Antigen Discovery for Next-Generation Pertussis Vaccines Using Immunoproteomics and Transposon-Directed Insertion Sequencing.

BACKGROUND: Despite high vaccination rates, the United States has experienced a resurgence in reported cases of pertussis after switching to the acellular pertussis vaccine, indicating a need for improved vaccines that enhance infection control. METHODS: Bordetella pertussis antigens recognized by convalescent-baboon serum and nasopharyngeal wash were identified by immunoproteomics and their subcellular localization predicted. Genes essential or important for persistence in the baboon airway were identified by transposon-directed insertion-site sequencing (TraDIS) analysis. RESULTS: In total, 314 B. pertussis antigens were identified by convalescent baboon serum and 748 by nasopharyngeal wash. Thirteen antigens were identified as immunogenic in baboons, essential for persistence in the airway by TraDIS, and membrane-localized: BP0840 (OmpP), Pal, OmpA2, BP1485, BamA, Pcp, MlaA, YfgL, BP2197, BP1569, MlaD, ComL, and BP0183. CONCLUSIONS: The B. pertussis antigens identified as immunogenic, essential for persistence in the airway, and membrane-localized warrant further investigation for inclusion in vaccines designed to reduce or prevent carriage of bacteria in the airway of vaccinated individuals.

Exploiting genomics to mitigate the public health impact of antimicrobial resistance.

Antimicrobial resistance (AMR) is a major global public health threat, which has been largely driven by the excessive use of antimicrobials. Control measures are urgently needed to slow the trajectory of AMR but are hampered by an incomplete understanding of the interplay between pathogens, AMR encoding genes, and mobile genetic elements at a microbial level. These factors, combined with the human, animal, and environmental interactions that underlie AMR dissemination at a population level, make for a highly complex landscape. Whole-genome sequencing (WGS) and, more recently, metagenomic analyses have greatly enhanced our understanding of these processes, and these approaches are informing mitigation strategies for how we better understand and control AMR. This review explores how WGS techniques have advanced global, national, and local AMR surveillance, and how this improved understanding is being applied to inform solutions, such as novel diagnostic methods that allow antimicrobial use to be optimised and vaccination strategies for better controlling AMR. We highlight some future opportunities for AMR control informed by genomic sequencing, along with the remaining challenges that must be overcome to fully realise the potential of WGS approaches for international AMR control.

Clinical and laboratory factors associated with neonatal sepsis mortality at a major Vietnamese children's hospital.

Sepsis is a major cause of neonatal mortality and children born in low- and middle-income countries (LMICs) are at greater risk of severe neonatal infections than those in higher-income countries. Despite this disparity, there are limited contemporaneous data linking the clinical features of neonatal sepsis with outcome in LMICs. Here, we aimed to identify factors associated with mortality from neonatal sepsis in Vietnam. We conducted a prospective, observational study to describe the clinical features, laboratory characteristics, and mortality rate of neonatal sepsis at a major children's hospital in Ho Chi Minh City. All in-patient neonates clinically diagnosed with probable or culture-confirmed sepsis meeting inclusion criteria from January 2017 to June 2018 were enrolled. We performed univariable analysis and logistic regression to identify factors independently associated with mortality. 524 neonates were recruited. Most cases were defined as late-onset neonatal sepsis and were hospital-acquired (91.4% and 73.3%, respectively). The median (IQR) duration of hospital stay was 23 (13-41) days, 344/524 (65.6%) had a positive blood culture (of which 393 non-contaminant organisms were isolated), and 69/524 (13.2%) patients died. Coagulase-negative staphylococci (232/405; 57.3%), Klebsiella spp. (28/405; 6.9%), and Escherichia coli (27/405; 6.7%) were the most isolated organisms. Sclerema (OR = 11.4), leukopenia <4,000/mm3 (OR = 7.8), thrombocytopenia <100,000/mm3 (OR = 3.7), base excess < -20 mEq/L (OR = 3.6), serum lactate >4 mmol/L (OR = 3.4), extremely low birth weight (OR = 3.2), and hyperglycaemia >180 mg/dL (OR = 2.6) were all significantly (p<0.05) associated with mortality. The identified risk factors can be adopted as prognostic factors for the diagnosis and treatment of neonatal sepsis and enable early risk stratification and interventions appropriate to reduce neonatal sepsis in LMIC settings.

