- Volume 2, Issue 1, 2020
Volume 2, Issue 1, 2020
- Abstracts from the Microbes in Medicine Meeting 2019
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- Oral Abstract
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Experimental evolution selects clinically relevant antibiotic resistance in biofilms but with collateral tradeoffs
The widespread usage of antimicrobials in modern clinical, veterinary and industrial practices has selected for the emergence of antibiotic-resistant bacteria, which are increasingly hard to treat with currently available antibiotics. Most bacteria in nature exist in aggregated communities known as biofilms, which are inherently highly tolerant to antibiotics. There is currently a limited understanding of how biofilms evolve in response to antimicrobial pressure. Here we used a biofilm evolution model as a tool to study the effects of antimicrobial exposure on biofilms compared to planktonic cultures. We showed that biofilms of the model foodborne pathogen, Salmonella Typhimurium rapidly evolve in response to exposure to three clinically important antibiotics. Adaptation to antibiotic stress imposed a marked cost in biofilm formation, particularly evident for populations exposed to cefotaxime and azithromycin. By pairing the evolution model with whole-genome sequencing, we were able to identify and characterise two distinct mechanisms of resistance to cefotaxime and azithromycin. Among others, we identified novel substitutions within the multidrug efflux transporter, AcrB (R717L and Q176K) and validated their impact in drug export as well as changes in regulators of this efflux system. We showed that the model biofilm system selects clinically-important mechanisms of resistance and can be used to help predict how biofilms evolve under antimicrobial pressure.
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Factors governing orthologous RpoD and H-NS evolution in Salmonella enterica Serovar Typhimurium and Escherichia coli
More LessPathogenicity island acquisition is considered to be a costly process that in turn allows bacteria to explore new niches. While pathogenicity islands are known to be well integrated into existing regulatory networks in Salmonella, we do not understand whether these islands have influenced the evolution of transcription factor coding sequence itself. Here, we exchanged two antagonisticly-acting bacterial transcription factors, RpoD (σ70) and H-NS, singly or combinatorially, between the related Gram-negative bacteria Salmonella enterica Serovar Typhimurium SL1344 and Escherichia coli str. K12 substr. MG1655, to understand their functional divergence in relation to pathogenicity island expression in Salmonella. Exchanging rpoD resulted in a small growth defect in Salmonella, while exchanging hns did not. We saw a strong upregulation of pathogenicity island expression in response to rpoD exchange, while only a very weak upregulation in response to HNS exchange. Exchanging both rpoD and hns in Salmonella resulted in a further upregulation of these islands. Using two different versions of the SPI1 regulatory knockout, ΔhilA and ΔhilD, where ΔhilA knocks out only SPI1 expression and ΔhilD knocks out both SPI1 and SPI2 expression, we show that both SPI1 and SPI2 expression are costly, and that their up-regulation is the cause of the growth defect in response to rpoD exchange. Thus, we show that both RpoD and H-NS sequence evolution in Salmonella were geared towards constraining pathogenicity island expression, the expression cost of these islands very clearly being a driver of RpoD evolution.
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Short-term consumption of a high-fat diet increases host susceptibility to Listeria monocytogenes infection
A Westernized-diet comprising a high caloric intake from animal fats is known to influence the development of pathological inflammatory conditions. However, there has been relatively little focus upon the implications of such diets for the progression of infectious disease. Here we investigated the influence of a high-fat (HF) diet upon parameters that influence Listeria monocytogenes infection in mice.
We determined that short-term administration of a HF diet increases the number of goblet cells, a known binding site for the pathogen in the gut, and also induces profound changes to the microbiota and promotes a pro-inflammatory gene expression profile in the host. Host physiological changes were concordant with significantly increased susceptibility to oral L. monocytogenes infection in mice fed a HF diet relative to low-fat (LF) or chow-fed animals. Prior to Listeria infection short-term consumption of HF diet elevated faecal levels of Firmicutes. During active infection with L. monocytogenes microbiota changes were further exacerbated but host inflammatory responses were significantly down-regulated relative to Listeria-infected LF or chow-fed groups, suggestive of a profound tampering of the host response influenced by infection in the context of a HF diet.
