159 prophages, belonged to the Inoviridae, Myoviridae, and Siphoviridae phage families. The remaining 184 prophages were uncharacterised and may therefore represent novel prophages. Transcriptional regulators with potential virulence effects were identified in three prophage clusters, including one uncharacterised cluster. These prophage clusters were unequally distributed throughout the R. solanacearum population being host genotype specific.
This research demonstrates that R. solanacearum contains a high level of uncharacterised prophage diversity and highlights novel prophages that could contribute to pathogen virulence. Given their potential host-genotype-specific virulence effects, R. solanacearumprophages could be co-evolving with their hosts, and may contribute to global variation in R. solanacearum virulence.
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Bacteriological and Physicochemical Assessment of Sachet and Bottled Drinking Water Sold in Makurdi Metropolis, Benue State, Nigeria
More LessBackgroundPackaged drinking water popularly known as “sachet and bottled water” serves a large percentage in increasing access to clean drinking water in Nigeria. But little attention is given to investigate the bacteriological assessment of these water which may be harmful for human consumption.
ObjectiveThis study was to assess the bacteriological and physicochemical characteristics of sachet and bottled water sold in Makurdi metropolis.
MethodologyA cross sectional study was carried out with a total of one hundred and sixty-five samples collected. These comprised of triplicates of fifty sachet water and five bottled water brands purchased using simple random sampling from street vendors within Makurdi metropolis and analyzed using standard methods and results were compared with the recommended guidelines for water quality.
ResultsThe presence of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus sp., Klebsiella sp., Salmonella sp. and Shigella sp. were detected in the water samples with a total bacteria count (MPN/100ml) ranging from 0 – 1100 MPN/100ml. Escherichia coli had the highest incidence of 80% followed by Staphylococcus aureus 66%, Salmonella species 48%, Pseudomonas aeruginosa 46%, Streptococcus sp 36%, Shigella species 36%, Klebsiella species 22%. The pH, conductivity, total dissolved solids, dissolved oxygen and sodium chloride levels ranged from 6.0 to 7.6, 8.4 to 188.4μS/cm, 4.2 to 94.2mg/l, 0.08 to 0.16mg/l and 0 to 0.4 respectively.
ConclusionThere is urgent need to intensify the monitoring of activities of this rapidly expanding industry and enforcing strict hygienic measures with a view to raising standards to improve packaged water quality.
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The mutational variety of the live-attenuated influenza vaccine proteome
More LessInfluenza viruses evolve rapidly, and for this reason the influenza vaccine needs to be updated every year. It is therefore important to identify where new mutations could be tolerated. To assess this, we asked which mutations could be identified in the proteins that had passed quality checks by being correctly folded, transported and assembled into influenza virus particles. We re-analysed mass spectrometry proteomics data obtained from the virus particles of genetically well-defined vaccine strains and identified point mutations within viral proteins. Point mutations were tolerated in virus particles at appreciable frequencies in proteins of both influenza A and B viruses, including HA, NA, M1, NP, NS1, PA and PB2. Structural analyses were used to assess the likely impact of this protein diversity on the molecular biology of the virus. As would be expected, mutations that were tolerated the virus particle generally occurred at sites that would not be expected to perturb protein function. We suggest that using proteomics to identify sites in viral proteins that either can or cannot tolerate mutations could inform influenza vaccine development by highlighting areas that have the potential for antigenic drift.