A Global Crowdsourcing Open Call to Improve Research Mentorship in Low- and Middle-Income Countries: A Mixed Methods Analysis.

Research mentoring programs are limited in many low- and middle-income countries (LMICs). The TDR Global initiated a global crowdsourcing open call soliciting proposals on how to improve research mentorship in LMICs. The purpose of this study is to examine ideas submitted to this open call to identify the ways to improve research mentorship in LMICs. Open calls have a group of individuals solve all or part of a problem and then share solutions. A WHO/TDR/SESH crowdsourcing guide was used to structure the open call. Each submission was judged by three independent individuals on a 1-10 scale. Textual submissions were extracted from eligible proposals and qualitatively analyzed via inductive and deductive coding techniques to identify themes. The open call received 123 submissions from 40 countries in Asia (49), Africa (38), Latin America (26), and Europe (10). Among all participants, 108 (87%) had research experience. A total of 21 submissions received a mean score of 7/10 or higher. Our thematic analysis identified three overarching themes related to prementoring, facilitation, and evaluation. Prementoring establishes mentor-mentee compatibility to lay foundations for mentorship. Facilitation involves iterative cycles of planning, communication, and skill improvement. Evaluation creates commitment and accountability within a framework of monitoring. This global crowdsourcing open call generated numerous mentorship ideas, including LMIC-contextualized facilitation tools. The open call demonstrates a need for greater focus on mentorship. Our data may inform the development of formal and informal mentoring programs in LMIC settings.

Long-read-sequenced reference genomes of the seven major lineages of enterotoxigenic Escherichia coli (ETEC) circulating in modern time.

Enterotoxigenic Escherichia coli (ETEC) is an enteric pathogen responsible for the majority of diarrheal cases worldwide. ETEC infections are estimated to cause 80,000 deaths annually, with the highest rates of burden, ca 75 million cases per year, amongst children under 5 years of age in resource-poor countries. It is also the leading cause of diarrhoea in travellers. Previous large-scale sequencing studies have found seven major ETEC lineages currently in circulation worldwide. We used PacBio long-read sequencing combined with Illumina sequencing to create high-quality complete reference genomes for each of the major lineages with manually curated chromosomes and plasmids. We confirm that the major ETEC lineages all harbour conserved plasmids that have been associated with their respective background genomes for decades, suggesting that the plasmids and chromosomes of ETEC are both crucial for ETEC virulence and success as pathogens. The in-depth analysis of gene content, synteny and correct annotations of plasmids will elucidate other plasmids with and without virulence factors in related bacterial species. These reference genomes allow for fast and accurate comparison between different ETEC strains, and these data will form the foundation of ETEC genomics research for years to come.

Molecular and epidemiological surveillance of polymyxin-resistant Klebsiella pneumoniae strains isolated from Brazil with multiple mgrB gene mutations.

The prevalence of polymyxin-resistant Enterobacteriaceae is increasing worldwide. Their emergence is worrisome and limits therapeutic options for severely ill patients. We aimed to investigate the molecular and epidemiological characteristics of polymyxin-resistant Klebsiella pneumoniae circulating in Brazilian hospitals. Polymyxin-resistant K. pneumoniae isolates from two Brazilian healthcare facilities were characterized phenotypically and subjected to whole genome sequencing (WGS). Using the WGS data we determined their sequence type, resistance gene content (resistome), their composition of virulence genes and plasmids. ST11 was the most common (80 %) sequence type among the isolates followed by ST345, ST15 and ST258. A resistome analysis revealed the common presence of blaKPC-2 and less frequently blaSHV-11, blaTEM-1, blaCTX-M-15, and blaOXA-9. Genes conferring resistance to aminoglycosides, fluoroquinolones, phenicols, sulphonamides, tetracyclines, trimethoprim and macrolide-lincosamide-streptogramin were also detected. We observed a clonal spread of polymyxin-resistant K. pneumoniae isolates, with polymyxin-resistance associated with various alterations in the mgrB gene including inactivation by an insertion sequence and nonsense point mutations. We additionally identified a novel 78-bp repeat sequence, encoding a MgrB protein with 26 amino acids duplicated in six isolates. This is the first observation of this type of alteration being associated with polymyxin resistance. Our findings demonstrate that mgrB alterations were the most common source of polymyxin-resistance in Brazilian clinical settings. Interestingly, distinct genetic events were identified among clonally related isolates, including a new amino acid alteration. The clinical implications and investigation of the resistance mechanisms is of great importance to patient safety and control of these infections, particularly in long-term care facilities.

