The effect of antibiotic selection on collateral effects and evolvability of uropathogenic Escherichia coli.

Trimethoprim is recommended as a first-line treatment of urinary tract infections (UTIs) in the UK. In 2018, 31.4% of Escherichia coli isolated from UTIs in England were trimethoprim-resistant, leading to overreliance on other first and second-line antibiotics. Here, we assessed whether, in principle, prior selection with trimethoprim results in collateral effects to other antibiotics recommended for the treatment of UTIs. As collateral effects, we considered changes in susceptibility, mutation-selection window and population establishment probability. We selected 10 trimethoprim-resistant derivatives from three clinical isolates of uropathogenic Escherichia coli. We found that mutations conferring trimethoprim resistance did not have any collateral effects on fosfomycin. In contrast, resistance to trimethoprim resulted in decreased susceptibility (collateral resistance) to nitrofurantoin, below the clinical breakpoint and narrowed the mutation-selection window, thereby reducing the maximum concentration for selection of nitrofurantoin resistance mutations. Our analyses demonstrate that multiple collateral responses should be accounted for when predicting and optimising antibiotic use, limiting future antimicrobial resistance emergence.

Analysis of the limited M. tuberculosis accessory genome reveals potential pitfalls of pan-genome analysis approaches.

Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 Mycobacterium tuberculosis ( Mtb ) isolates. Mtb is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in Mtb originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated Mtb accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to Mtb and E. coli pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.

Diversity of antimicrobial-resistant bacteria isolated from Australian chicken and pork meat.

Antimicrobial-resistant bacteria are frequently isolated from retail meat and may infect humans. To determine the diversity of antimicrobial-resistant bacteria in Australian retail meat, bacteria were cultured on selective media from raw chicken (n = 244) and pork (n = 160) meat samples obtained from all four major supermarket chains in the ACT/NSW, Australia, between March and June 2021. Antimicrobial susceptibility testing (AST) was performed for 13 critically and 4 highly important antibiotics as categorised by the World Health Organization (WHO) for a wide range of species detected in the meat samples. A total of 288 isolates underwent whole-genome sequencing (WGS) to identify the presence of antimicrobial resistance (AMR) genes, virulence genes, and plasmids. AST testing revealed that 35/288 (12%) of the isolates were found to be multidrug-resistant (MDR). Using WGS data, 232/288 (81%) of the isolates were found to harbour resistance genes for critically or highly important antibiotics. This study reveals a greater diversity of AMR genes in bacteria isolated from retail meat in Australia than previous studies have shown, emphasising the importance of monitoring AMR in not only foodborne pathogenic bacteria, but other species that are capable of transferring AMR genes to pathogenic bacteria.

Towards personalised anti-microbial and immune approaches to infections in acute care. Can real-time genomic-informed diagnosis of pathogens, and immune-focused therapies improve outcomes for patients? An observational, experimental study protocol.

INTRODUCTION: Infection causes a vast burden of disease, with significant mortality, morbidity and costs to health-care systems. However, identifying the pathogen causative infection can be challenging, resulting in high use of broad-spectrum antibiotics, much of which may be inappropriate. Novel metagenomic methods have potential to rapidly identify pathogens, however their clinical utility for many infections is currently unclear. Outcome from infection is also impacted by the effectiveness of immune responses, which can be impaired by age, co-morbidity and the infection itself. The aims of this study are twofold: To compare diversity of organisms identified and time-to-result using metagenomic methods versus traditional culture -based techniques, to explore the potential clinical role of metagenomic approaches to pathogen identification in a range of infections.To characterise the ex vivo function of immune cells from patients with acute infection, exploring host and pathogen-specific factors which may affect immune function and overall outcomes. METHODS: This is a prospective observational study of patients with acute infection. Patients with symptoms suggestive of an acute infection will be recruited, and blood and bodily fluid relevant to the site of infection collected (for example, sputum and naso-oropharyngeal swabs for respiratory tract infections, or urine for a suspected urinary tract infection). Metagenomic analysis of samples will be compared to traditional microbiology, alongside the antimicrobials received. Blood and respiratory samples such as bronchoalveolar lavage will be used to isolate immune cells and interrogate immune cell function. Where possible, similar samples will be collected from matched participants without a suspected infection to determine the impact of infection on both microbiome and immune cell function.

Borderline rpoB mutations transmit at the same rate as common rpoB mutations in a tuberculosis cohort in Bangladesh.

The spread of multidrug-resistant tuberculosis (MDR-TB) is a growing problem in many countries worldwide. Resistance to one of the primary first-line drugs, rifampicin, is caused by mutations in the Mycobacterium tuberculosis rpoB gene. So-called borderline rpoB mutations confer low-level resistance, in contrast to more common rpoB mutations which confer high-level resistance. While some borderline mutations show lower fitness in vitro than common mutations, their in vivo fitness is currently unknown. We used a dataset of 394 whole genome sequenced MDR-TB isolates from Bangladesh, representing around 44 % of notified MDR-TB cases over 6 years, to look at differences in transmission clustering between isolates with borderline rpoB mutations and those with common rpoB mutations. We found a relatively low percentage of transmission clustering in the dataset (34.8 %) but no difference in clustering between different types of rpoB mutations. Compensatory mutations in rpoA, rpoB, and rpoC were associated with higher levels of transmission clustering as were lineages two, three, and four relative to lineage one. Young people as well as patients with high sputum smear positive TB were more likely to be in a transmission cluster. Our findings show that although borderline rpoB mutations have lower in vitro growth potential this does not translate into lower transmission potential or in vivo fitness. Proper detection of these mutations is crucial to ensure they do not go unnoticed and spread MDR-TB within communities.

