Purine and carbohydrate availability drive Enterococcus faecalis fitness during wound and urinary tract infections.

IMPORTANCE: Although E. faecalis is a common wound pathogen, its pathogenic mechanisms during wound infection are unexplored. Here, combining a mouse wound infection model with in vivo transposon and RNA sequencing approaches, we identified the E. faecalis purine biosynthetic pathway and galactose/mannose MptABCD phosphotransferase system as essential for E. faecalis acute replication and persistence during wound infection, respectively. The essentiality of purine biosynthesis and the MptABCD PTS is driven by the consumption of purine metabolites by E. faecalis during acute replication and changing carbohydrate availability during the course of wound infection. Overall, our findings reveal the importance of the wound microenvironment in E. faecalis wound pathogenesis and how these metabolic pathways can be targeted to better control wound infections.

Evaluation of a new antiresistic strategy to manage antibiotic resistance.

OBJECTIVES: Systemic strategies for combating antimicrobial resistance (AMR) currently focus on limiting antibiotic use and have been generally insufficient in preventing the rise of AMR. Additionally, they often generate other adverse incentives, such as discouraging pharmaceutical companies from investing in research and development of new antibiotics, further exacerbating the problem. This paper proposes a novel systemic strategy for tackling AMR, which we term 'antiresistics': any intervention (whether a small molecule, genetic element, phage, or whole organism) that reduces resistance rates in pathogen populations. A prime example of an antiresistic would be a small molecule that specifically disrupts the maintenance of antibiotic resistance plasmids. Of note, an antiresistic would be expected to have a population-level effect and not necessarily be useful on a time scale relevant to individual patients. METHODS: We developed a mathematical model to assess the effect of antiresistics on population resistance levels and calibrated it to longitudinal data available at the country level. We also estimated potential effects on idealised rates for the introduction of new antibiotics. RESULTS: The model shows that greater use of antiresistics allows for greater usage of existing antibiotics. This leads to an ability to maintain a constant overall rate of antibiotic efficacy with a slower rate of developing new antibiotics; subsequently, antiresistics have a positive benefit on the effective lifetime and thus profitability of antibiotics. CONCLUSIONS: By directly reducing resistance rates, antiresistics can provide clear qualitative benefits (which may be quantitatively large) in terms of existing antibiotic efficacy, longevity, and alignment of incentives.

Depleting Cationic Lipids Involved in Antimicrobial Resistance Drives Adaptive Lipid Remodeling in Enterococcus faecalis.

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human ß-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while ΔmprF2 and ΔmprF1 ΔmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2 and ΔmprF1 ΔmprF2. In the mprF mutants, particularly ΔmprF1 ΔmprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the ΔmprF1 ΔmprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both ΔmprF2 and ΔmprF1 ΔmprF2, compared to the wild type and ΔmprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. IMPORTANCE The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

Invasive Streptococcus agalactiae ST283 infection after fish consumption in two sisters, Lao PDR.

Background: Streptococcus agalactiae is a normal commensal of the human gastro-intestinal and female genital tracts. It causes serious disease in neonates and pregnant women, as well as non-pregnant adults. Food-borne outbreaks have also been described. A link between invasive Group B streptococcus (GBS) infection in humans caused by S. agalactiae serotype III-4, sequence type 283 (ST283) and the consumption of raw fresh-water fish was first described in Singapore in 2015. Case presentation: We report the simultaneous occurrence of acute fever and myalgia in two sisters who were visiting Laos. Both were found to have invasive GBS ST283 infection, confirmed by blood culture. Infection was temporally linked to fish consumption. They responded well to intravenous antibiotics within 48 hours. Conclusions: Food-borne transmission of Streptococcus agalactiae is an important and under-recognised source of serious human disease throughout Southeast Asia and possibly beyond.

Development of mass allelic exchange, a technique to enable sexual genetics in Escherichia coli.

Despite dramatic advances in genomics, connecting genotypes to phenotypes is still challenging. Sexual genetics combined with linkage analysis is a powerful solution to this problem but generally unavailable in bacteria. We build upon a strong negative selection system to invent mass allelic exchange (MAE), which enables hybridization of arbitrary (including pathogenic) strains of Escherichia coli. MAE reimplements the natural phenomenon of random cross-overs, enabling classical linkage analysis. We demonstrate the utility of MAE with virulence-related gain-of-function screens, discovering that transfer of a single operon from a uropathogenic strain is sufficient for enabling a commensal E. coli to form large intracellular bacterial collections within bladder epithelial cells. MAE thus enables assaying natural allelic variation in E. coli (and potentially other bacteria), complementing existing loss-of-function genomic techniques.