Overall the results indicate that short-term consumption of a Westernized-diet has the capacity to significantly alter host susceptibility to L. monocytogenes infection concomitant with changes to the host physiological landscape. The findings suggest that diet should be a consideration when developing models that reflect human infectious disease.
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Transposable temperate phages promote the evolution of divergent social strategies in Pseudomonas aeruginosa populations
More LessTransposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments, where the cost of producing cooperative iron-chelating siderophores is high and low, respectively, we show that transposable phages promote divergence into extreme siderophore production phenotypes. Iron-limited populations with transposable phages evolved siderophore over-producing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvdgenes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signaling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.
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Pharmacological modulation of the autophagy pathway enhances Mtb killing
More LessMycobacterium tuberculosis (Mtb) is the causative agent for tuberculosis which remains a serious health concern globally. The emergence of multi-drug resistant strains and the failure of BCG vaccine to control the epidemic highlight the need to achieve a better understanding of the host immune response and develop new therapies. Autophagy has been identified as an important element in both innate and adaptive immunity against tuberculosis. However Mtb employs an array of immune modulators to invade and thrive in macrophages including by inhibiting autophagosome fusion with lysosomes. Our aim is to identify novel modulators of the autophagy pathway. To do this we have screened a number of FDA approved autophagy-enhancing drugs to reverse the Mtb-induced block of autophagic flux and boost host immune responses.
Murine BMDMs expressing EGFP-LC3 were infected with H37Ra and treated with or without autophagy-enhancing drugs. The number of GFP-LC3-positive puncta per cell was quantified by high content analysis to assess autophagic induction and flux, thereby identifying compounds capable of overcoming the Mtb-induced block in autophagy. We identified three compounds, Sorafenib, Valproic Acid and Carbamazepine, which promote autophagic flux in Mtb-infected cells. Human MDMs were infected with H37Ra and treated with or without these drugs to determine their ability to promote intracellular killing of Mtb and boost the innate immune response. Our data show that Sorafenib, Valproic Acid and Carbamazepine are capable of overcoming the Mtb-induced block in autophagy and reducing bacterial burden in MDMs. This work highlights the potential for autophagy-enhancing drugs as adjunctive treatment for tuberculosis.
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The role of mature mycolic acids in mycobacterial biofilm formation and interactions with the human complement system
Tuberculosis, causative agent Mycobacterium tuberculosis, caused an estimated 10 million new cases and 1.6 million deaths in 2017.
Mycobacterial cell wall components are important for host-pathogen interactions and modulating the innate and adaptive immune response. Much is understood about the interplay between bacterial phenotype, cell wall modulation and interaction with aspects of the immune response. However, the impact of phenotype, such as biofilms on complement system activation, has not been studied in-depth.
M. tuberculosis may persist as biofilms in a chronic, non-replicating state. Mycolic acids are a major cell wall lipid and their accumulation is a hallmark of biofilm formation. M. smegmatis was mutated in gene MSMEG4722(orthologof M. tuberculosis Rv2509) (ΔMSMEG4722)which encodes a reductase involved in the final step of mycolic acid biosynthesis.
Using ΔMSMEG4722,this project explored the role of mycolic acid maturation in biofilm development, determined the relationship between lipids and complement activity with planktonic or biofilm bacteria.
Thin-layer chromatography of ΔMSMEG4722 biofilms displayed a loss of mature free mycolic acid compared to M. smegmatis, but accumulated un-reduced free mycolate. In both biofilm and planktonicΔMSMEG4722, distribution of the virulence factor trehalose dimycolate changed throughout the cell wall compared to M. smegmatis.
Flow cytometry analysis of the deposition of complement opsonin C3b revealed increased C3 on planktonic ΔMSMEG4722 compared to M. smegmatis, but this difference was not observed on bacteria from biofilms.