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Exploring foot-and-mouth disease virus antibody interactions using biolayer interferometry
Foot-and-mouth disease virus (FMDV) vaccines protect animals from infection by inducing antibodies. The level of neutralising antibody induced in response to vaccination (or infection), as measured by a virus neutralisation test, is an important parameter with regards to the level of protection afforded against subsequent challenge. However, in addition to overall titre, antibody avidity also represents a crucial metric when assessing the protective efficacy of antibodies. In this project we investigated the use of biolayer interferometry (BLI) to measure the avidity of FMDV antibodies to FMDV antigens. Antibodies targeting site I of the FMDV particle were detected using a commercially synthesised biotinylated peptide. In contrast, the entire antigenic landscape of the FMDV particle was represented by biotinylated FMDV capsids. The antigens were loaded onto Octet streptavidin biosensors at an optimal concentration prior to dipping into antibodies. The sera from different animals varied in avidity, reflecting the quantitative differences in avidity that exist between individual animals in response to FMDV vaccines. Interestingly, the Kdis values obtained for site I vs the entire capsid were different, supporting the importance of other sites beyond site I. Similarly, monoclonal antibodies targeting distinct, known antigenic sites on the capsid surface also resulted in different avidities. The BLI methodology reported here offers a useful approach by which to investigate the strength of antibody interactions at specific sites. In conjunction with recombinant technology, BLI will help aid in investigations into the relative importance of the different antigenic sites with regards to inducing a protective response.
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Genomic diversity of Bacillus anthracis in endemic settings: novel approaches and data
Anthrax, caused by the spore-forming bacterium Bacillus anthracis, remains endemic in many developing countries where it has significant impacts on the health and livelihoods of livestock-keeping communities. While the global genomic diversity of B. anthracisis is well characterised, few studies have quantified its diversity in endemic settings at more local scales, where this information could be critical for elucidating transmission dynamics and guiding control efforts. We collected samples from 526 anthrax-suspected animal carcasses between 2016 and 2018 in the Ngorongoro Conservation Area in northern Tanzania. Seventy five percent were confirmed positive through qPCR, suggesting that anthrax is responsible for a large proportion of sudden deaths in livestock in this area. A subset of positive samples were cultured for whole genome sequencing (n = 73), including multiple isolates from individual carcasses. All sequenced isolates belonged to the Ancient A lineage of B. anthracis, a common strain in southeastern Africa. No clear spatial clustering was observed, possibly reflecting extensive animal movement related to local nomadic practices. Moreover, high levels of within-host diversity were observed which suggests that cases commonly result from simultaneous infection with multiple strains. Additionally, we trialed a targeted sequence capture approach on 93 samples. This was successful in recovering >80% of the chromosomal genome at > 15X coverage from 60% of samples tested, thus representing a valuable tool for culture-free sequencing of this high-risk bacterium. Our work paves the way for integrating genomic data for B. anthracis into epidemiological studies and monitoring of control programs in endemic areas worldwide.
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Piperacillin/tazobactam resistance in a clinical isolate of Escherichia colidue to IS26-mediated amplification of blaTEM-1B
We identified a clinical isolate of Escherichia coli displaying an unusual, emerging phenotype; piperacillin/tazobactam (TZP)-resistant, 3rd generation cephalosporin-susceptible. Prior to treatment with TZP, a TZP-susceptible E. coli isolate was isolated from the same patient. Hyperproduction of a class A β-lactamase has previously been linked to this phenotype, but the mechanism of hyperproduction in isolates lacking promoter region mutations is not well understood.
Clonality of the two isolates was initially assessed with RFLP, and β-lactamase activity was determined using a nitrocefin assay. Both isolates were sequenced on Illumina and Oxford Nanopore Technology platforms and fitness assessed competitively. A plasmid construct containing the insertion sequence IS26was used to capture a translocatable unit (TU) in vitro.
The two E. coli clinical isolates were confirmed to be clonal, with the TZP-resistant isolate hyperproducing blaTEM-1. However, no promoter region mutations were identified in the TZP-resistant isolate. Hybrid assembly revealed that an ~11kb segment of DNA was excised from a IS26 flanked pseudo-compound transposon in the TZP-resistant isolate, forming a circular TU containing blaTEM-1. Multiple re-insertion events of the TU, mediated by IS26, led to tandem repeats of the TU within the chromosome, increasing the copy number of blaTEM-1. Excision and insertion events were confirmed via capture of the TU. Amplification of the TU in the TZP-resistant isolate incurred no significant change in fitness in different environmental conditions.
This study improves the understanding of the TZP-resistant, 3rd generation cephalosporin-susceptible phenotype in E. coli and antimicrobial resistance prediction of this phenotype from genotypic data.
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