Long-term ecological and evolutionary dynamics in the gut microbiomes of carbapenemase-producing Enterobacteriaceae colonized subjects.

Long-term colonization of the gut microbiome by carbapenemase-producing Enterobacteriaceae (CPE) is a growing area of public health concern as it can lead to community transmission and rapid increase in cases of life-threatening CPE infections. Here, leveraging the observation that many subjects are decolonized without interventions within a year, we used longitudinal shotgun metagenomics (up to 12 timepoints) for detailed characterization of ecological and evolutionary dynamics in the gut microbiome of a cohort of CPE-colonized subjects and family members (n = 46; 361 samples). Subjects who underwent decolonization exhibited a distinct ecological shift marked by recovery of microbial diversity, key commensals and anti-inflammatory pathways. In addition, colonization was marked by elevated but unstable Enterobacteriaceae abundances, which exhibited distinct strain-level dynamics for different species (Escherichia coli and Klebsiella pneumoniae). Finally, comparative analysis with whole-genome sequencing data from CPE isolates (n = 159) helped identify substrain variation in key functional genes and the presence of highly similar E. coli and K. pneumoniae strains with variable resistance profiles and plasmid sharing. These results provide an enhanced view into how colonization by multi-drug-resistant bacteria associates with altered gut ecology and can enable transfer of resistance genes, even in the absence of overt infection and antibiotic usage.

Characterisation of Salmonella Enteritidis ST11 and ST1925 Associated with Human Intestinal and Extra-Intestinal Infections in Singapore.

Salmonella Enteritidis is a major foodborne pathogen worldwide. In this study, a total of 276 S. enteritidis isolates, collected between 2016 and 2017 from human, food and farm/slaughterhouse samples, were studied to enhance the understanding of the epidemiology of human salmonellosis in Singapore. Results showed all 276 isolates belonged either to ST1925 (70.3%) or ST11 (29.7%), with ST11 being significantly more frequent in extra-intestinal isolates and chicken isolates. Food isolates, most of which were from poultry, showed the highest prevalence of resistance (33-37%) against beta-lactams or beta-lactams/beta-lactamase inhibitor combination (ampicillin, piperacillin and ampicillin/sulbactam). The analysis showed the detection of genes associated with resistance to aminoglycoside genes (99.6%), tetracycline (55.1%), and beta-lactams (14.9%) of all isolates. Nine types of plasmids were found in 266 isolates; the most common incompatibility group profiles were IncFIB(S)-IncFII(S)-IncX1 (72.2%) and IncFIB(S)-IncFII(S) (15.8%). Most plasmid harbouring isolates from chicken (63.6%, 14/22) and from human (73.8%, 175/237) shared the same plasmid profile (IncFIB(S)-IncFII(S)-IncX1). SNP analysis showed clustering of several isolates from poultry food products and human isolates, suggesting phylogenetic relatedness among these isolates. Lastly, this study provides important epidemiological insights on the application of phenotypic and next-generation sequencing (NGS) tools for improved food safety and public health surveillance and outbreak investigation of S.enteritidis.

Transitional premonocytes emerge in the periphery for host defense against bacterial infections.

Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes.

GBS-SBG - GBS Serotyping by Genome Sequencing.

Group B Streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal meningitis and a rising cause of sepsis in adults. Recently, it has also been shown to cause foodborne disease. As with many other bacteria, the polysaccharide capsule of GBS is antigenic, enabling its use for strain serotyping. Recent advances in DNA sequencing have made sequence-based typing attractive (as has been implemented for several other bacteria, including Escherichia coli, Klebsiella pneumoniae species complex, Streptococcus pyogenes, and others). For GBS, existing WGS-based serotyping systems do not provide complete coverage of all known GBS serotypes (specifically including subtypes of serotype III), and none are simultaneously compatible with the two most common data types, raw short reads and assembled sequences. Here, we create a serotyping database (GBS-SBG, GBS Serotyping by Genome Sequencing), with associated scripts and running instructions, that can be used to call all currently described GBS serotypes, including subtypes of serotype III, using both direct short-read- and assembly-based typing. We achieved higher concordance using GBS-SBG on a previously reported data set of 790 strains. We further validated GBS-SBG on a new set of 572 strains, achieving 99.8% concordance with PCR-based molecular serotyping using either short-read- or assembly-based typing. The GBS-SBG package is publicly available and will hopefully accelerate and simplify serotyping by sequencing for GBS.