Evidence from this work suggests mycolic acid maturation is important for biofilm development and plays a role in complement activation on mycobacteria.
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- Poster Presentation
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Defining the link between efflux pumps and biofilm formation
More LessBiofilms are intrinsically important to our understanding of bacterial pathogens. The majority of bacterial life exists as part of a biofilm, as despite this, we have very little understanding of the genetic pathways that facilitate and drive biofilm formation. Work in our group has outlined a phenotypic link between efflux pump activity and biofilm formation, both of which have been implicated in decreased susceptibility to multiple antibiotics. Genetic or chemical inactivation of efflux results in a substantial decrease in biofilm formation. We have determined that this is due to transcriptional repression of one of the main components of the biofilm: the amyloid fibrous protein, curli. This relationship between efflux activity and biofilm formation has been identified in many Gram-negative and Gram-positive bacterial pathogens, but the regulatory network through which these phenotypes are linked is unknown. My PhD project aims to determine the pathway that links efflux pump activity and biofilm formation. I will investigate this using TraDIS, which is a large-scale transposon mutagenesis approach that will be used to determine all of the genes responsible for efflux activity and biofilm formation. My poster will present preliminary results from this genome-wide screen in E. coli. I will outline the further work to be undertaken, including repeating this experiment in Salmonella Typhimurium and formulating and testing hypotheses as to how efflux activity and biofilm formation are linked in both species. This will overall improve our understanding of important mechanisms of antimicrobial resistance in human pathogens.
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Carbapenem-resistant Enterobacteriaceae : A serious concern in cancer patients
More LessIntroduction. Gram-negative bacteria, including Enterobacteriaceae, are an important cause of infections in cancer patients. Members of Enterobacteriaceae are commonly isolated from bloodstream infections, surgical site infections, urinary tract infections, and lower respiratory tract infections. There has been an increase in the isolation of gram-negative MDRO’s over the years with ESBL’s and for the last few years with carbapenem-resistant Enterobacteriaceae (CRE). CRE’s have become a serious threat to cancer patients.
Material & Methods. A total of 61331 clinical samples from 19015 patients were received in the Dept of Microbiology, Tata Memorial Center, Mumbai, India during January 2018 to May 2019. All the samples were processed as per routine microbiological procedures and antimicrobial susceptibility testing was performed as per CLSI guidelines.
Results. Blood was the commonest sample received followed by urine, respiratory tract samples, surgical site samples and sterile body fluids. E. coli was the commonest microorganism isolated followed by Klebsiella pneumoniae, P. aeruginosa, S. aureus, Acinetobacter sp., Enterobacter sp. and Enterococci. Colistin was the most susceptible antibiotic for gram negative organisms followed by tigecycline, aminoglycosides, cefoperazone-sulbactam and piperacillin-tazobactam. Overall 77.8% CRE’s were detected whereas it was 81.8% in LRTI, followed by 82.5% in UTI, 79.1% in BSI and 75% in SSI.
Conclusion. The increasing prevalence of CRE infections represents a major threat to cancer patients. With the high mortality of CRE infections and increasing resistance to available antibiotics, it is urgent for the medical community to develop new and effective therapeutic strategies. Robust anti-microbial stewardship is the need of the hour.
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The (p)ppGpp-binding GTPase Era promotes rRNA processing and cold adaptation in Staphylococcus aureus
More LessRibosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase Rel Sau , with Rel Sau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway.