An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria.

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.

MICHELINdb: a web-based tool for mining of helminth-microbiota interaction datasets, and a meta-analysis of current research.

BACKGROUND: The complex network of interactions occurring between gastrointestinal (GI) and extra-intestinal (EI) parasitic helminths of humans and animals and the resident gut microbial flora is attracting increasing attention from biomedical researchers, because of the likely implications for the pathophysiology of helminth infection and disease. Nevertheless, the vast heterogeneity of study designs and microbial community profiling strategies, and of bioinformatic and biostatistical approaches for analyses of metagenomic sequence datasets hinder the identification of bacterial targets for follow-up experimental investigations of helminth-microbiota cross-talk. Furthermore, comparative analyses of published datasets are made difficult by the unavailability of a unique repository for metagenomic sequence data and associated metadata linked to studies aimed to explore potential changes in the composition of the vertebrate gut microbiota in response to GI and/or EI helminth infections. RESULTS: Here, we undertake a meta-analysis of available metagenomic sequence data linked to published studies on helminth-microbiota cross-talk in humans and veterinary species using a single bioinformatic pipeline, and introduce the 'MICrobiome HELminth INteractions database' (MICHELINdb), an online resource for mining of published sequence datasets, and corresponding metadata, generated in these investigations. CONCLUSIONS: By increasing data accessibility, we aim to provide the scientific community with a platform to identify gut microbial populations with potential roles in the pathophysiology of helminth disease and parasite-mediated suppression of host inflammatory responses, and facilitate the design of experiments aimed to disentangle the cause(s) and effect(s) of helminth-microbiota relationships. Video abstract.

Commensal Escherichia coli are a reservoir for the transfer of XDR plasmids into epidemic fluoroquinolone-resistant Shigella sonnei.

Despite the sporadic detection of fluoroquinolone-resistant Shigella in Asia in the early 2000s and the subsequent global spread of ciprofloxacin-resistant (cipR) Shigella sonnei from 2010, fluoroquinolones remain the recommended therapy for shigellosis1-7. The potential for cipR S. sonnei to develop resistance to alternative second-line drugs may further limit future treatment options8. Here, we aim to understand the evolution of novel antimicrobial resistant (AMR) S. sonnei variants after introduction into Vietnam. We found that cipR S. sonnei displaced the resident ciprofloxacin-susceptible (cipS) lineage while rapidly acquiring additional resistance to multiple alternative antimicrobial classes. We identified several independent acquisitions of extensively drug-resistant/multidrug-resistant-inducing plasmids, probably facilitated by horizontal transfer from commensals in the human gut. By characterizing commensal Escherichia coli from Shigella-infected and healthy children, we identified an extensive array of AMR genes and plasmids, including an identical multidrug-resistant plasmid isolated from both S. sonnei and E. coli in the gut of a single child. We additionally found that antimicrobial usage may impact plasmid transfer between commensal E. coli and S. sonnei. These results suggest that, in a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines.

Risk factors for polymyxin-resistant carbapenemase-producing Enterobacteriaceae in critically ill patients: An epidemiological and clinical study.