DNA methylation by three Type I restriction modification systems of Escherichia coli does not influence gene regulation of the host bacterium.

DNA methylation is a common epigenetic mark that influences transcriptional regulation, and therefore cellular phenotype, across all domains of life. In particular, both orphan methyltransferases and those from phasevariable restriction modification systems (RMSs) have been co-opted to regulate virulence epigenetically in many bacteria. We now show that three distinct non-phasevariable Type I RMSs in Escherichia coli have no measurable impact on gene expression, in vivo virulence, or any of 1190 in vitro growth phenotypes. We demonstrated this using both Type I RMS knockout mutants as well as heterologous installation of Type I RMSs into two E. coli strains. These data provide three clear and currently rare examples of restriction modification systems that have no impact on their host organism's gene regulation. This leads to the possibility that other such nonregulatory methylation systems may exist, broadening our view of the potential role that RMSs may play in bacterial evolution.

Whole-Genome Sequencing Analysis of Salmonella Isolates from Poultry Farms, a Slaughterhouse, and Retail Stalls in Thailand.

The draft genome sequences of 21 Salmonella isolates obtained from poultry production chains in Hat Yai City, Songkhla Province, Thailand, are reported in this study. Our study revealed that there was Salmonella environmental contamination along poultry production chains and cross-contamination among poultry through inanimate surfaces in the environment.

Epidemiology and Transmission of Carbapenemase-Producing Enterobacteriaceae in a Health Care Network of an Acute-Care Hospital and Its Affiliated Intermediate- and Long-Term-Care Facilities in Singapore.

Movement of patients in a health care network poses challenges for the control of carbapenemase-producing Enterobacteriaceae (CPE). We aimed to identify intra- and interfacility transmission events and facility type-specific risk factors of CPE in an acute-care hospital (ACH) and its intermediate-term and long-term-care facilities (ILTCFs). Serial cross-sectional studies were conducted in June and July of 2014 to 2016 to screen for CPE. Whole-genome sequencing was done to identify strain relatedness and CPE genes (blaIMI, blaIMP-1, blaKPC-2, blaNDM-1, and blaOXA-48). Multivariable logistic regression models, stratified by facility type, were used to determine independent risk factors. Of 5,357 patients, half (55%) were from the ACH. CPE prevalence was 1.3% in the ACH and 0.7% in ILTCFs (P = 0.029). After adjusting for sociodemographics, screening year, and facility type, the odds of CPE colonization increased significantly with a hospital stay of ≥3 weeks (adjusted odds ratio [aOR], 2.67; 95% confidence interval [CI], 1.17 to 6.05), penicillin use (aOR, 3.00; 95% CI, 1.05 to 8.56), proton pump inhibitor use (aOR, 3.20; 95% CI, 1.05 to 9.80), dementia (aOR, 3.42; 95% CI, 1.38 to 8.49), connective tissue disease (aOR, 5.10; 95% CI, 1.19 to 21.81), and prior carbapenem-resistant Enterobacteriaceae (CRE) carriage (aOR, 109.02; 95% CI, 28.47 to 417.44) in the ACH. For ILTCFs, presence of wounds (aOR, 5.30; 95% CI, 1.01 to 27.72), respiratory procedures (aOR, 4.97; 95% CI, 1.09 to 22.71), vancomycin-resistant enterococcus carriage (aOR, 16.42; 95% CI, 1.52 to 177.48), and CRE carriage (aOR, 758.30; 95% CI, 33.86 to 16,982.52) showed significant association. Genomic analysis revealed only possible intra-ACH transmission and no evidence for ACH-to-ILTCF transmission. Although CPE colonization was predominantly in the ACH, risk factors varied between facilities. Targeted screening and precautionary measures are warranted.

Household transmission of carbapenemase-producing Enterobacteriaceae: a prospective cohort study.