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The antibiofilm effects of docosahexaenoic acid
More LessThe ability of bacteria to form biofilms allows for recalcitrance against conventional antibiotic therapies (Potera, 1999). Therefore, this has contributed to the prevalence of biofilm acquired infections (BAI) clinically (Percival and Kite, 2018) which has resulted in increased morbidity and mortality amongst patients, with immunocompromised patients at risk in particular (Donlan, 2002). Biofilm formation on abiotic and biotic surfaces has enabled bacteria to colonise major organs and medical devices, such as within the lungs, on implant surfaces, contact lenses, and urinary catheters (Vinh and Embil, 2005; Percival et al., 2012; Seth et al., 2012). Docosahexaenoic acid (DHA) is a poly-unsaturated fatty acid known to exhibit antibiofilm and antimicrobial effects (Sun et al., 2017; Kim et al., 2018). Our research has found that DHA possesses strong anti-biofilm effects against Klebsiella pneumoniae and Enterococcus faecalis at low mM concentrations. DHA was capable of reducing biofilm formation by both K. pneumoniae and E. faecalis by approximately 60%. It is believed that this is the first time these effects have been reported. We have also have evidence that DHA in conjunction with an antibiotic is better at reducing biofilm formation by these strains better then either alone.
To date, there is much debate into how DHA exerts these anti-biofilm effects. DHA has been reported to distort bacterial membrane’s therefore, impacting biofilm formation (Sun et al., 2017). We aim to elucidate the precise mechanism of action by utilising a number of genetic approaches alongside the dynamic flow cell system and confocal microscopy.
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Antimicrobial resistance in bacteria associated with childhood pneumonia in Malawi
More LessCommunity acquired pneumonia (CAP) is a disease caused by an infection (by bacteria, viruses or fungi) that in cases of chronic infection lead to high mortality rates particularly in children. Pneumonia is the leading cause of death in children under the age of five worldwide with over 2 million deaths reported in 2016 by the World Health Organisation. Developing countries in sub Saharan Africa such as Malawi see some of the highest rates of the disease. CAP infections are responsible for 20% of deaths of new-borns in Malawi. Antibiotic resistance in disease causing organisms is of growing concern in countries like Malawi.
We obtained bacterial isolates from blood samples taken from Malawian children, aged 18 -60 months, diagnosed with CAP. Streptococcus pneumonia is the primary pathogen in CAP but associated ESKAPE pathogens like Pseudomonas and Staphylococci spp. and Escherichia coli are of growing importance. These were the most common bacteria purified, isolated and characterised from these samples resulting in a unique culture collection. These pathogens have been screened for antimicrobial resistance with a variety of antibiotics to generate a resistance profile based on the minimal inhibitory concentrations of selected isolates using antibiotic susceptibility disks and by broth dilution method. Initial assessment shows current front-line therapies used by the Malawian ministry of health are ineffective in treating CAP.
Our research aims to fully characterise these strains at the genetic level to better understand the mechanisms behind how antibiotic resistance develops to aid in combating this growing issue in countries like Malawi.
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Significance of the chromosomal positions of the genes encoding DNA gyrase in Salmonella enterica Serovar Typhimurium
More LessChanging positions of genes on a chromosome is an informative way of learning about why an existing chromosome structure and gene order was selected by evolution in bacteria. In Salmonella gyrA and gyrB – genes encoding DNA gyrase – enzyme that introduces negative supercoils in DNA, are located almost at opposite poles of a chromosome. However, in many other bacteria they are arranged in a gyrBA operon. In order to investigate the significance of this fact, gyrBA operon was made in Salmonella by bringing gyrA open reading frame and its ribosome binding site directly downstream of gyrB and under a control of its regulatory regions, the original copy of gyrA was deleted. The gyrBA strain obtained as a result exhibits no differences from the wild type in growth and morphology, however, the ability to supercoil DNA is altered between gyrBA and the WT. This is specifically important at conditions mimicking environment inside a macrophage in terms of Mg2+ concentration, as it may suggest alteration of gyrBA survival inside a macrophage. An attempt to make strain with gyrAB operon was not successful, as it was not possible to delete the original gyrB, suggesting particular importance of its position.
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Understanding selection of antimicrobial resistance in biofilms through experimental evolution
More LessBackground. Biofilms possess several important properties which differentiate them from free-living cells, including their response to selective pressures. This work demonstrates a link between exposure to non-therapeutic antimicrobials and selection for antibiotic resistance in experimentally evolved biofilms of Pseudomonas aeruginosa.