This study aimed to assess the clinical impact and potential risk factors associated with polymyxin-resistant Enterobacteriaceae strains isolated from patients hospitalized in adult and neonatal intensive care units. A case-control study was conducted from September 2015 to January 2017. Antimicrobial susceptibility of polymyxin-resistant Enterobacteriaceae strains was determined by broth microdilution. The presence of resistance genes was evaluated by polymerase chain reaction and DNA sequencing. Renal failure [P=0.02, odds ratio (OR) 11.37, 95% confidence interval (CI) 1.0-128.63], use of a urinary catheter (P<0.01, OR 4.16, 95% CI 38.82-366.07), transfer between hospital units (P=0.03, OR 9.98, 95% CI 1.01-98.42), carbapenem use (P<0.01, OR 45.49, 95% CI 6.93-298.62) and surgical procedure (P<0.01, OR 16.52, 95% CI 2.83-96.32) were found to be risk factors for the acquisition of polymyxin-resistant strains in adult patients. For neonatal patients, use of a central venous catheter (P<0.01, OR 69.59, 95% CI 7.33-660.30) was the only risk factor associated with the acquisition of polymyxin-resistant strains. Analysis of the outcomes revealed that the mortality rate was significantly higher in adult (66.6%) and neonatal (23.5%) patients with polymyxin-resistant strains than in those with polymyxin-susceptible strains. In addition, carbapenem exposure (P<0.01, OR 50.93, 95% CI 2.26->999.999) was strongly associated with mortality. On the other hand, aminoglycoside use (P<0.03, OR 0.06, 95% CI 0.004-0.97) was a protective factor against mortality from polymyxin-resistant strains. Several risk factors were associated with polymyxin-resistant strains. The high mortality rates showed that acquisition of these strains is a predictor for unfavourable outcomes. Combination treatment with an aminoglycoside and polymyxin might be a better combination to improve patient outcomes.

High-resolution sweep metagenomics using fast probabilistic inference.

Determining the composition of bacterial communities beyond the level of a genus or species is challenging because of the considerable overlap between genomes representing close relatives. Here, we present the mSWEEP pipeline for identifying and estimating the relative sequence abundances of bacterial lineages from plate sweeps of enrichment cultures. mSWEEP leverages biologically grouped sequence assembly databases, applying probabilistic modelling, and provides controls for false positive results. Using sequencing data from major pathogens, we demonstrate significant improvements in lineage quantification and detection accuracy. Our pipeline facilitates investigating cultures comprising mixtures of bacteria, and opens up a new field of plate sweep metagenomics.

Dissecting the molecular evolution of fluoroquinolone-resistant Shigella sonnei.

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400 S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei. We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations (gyrA-S83L, parC-S80I, and gyrA-D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance.

Contrasting patterns of longitudinal population dynamics and antimicrobial resistance mechanisms in two priority bacterial pathogens over 7 years in a single center.

BACKGROUND: Two of the most important pathogens contributing to the global rise in antimicrobial resistance (AMR) are Klebsiella pneumoniae and Enterobacter cloacae. Despite this, most of our knowledge about the changing patterns of disease caused by these two pathogens is based on studies with limited timeframes that provide few insights into their population dynamics or the dynamics in AMR elements that they can carry. RESULTS: We investigate the population dynamics of two priority AMR pathogens over 7 years between 2007 and 2012 in a major UK hospital, spanning changes made to UK national antimicrobial prescribing policy in 2007. Between 2006 and 2012, K. pneumoniae showed epidemiological cycles of multi-drug-resistant (MDR) lineages being replaced approximately every 2 years. This contrasted E. cloacae where there was no temporally changing pattern, but a continuous presence of the mixed population. CONCLUSIONS: The differing patterns of clonal replacement and acquisition of mobile elements shows that the flux in the K. pneumoniae population was linked to the introduction of globally recognized MDR clones carrying drug resistance markers on mobile elements. However, E. cloacae carries a chromosomally encoded ampC conferring resistance to front-line treatments and shows that MDR plasmid acquisition in E. cloacae was not indicative of success in the hospital. This led to markedly different dynamics in the AMR populations of these two pathogens and shows that the mechanism of the resistance and its location in the genome or mobile elements is crucial to predict population dynamics of opportunistic pathogens in clinical settings.

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions. A significant fraction of changes in gene expression can be explained by positive selection on single mutations that either create or disrupt transcriptional start sites (TSS). Furthermore, we show that clinical strains have different methyltransferases inactivated and thus different methylation patterns. Under the tested conditions, differential methylation has a minor direct role on transcriptomic differences between strains. However, disruption of a methyltransferase in one clinical strain revealed important expression differences suggesting indirect mechanisms of expression regulation. Our study demonstrates that variation in transcriptional profiles are mainly due to TSS mutations and have likely evolved due to differences in host characteristics.

Clinical and laboratory-induced colistin-resistance mechanisms in Acinetobacter baumannii.