Molecular and metabolic characteristics of wastewater associated Escherichia coli strains.

We previously characterized the genetic diversity of Escherichia coli strains isolated from septic tanks in the Canberra region, Australia. In this study, we used repetitive element palindromic (REP) PCR fingerprinting to identify dominant REP-types belonging to phylogroups A and B1 strains across septic tanks. Subsequently, 76 E. coli strains were selected for whole-genome sequencing and phenotype microarrays. Comparative genome analysis was performed to compare septic tank E. coli genomes with a collection of 433 E. coli isolates from different hosts and freshwater. Clonal complexes (CCs) 10 (n = 15) and 399 (n = 10) along with sequence type (ST) 401 (n = 9) were the common lineages in septic tanks. CC10 strains have been detected from animal hosts and freshwater, whereas CC399 and ST401 strains appeared to be associated with septic tanks as they were uncommon in isolates from other sources. Comparative genome analysis revealed that CC399 and ST401 were genetically distinct from other isolates and carried an abundance of niche-specific traits involved in environmental adaptation. These strains also showed distinct metabolic characteristics, such as the ability to utilize pectin, which may provide a fitness advantage under nutrient-limited conditions. The results of this study characterized the adaptive mechanisms allowing E. coli to persist in wastewater.

Decreased Susceptibility of Shigella Isolates to Azithromycin in Children in Tehran, Iran.

Azithromycin (AZT) has widely been used for the treatment of shigellosis in children. Recent studies showed a high rate of decreased susceptibility to azithromycin due to different mechanisms of resistance in Shigella isolates. Accordingly, the purpose of this study was to investigate the role of azithromycin resistance mechanisms of Shigella isolates in Iran during a two-year period. In this study, we investigated the mechanisms of resistance among Shigella spp. that were isolated from children with shigellosis. The minimum inhibitory concentration (MIC) of Shigella isolates to azithromycin was determined by the agar dilution method in the presence and absence of Phe-Arg-ß-naphthylamide inhibitor. The presence of 12 macrolide resistance genes was investigated for all isolates by PCR for the first time in Tehran province in Iran. Among the 120 Shigella spp., only the mph(A) gene (49.2%) was detected and other macrolide resistance genes were absent. The phenotypic activity of efflux pump was observed in 1.9% of isolates which were associated with over expression of both omp(A) and omp(W) genes. The high prevalence of the mph(A) gene among DSA isolates may indicate that azithromycin resistance has evolved as a result of antimicrobial selection pressures and inappropriate use of azithromycin.

Prevalence, diversity and genetic structure of Escherichia coli isolates from septic tanks.

The present study investigated the diversity and genetic structure of Escherichia coli isolates from 100 septic tanks in the Canberra region, Australia. The physicochemical characteristics of the septic tanks were determined to examine the extent to which environmental factors might influence E. coli prevalence, diversity and population structure. The results of this study indicated that the temperature of the septic tank could explain some of the variation observed in the number of E. coli isolates recovered per septic tank, whereas pH was an important driver of E. coli diversity. Conductivity, pH and household size had a significant impact on E. coli population structure, and household size significantly affected the probability of detecting human-associated E. coli lineages [sequence types (STs) 69, 73, 95 and 131] in septic tanks. Phylogroup A and B1 strains were not randomly distributed among septic tanks, and the strong negative association between them may indicate intraspecific competition. The findings of this study suggest that the combination of environmental factors and intraspecific interactions may influence the distribution and genetic structure of E. coli in the environment.

Molecular docking based screening of predicted potential inhibitors for VP40 from Ebola virus.

Ebola virus is a member of Filoviridae and cause severe human disease with 90 percent mortality. The life cycle of Ebola contains an assembly stage which is mediated by VP40 proteins. VP40 subunits oligomerize and form ring-structures which are either octamers or hexamers. Prevention of VP40 matrix protein assembly prevents virus particle formation as well as virus budding. In the present study we simulated the biological condition for a single VP40 subunit. Then a library containing 120.000 drugs like chemicals was used as the virtual screening database. Top 10 successive hits were then analyzed regarding absorption, distribution, metabolism, and excretion properties. Moreover probable accessorial human protein targets and toxicity properties of successive hits were analyzed by in silico tools. We found 4 chemicals that could bind VP40 subunits in a manner that by making an interfering steric condense prevents matrix protein oligomerization. The pharmacokinetic and toxicity studies also validated the potential of 4 finlay successive hits to be considered as a new anti-Ebola drugs.