Methods. Biofilms of P. aeruginosa PA14 were experimentally evolved through successive subculture under periodically increasing antimicrobial stress. Beginning at 0.25-times MIC, biofilms were exposed to benzalkonium chloride, ciprofloxacin, colistin, zinc or copper sulfate. When the antimicrobial stress resulted in growth failure, the last successful passage was phenotyped. Biofilm formation was quantified via crystal violet uptake and MICs determined by broth microdilution according to EUCAST guidelines.
Results. Experimental evolution selected for mutants which produced approximately four-fold more biomass than the parenteral strain. Both ciprofloxacin and chloramphenicol readily selected for mutants able to survive >1024-times MIC. Conversely, no change in susceptibility was observed for either metal. However, despite zinc sulfate not selecting for decreased susceptibility to itself, cross-resistance to both colistin and meropenem was observed in the zinc-passaged lineages. Benzalkonium chloride did not select for a significant change in susceptibility when grown planktonically. However, when assayed as a biofilm, a 5-fold decrease in benzalkonium chloride susceptibility was observed, indicating a biofilm-specific mechanism of antimicrobial tolerance.
Conclusion. Mutants with an increased capacity to form biofilms and decreased antimicrobial susceptibility were successfully selected. Furthermore, zinc sulfate, a common feed additive, was able to select for decreased susceptibility to clinically-relevant antibiotics, lending to concerns that ubiquitous non-therapeutic antimicrobials may act as non-canonical drivers of antibiotic resistance.
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The evolution of Pseudomonas aeruginosa during short-term acute respiratory infections
Background. The emergence and spread of bacteria with resistance to multiple antimicrobial agents is raising the mortality rates associated with bacterial infections, and poses a fundamental threat to human health. Pseudomonas aeruginosais an opportunistic human pathogen that is responsible for 10-15% of health-care associated infections worldwide, and is known for its ability to rapidly develop enhanced resistance during the course of treating an infection.
Methods. P. aeruginosa isolates have been collected from ICU patients in hospitals across Europe over a longitudinal sampling of short-term acute respiratory infection. Twelve isolates per patient per sampling of infection have been acquired. Genome sequencing and high-throughput phenotyping of growth rate and antibiotic resistance profile has been carried out.
Results and conclusions. Using high-throughput phenotyping and genome sequence data we have been able to characterise population diversity and variation in phenotypic resistance profile, growth rate and acquired resistance gene content of these isolates. A gradient of diversity was found within patient infections, ranging from monoclonal to multiple strains present. Large within-patient population diversity in phenotypic resistance profile and acquired resistance gene content could be observed in multiple sequence-type infections.
This research has been carried out in collaboration with the COMBACTE-MAGNET research consortium (Combatting Bacterial Resistance in Europe – Molecules against Gram-Negative Infections).
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Plasma activated liquids: New decontamination solutions
More LessIn the field of new decontaminants, there is an increasing consensus that improved disinfection of environmental surfaces is needed in patient care facilities as an important component in the overall strategy for prevention of HAI. The interaction of cold atmospheric plasma, i.e. an ionized gas, with liquids results in complex physical and chemical processes, which offer a source of short-lived and long-lived reactive chemical species that are critical for microbial inactivation. These solutions may fulfil the urgent need for new decontaminant solutions for special purposes such as disinfection of surfaces, or use as an antiseptic for body surfaces.
In this study, we explored the bactericidal effects of plasma activated liquids (PALs) on E.coli and S. aureus strains and investigated factors which influence PALs stability over time. Liquids of interest were non-complex solutions such as water and saline. An atmospheric cold plasma system using air was employed for the generation of PALs. The solutions were compared with respect to their content of long-lived reactive chemical species and bactericidal effects.