The increasing incidence and emergence of multi-drug resistant (MDR) Acinetobacter baumannii has become a major global health concern. Colistin is a historic antimicrobial that has become commonly used as a treatment for MDR A. baumannii infections. The increase in colistin usage has been mirrored by an increase in colistin resistance. We aimed to identify the mechanisms associated with colistin resistance in A. baumannii using multiple high-throughput-sequencing technologies, including transposon-directed insertion site sequencing (TraDIS), RNA sequencing (RNAseq) and whole-genome sequencing (WGS) to investigate the genotypic changes of colistin resistance in A. baumannii. Using TraDIS, we found that genes involved in drug efflux (adeIJK), and phospholipid (mlaC, mlaF and mlaD) and lipooligosaccharide synthesis (lpxC and lpsO) were required for survival in sub-inhibitory concentrations of colistin. Transcriptomic (RNAseq) analysis revealed that expression of genes encoding efflux proteins (adeI, adeC, emrB, mexB and macAB) was enhanced in in vitro generated colistin-resistant strains. WGS of these organisms identified disruptions in genes involved in lipid A (lpxC) and phospholipid synthesis (mlaA), and in the baeS/R two-component system (TCS). We additionally found that mutations in the pmrB TCS genes were the primary colistin-resistance-associated mechanisms in three Vietnamese clinical colistin-resistant A. baumannii strains. Our results outline the entire range of mechanisms employed in A. baumannii for resistance against colistin, including drug extrusion and the loss of lipid A moieties by gene disruption or modification.

O-Antigen-Dependent Colicin Insensitivity of Uropathogenic Escherichia coli.

The outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted by Escherichia coli, can target other E. coli cells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of various E. coli strains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why do E. coli strains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenic E. coli sequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen into E. coli K-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizing E. coli toward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coli infections can be a major health burden, especially with the organism becoming increasingly resistant to "last-resort" antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenic E. coli strain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity among E. coli organisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenic E. coli such as uropathogenic E. coli (UPEC).

The phylogeography and incidence of multi-drug resistant typhoid fever in sub-Saharan Africa.

There is paucity of data regarding the geographical distribution, incidence, and phylogenetics of multi-drug resistant (MDR) Salmonella Typhi in sub-Saharan Africa. Here we present a phylogenetic reconstruction of whole genome sequenced 249 contemporaneous S. Typhi isolated between 2008-2015 in 11 sub-Saharan African countries, in context of the 2,057 global S. Typhi genomic framework. Despite the broad genetic diversity, the majority of organisms (225/249; 90%) belong to only three genotypes, 4.3.1 (H58) (99/249; 40%), 3.1.1 (97/249; 39%), and 2.3.2 (29/249; 12%). Genotypes 4.3.1 and 3.1.1 are confined within East and West Africa, respectively. MDR phenotype is found in over 50% of organisms restricted within these dominant genotypes. High incidences of MDR S. Typhi are calculated in locations with a high burden of typhoid, specifically in children aged <15 years. Antimicrobial stewardship, MDR surveillance, and the introduction of typhoid conjugate vaccines will be critical for the control of MDR typhoid in Africa.

A global genomic approach uncovers novel components for twitching motility-mediated biofilm expansion in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an extremely successful pathogen able to cause both acute and chronic infections in a range of hosts, utilizing a diverse arsenal of cell-associated and secreted virulence factors. A major cell-associated virulence factor, the Type IV pilus (T4P), is required for epithelial cell adherence and mediates a form of surface translocation termed twitching motility, which is necessary to establish a mature biofilm and actively expand these biofilms. P. aeruginosa twitching motility-mediated biofilm expansion is a coordinated, multicellular behaviour, allowing cells to rapidly colonize surfaces, including implanted medical devices. Although at least 44 proteins are known to be involved in the biogenesis, assembly and regulation of the T4P, with additional regulatory components and pathways implicated, it is unclear how these components and pathways interact to control these processes. In the current study, we used a global genomics-based random-mutagenesis technique, transposon directed insertion-site sequencing (TraDIS), coupled with a physical segregation approach, to identify all genes implicated in twitching motility-mediated biofilm expansion in P. aeruginosa. Our approach allowed identification of both known and novel genes, providing new insight into the complex molecular network that regulates this process in P. aeruginosa. Additionally, our data suggest that the flagellum-associated gene products have a differential effect on twitching motility, based on whether components are intra- or extracellular. Overall the success of our TraDIS approach supports the use of this global genomic technique for investigating virulence genes in bacterial pathogens.