OBJECTIVES: To estimate the transmission rate of carbapenemase-producing Enterobacteriaceae (CPE) in households with recently hospitalized CPE carriers. METHODS: We conducted a prospective case-ascertained cohort study. We identified the presence of CPE in stool samples from index subjects, household contacts and companion animals and environmental samples at regular intervals. Linked transmissions were identified by WGS. A Markov model was constructed to estimate the household transmission potential of CPE. RESULTS: Ten recently hospitalized index patients and 14 household contacts were included. There were seven households with one contact, two households with two contacts, and one household with three contacts. Index patients were colonized with blaOXA-48-like (n = 4), blaKPC-2 (n = 3), blaIMP (n = 2), and blaNDM-1 (n = 1), distributed among divergent species of Enterobacteriaceae. After a cumulative follow-up time of 9.0 years, three family members (21.4%, 3/14) acquired four different types of CPE in the community (hazard rate of 0.22/year). The probability of CPE transmission from an index patient to a household contact was 10% (95% CI 4%-26%). CONCLUSIONS: We observed limited transmission of CPE from an index patient to household contacts. Larger studies are needed to understand the factors associated with household transmission of CPE and identify preventive strategies.

Active bacterial modification of the host environment through RNA polymerase II inhibition.

Unlike pathogens, which attack the host, commensal bacteria create a state of friendly coexistence. Here, we identified a mechanism of bacterial adaptation to the host niche, where they reside. Asymptomatic carrier strains were shown to inhibit RNA polymerase II (Pol II) in host cells by targeting Ser2 phosphorylation, a step required for productive mRNA elongation. Assisted by a rare, spontaneous loss-of-function mutant from a human carrier, the bacterial NlpD protein was identified as a Pol II inhibitor. After internalization by host cells, NlpD was shown to target constituents of the Pol II phosphorylation complex (RPB1 and PAF1C), attenuating host gene expression. Therapeutic efficacy of a recombinant NlpD protein was demonstrated in a urinary tract infection model, by reduced tissue pathology, accelerated bacterial clearance, and attenuated Pol II-dependent gene expression. The findings suggest an intriguing, evolutionarily conserved mechanism for bacterial modulation of host gene expression, with a remarkable therapeutic potential.

Sequencing of E. coli strain UTI89 on multiple sequencing platforms.

OBJECTIVES: The availability of matched sequencing data for the same sample across different sequencing platforms is a necessity for validation and effective comparison of sequencing platforms. A commonly sequenced sample is the lab-adapted MG1655 strain of Escherichia coli; however, this strain is not fully representative of more complex and dynamic genomes of pathogenic E. coli strains. DATA DESCRIPTION: We present six new sequencing data sets for another E. coli strain, UTI89, which is an extraintestinal pathogenic strain isolated from a patient suffering from a urinary tract infection. We now provide matched whole genome sequencing data generated using the PacBio RSII, Oxford Nanopore MinION R9.4, Ion Torrent, ABI SOLiD, and Illumina NextSeq sequencers. Together with other publically available datasets, UTI89 has a nearly complete suite of data generated on most second- and third-generation sequencers. These data can be used as an additional validation set for new sequencing technologies and analytical methods. More than being another E. coli strain, however, UTI89 is pathogenic, with a 10% larger genome, additional pathogenicity islands, and a large plasmid, features that are common among other naturally occurring and disease-causing E. coli isolates. These data therefore provide a more medically relevant test set for development of algorithms.

Adaptation of Arginine Synthesis among Uropathogenic Branches of the Escherichia coli Phylogeny Reveals Adjustment to the Urinary Tract Habitat.

