- Volume 4, Issue 5, 2022

Background. 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.

We 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.

When 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.

We 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.

The 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.

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 .

Introduction. 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.

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.

Capnocytophaga 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.

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.

Microbial 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.

Zika 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.

Exoribonucleases 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.

Only 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.

In 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.