Changing Antimicrobial Resistance Trends in Kathmandu, Nepal: A 23-Year Retrospective Analysis of Bacteraemia.

A comprehensive longitudinal understanding of the changing epidemiology of the agents causing bacteraemia and their AMR profiles in key locations is crucial for assessing the progression and magnitude of the global AMR crisis. We performed a retrospective analysis of routine microbiological data from April 1992 to December 2014, studying the time trends of non-Salmonella associated bacteraemia at a single Kathmandu healthcare facility. The distribution of aetiological agents, their antimicrobial susceptibility profiles, and the hospital ward of isolation were assessed. Two hundred twenty-four thousand seven hundred forty-one blood cultures were performed over the study period, of which, 30,353 (13.5%) exhibited growth for non-contaminant bacteria. We observed a significant increasing trend in the proportion of MDR non-Salmonella Enterobacteriaceae (p < 0.001), other Gram-negative organisms (p = 0.006), and Gram-positive organisms (p = 0.006) over time. Additionally, there was an annual increasing trend in the proportion of MDR organisms in bacteria-positive blood cultures originating from patients attending the emergency ward (p = 0.006) and the outpatient department (p = 0.006). This unique dataset demonstrates that community acquired non-Salmonella bacteraemia has become an increasingly important cause of hospital admission in Kathmandu. An increasing burden of bacteraemia associated with MDR organisms in the community underscores the need for preventing the circulation of MDR bacteria within the local population.

Investigating the Campylobacter jejuni Transcriptional Response to Host Intestinal Extracts Reveals the Involvement of a Widely Conserved Iron Uptake System.

Campylobacter jejuni is a pathogenic bacterium that causes gastroenteritis in humans yet is a widespread commensal in wild and domestic animals, particularly poultry. Using RNA sequencing, we assessed C. jejuni transcriptional responses to medium supplemented with human fecal versus chicken cecal extracts and in extract-supplemented medium versus medium alone. C. jejuni exposed to extracts had altered expression of 40 genes related to iron uptake, metabolism, chemotaxis, energy production, and osmotic stress response. In human fecal versus chicken cecal extracts, C. jejuni displayed higher expression of genes involved in respiration (fdhTU) and in known or putative iron uptake systems (cfbpA, ceuB, chuC, and CJJ81176_1649-1655 [here designated 1649-1655]). The 1649-1655 genes and downstream overlapping gene 1656 were investigated further. Uncharacterized homologues of this system were identified in 33 diverse bacterial species representing 6 different phyla, 21 of which are associated with human disease. The 1649 and 1650 (p19) genes encode an iron transporter and a periplasmic iron binding protein, respectively; however, the role of the downstream 1651-1656 genes was unknown. A Δ1651-1656 deletion strain had an iron-sensitive phenotype, consistent with a previously characterized Δp19 mutant, and showed reduced growth in acidic medium, increased sensitivity to streptomycin, and higher resistance to H2O2 stress. In iron-restricted medium, the 1651-1656 and p19 genes were required for optimal growth when using human fecal extracts as an iron source. Collectively, this implicates a function for the 1649-1656 gene cluster in C. jejuni iron scavenging and stress survival in the human intestinal environment.IMPORTANCE Direct comparative studies of C. jejuni infection of a zoonotic commensal host and a disease-susceptible host are crucial to understanding the causes of infection outcome in humans. These studies are hampered by the lack of a disease-susceptible animal model reliably displaying a similar pathology to human campylobacteriosis. In this work, we compared the phenotypic and transcriptional responses of C. jejuni to intestinal compositions of humans (disease-susceptible host) and chickens (zoonotic host) by using human fecal and chicken cecal extracts. The mammalian gut is a complex and dynamic system containing thousands of metabolites that contribute to host health and modulate pathogen activity. We identified C. jejuni genes more highly expressed during exposure to human fecal extracts in comparison to chicken cecal extracts and differentially expressed in extracts compared with medium alone, and targeted one specific iron uptake system for further molecular, genetic, and phenotypic study.