Urinary tract infections (UTIs) are predominantly caused by uropathogenic Escherichia coli (UPEC). UPEC pathogenesis requires passage through a severe population bottleneck involving intracellular bacterial communities (IBCs) that are clonal expansions of a single invading UPEC bacterium in a urothelial superficial facet cell. IBCs occur only during acute pathogenesis. The bacteria in IBCs form the founder population that develops into persistent extracellular infections. Only a small fraction of UPEC organisms proceed through the IBC cycle, regardless of the inoculum size. This dramatic reduction in population size precludes the utility of genomic mutagenesis technologies for identifying genes important for persistence. To circumvent this bottleneck, we previously identified 29 positively selected genes (PSGs) within UPEC and hypothesized that they contribute to virulence. Here, we show that 8 of these 29 PSGs are required for fitness during persistent bacteriuria. Conversely, 7/8 of these PSG mutants showed essentially no phenotype in acute UTI. Deletion of the PSG argI leads to arginine auxotrophy. Relative to the other arg genes, argI in the B2 clade (which comprises most UPEC strains) of E. coli has diverged from argI in other E. coli clades. Replacement of argI in a UPEC strain with a non-UPEC argI allele complemented the arginine auxotrophy but not the persistent bacteriuria defect, showing that the UPEC argI allele contributes to persistent infection. These results highlight the complex roles of metabolic pathways during infection and demonstrate that evolutionary approaches can identify infection-specific gene functions downstream of population bottlenecks, shedding light on virulence and the genetic evolution of pathogenesis.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the most common cause of human urinary tract infection (UTI). Population bottlenecks during early stages of UTI make high-throughput screens impractical for understanding clinically important later stages of UTI, such as persistence and recurrence. As UPEC is hypothesized to be adapted to these later pathogenic stages, we previously identified 29 genes evolving under positive selection in UPEC. Here, we found that 8 of these genes, including argI (which is involved in arginine biosynthesis), are important for persistence in a mouse model of UTI. Deletion of argI and other arginine synthesis genes resulted in (i) arginine auxotrophy and (ii) defects in persistent UTI. Replacement of a B2 clade argI with a non-B2 clade argI complemented arginine auxotrophy, but the resulting strain remained attenuated in its ability to cause persistent bacteriuria. Thus, argI may have a second function during UTI that is not related to simple arginine synthesis. This study demonstrates how variation in metabolic genes can impact virulence and provides insight into the mechanisms and evolution of bacterial virulence.

A Faster and More Accurate Algorithm for Calculating Population Genetics Statistics Requiring Sums of Stirling Numbers of the First Kind.

Ewen's sampling formula is a foundational theoretical result that connects probability and number theory with molecular genetics and molecular evolution; it was the analytical result required for testing the neutral theory of evolution, and has since been directly or indirectly utilized in a number of population genetics statistics. Ewen's sampling formula, in turn, is deeply connected to Stirling numbers of the first kind. Here, we explore the cumulative distribution function of these Stirling numbers, which enables a single direct estimate of the sum, using representations in terms of the incomplete beta function. This estimator enables an improved method for calculating an asymptotic estimate for one useful statistic, Fu's [Formula: see text] By reducing the calculation from a sum of terms involving Stirling numbers to a single estimate, we simultaneously improve accuracy and dramatically increase speed.

Duration of Carbapenemase-Producing Enterobacteriaceae Carriage in Hospital Patients.

To determine the duration of carbapenemase-producing Enterobacteriaceae (CPE) carriage, we studied 21 CPE carriers for ¼1 year. Mean carriage duration was 86 days; probability of decolonization in 1 year was 98.5%, suggesting that CPE-carriers' status can be reviewed yearly. Prolonged carriage was associated with use of antimicrobial drugs.

Metagenome-wide association analysis identifies microbial determinants of post-antibiotic ecological recovery in the gut.

Loss of diversity in the gut microbiome can persist for extended periods after antibiotic treatment, impacting microbiome function, antimicrobial resistance and probably host health. Despite widespread antibiotic use, our understanding of the species and metabolic functions contributing to gut microbiome recovery is limited. Using data from 4 discovery cohorts in 3 continents comprising >500 microbiome profiles from 117 individuals, we identified 21 bacterial species exhibiting robust association with ecological recovery post antibiotic therapy. Functional and growth-rate analysis showed that recovery is supported by enrichment in specific carbohydrate-degradation and energy-production pathways. Association rule mining on 782 microbiome profiles from the MEDUSA database enabled reconstruction of the gut microbial 'food web', identifying many recovery-associated bacteria as keystone species, with the ability to use host- and diet-derived energy sources, and support repopulation of other gut species. Experiments in a mouse model recapitulated the ability of recovery-associated bacteria (Bacteroides thetaiotaomicron and Bifidobacterium adolescentis) to promote recovery with synergistic effects, providing a boost of two orders of magnitude to microbial abundance in early time points and faster maturation of microbial diversity. The identification of specific species and metabolic functions promoting recovery opens up opportunities for rationally determining pre- and probiotic formulations offering protection from long-term consequences of frequent antibiotic usage.