Our results documented that PALs may carry different concentrations of chemical species and maintain diverse antimicrobial properties. The bactericidal activity of these solutions demonstrated high thermal stability and could be preserved over a 6 month period through specific sub-ambient storage conditions. Investigation of the inactivation processes in relation to the PALs’ chemical composition will enhance our knowledge on how prokaryotic and eukaryotic cells respond to these, and demonstrate how PALs could be a promising treatment method for future applications, with chemical and antimicrobial stability.
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A matter of life and death; identifying host genomic factors that determine susceptibility of chickens to highly pathogenic avian influenza
More LessDNA samples collected from survivors of recent outbreaks of highly pathogenic avian influenza (HPAI) in Mexico and the USA have provided a rare opportunity to study the genetic mechanisms underpinning susceptibility of chickens to this devastating and economically impactful disease which normally exhibits 70-100% mortality in the chicken host. Whole Genome Sequence (WGS) data has been used to perform Genome-Wide Association Studies (GWAS), which have highlighted single nucleotide polymorphisms (SNPs) segregating significantly between survivors and controls, with a pedigreed experimental group exhibiting a highly significant signal on chicken chromosome 2 in the region of a biologically relevant gene. Candidate SNPs for resilience are currently being validated using in vitro gene editing methodologiesthat modulate candidate gene expression to investigate the effect on viral replication and cellular response to HPAI infection. A detailed understanding of the genomic resilience to HPAI from this study will have implications for both the poultry industry and for public health.
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Characterising the urinary microbial community in patients with symptoms of urinary tract infection
More LessBackground. Every year 250 million individuals develop urinary tract infections (UTI), the diagnosis of which, usually involves the ‘gold standard’ midstream urine culture (MSU) test. Prompt detection aided by sensitive tests may increase the likelihood of successful treatment. However, recent evidence highlights the potential inadequacies of this test.
Methods. We assessed the UK’s MSU culture by comparison with enhanced culture techniques and 16S rRNA gene sequencing. With ethical approval, patients attending their first consultation at the Whittington Hospital Clinic and asymptomatic controls provided MSU specimens. Each specimen was submitted to the Hospital Microbiology laboratory for MSU culture. 16S rRNA gene sequencing was performed on 1ml unspun urine and 30ml of centrifuged urine.
Results. Urine specimens were analysed for 33 patients (mean age= 49 years, sd=16.5) and 29 controls (mean age=40.7 years, sd=15.7). Comparison of routine MSU cultures between patients and controls indicated that the test failed to discriminate between patients and controls(χ2= 0.539, df = 1, P = 0.674). Bacterial DNA was detected in 97.0% patients and 89.7% controls. Enterobacteriaceae was most abundant in patients, whereas Streptococcus dominated in controls. A higher distribution of the median number of observed taxa (centrifuged and unspun samples combined) was seen in patients (χ2 = 8.0, df = 2, P <0.05).
Conclusion. The MSU culture failed to discriminate between patients and controls and missed recognised uropathogens that were detected with bacterial DNA sequencing. Microbial communities characterised by sequencing warrant further investigation to understand the role of each member in UTI.
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Vorinostat (SAHA) promotes innate and adaptive immunity to Mycobacterium tuberculosis
More LessMycobacterium tuberculosis (Mtb) is increasingly resistant to antibiotics. Therefore, it is paramount to develop host-directed therapies (HDT) aimed at activating the host immune response to promote bacterial killing. Mtb is phagocytosed by alveolar macrophages (AM) and infiltrating monocyte-derived macrophages (MDM) in the lung which upregulate effector functions by epigenetic modifications to enable transcription. Suberanilohydroxamic acid (Vorinostat; SAHA), an FDA-approved histone deacetylase inhibitor, can modulate these changes and support immunity.
Human AM were purified from bronchoalveolar lavage. MDM were obtained from blood of healthy donors and patients with TB. Macrophages were infected with Mtb in the presence of SAHA. After 24 hours, cytokine secretion was quantified. Macrophages were washed and co-cultured with CFSE-labelled PBMC from IGRA positive donors. T-cell responses were analysed by flow cytometry and ELISA. Macrophages were lysed and colony forming units were enumerated.
SAHA increased IL-1β and decreased IL-10 in human AM and MDM infected with Mtb. Proliferating T-helper cells co-cultured with Mtb-infected, SAHA-treated AM or MDM exhibited enhanced IFN-γ and GMCSF co-production. SAHA promotes proinflammatory immune responses to Mtb infection in human AM and MDM, with a subsequent effect on T cell responses. SAHA may therefore be beneficial as a host-directed therapy or vaccine adjunct against TB.
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Development of a microbially-derived therapy against Fusobacterium nucleatum, a bacterial pathogen linked with colorectal cancer
More LessBackground. Specific bacterial species have been linked to several intestinal diseases, including colorectal cancer (CRC). In recent years, high abundances of an emerging pathogen, Fusobacterium nucleatum, have been identified in tumors and stool samples of CRC patients and it has been suggested that F. nucleatum contributes to CRC initiation and development. The possibility of suppressing the growth of F. nucleatum in the GI tract using antimicrobial-producing probiotic bacteria may reduce the overall risk of CRC development.
Methods. Here, we screen a collection of faecal samples from healthy donors against F. nucleatum in an effort to discover an antimicrobial-producing isolate capable of selectively inhibiting this emerging human pathogen. Potential isolates with anti-Fusobacterial activity were then further analysed for the ability to inhibit the pathogen in cell culture and in a faecal fermentation system, which simulates the dynamic conditions of the human colon.
Results. Culture-based screening of over 16,000 colonies of gastrointestinal origin resulted in the identification of one faecal isolate with probiotic potential displaying significant antagonistic activity against F. nucleatum initially in cell culture media and subsequently inhibition was confirmed in the simulated intestinal model.
Conclusion. This study reveals that, a novel gut isolate demonstrates inhibition against the CRC-associated F. nucleatum in vitro and suppresses its growth in a model of the human distal colon. This is an important finding, suggesting the potential of a natural gut bacterium to supress the growth of a bacterial pathogen associated with CRC, which may contribute to reducing the overall risk of developing the disease.
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Quorum quenching activity of Aerva lanata against Catheter Associated Urinary Tract Infections (CAUTI) Pathogens
More LessBackground. CAUTI is the most common hospital infection caused by Gram-negative bacterium owing to their ability to colonize UT mucosa. These uro-pathogens elaborate quorum sensing (QS) molecules that modify the expression and secretion of biofilm matrix compounds in response to rheological and other UT changes during catheterization. Quenching the biological effects of QS molecules will have substantial clinical value. This study identifies and evaluate the antibiofilm/anti-virulence properties of Aerva lanata plant extract against select CAUTI pathogens.
Methods. Methanolic extract of Aerva lanata was screened for its anti-urobacterial effects using Chromobacterium violaceum as the test strain. A. lanata extract impact on the QS molecule, acyl homoserine lactone (AHL) regulated bacterial functions essential for biofilm formation was investigated. It includes RT-PCR analysis of QS genes in the bacterium and in silico studies of bacterial compounds.
Results. Methanolic extract of A. lanata interfered with AHL regulated physiological functions such as biofilm formation, pellicle inhibition, flagellar motility and exopolysaccharides (EPS) production. In situ-visualization of biofilm with confocal microscopy showed concentration influenced reduction in bacterial biofilm formation in response to plant extract. GC-MS identified 11 compounds and molecular docking analysis predicted their respective host receptors. In silico analysis of plant extract revealed putative compounds with potential to inhibit the bacterial QS AHL system. RT-PCR analysis revealed down regulation of QS related virulence genes viz., las1, lasR, lasB, lasA.
Conclusion. Data obtained in this study strongly suggests that Aerva lanata should be further investigated for its therapeutic potentials to treat bacterial CAUTI.
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