Genetic and functional enrichments associated with Enterococcus faecalis isolated from the urinary tract.

IMPORTANCE: Urinary tract infection (UTI) is a global health issue that imposes a substantial burden on healthcare systems. Women are disproportionately affected by UTI, with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. How Enterococcus faecalis, a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinary E. faecalis isolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may be involved in E. faecalis survival in the urinary tract.

Lipid discovery enabled by sequence statistics and machine learning.

Bacterial membranes are complex and dynamic, arising from an array of evolutionary pressures. One enzyme that alters membrane compositions through covalent lipid modification is MprF. We recently identified that Streptococcus agalactiae MprF synthesizes lysyl-phosphatidylglycerol (Lys-PG) from anionic PG, and a novel cationic lipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), from neutral glycolipid Glc-DAG. This unexpected result prompted us to investigate whether Lys-Glc-DAG occurs in other MprF-containing bacteria, and whether other novel MprF products exist. Here, we studied protein sequence features determining MprF substrate specificity. First, pairwise analyses identified several streptococcal MprFs synthesizing Lys-Glc-DAG. Second, a restricted Boltzmann machine-guided approach led us to discover an entirely new substrate for MprF in Enterococcus , diglucosyl-diacylglycerol (Glc2-DAG), and an expanded set of organisms that modify glycolipid substrates using MprF. Overall, we combined the wealth of available sequence data with machine learning to model evolutionary constraints on MprF sequences across the bacterial domain, thereby identifying a novel cationic lipid.

CRISPR-Cas inhibits plasmid transfer and immunizes bacteria against antibiotic resistance acquisition in manure.

The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system CRISPR-Cas acts as a barrier to the spread of antibiotic resistance plasmids, and CRISPR-Cas-based antimicrobials can be effective to selectively deplete antibiotic-resistant bacteria. While significant surveillance efforts monitor the spread of antibiotic-resistant bacteria in the clinical context, a major, often overlooked aspect of the issue is resistance emergence in agriculture. Farm animals are commonly treated with antibiotics, and antibiotic resistance in agriculture is on the rise. Yet, CRISPR-Cas efficacy has not been investigated in this setting. Here, we evaluate the prevalence of CRISPR-Cas in agricultural Enterococcus faecalis strains and its anti-plasmid efficacy in an agricultural niche - manure. We show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. CRISPR-Cas was found to be an effective barrier against resistance plasmid transfer in manure, with improved effect as time progressed. CRISPR-based antimicrobials to cure resistant E. faecalis of erythromycin resistance was limited by delivery efficiency of the CRISPR antimicrobial in manure. However, immunization of bacteria against resistance gene acquisition in manure was highly effective. Together, our results show that E. faecalis CRISPR-Cas is prevalent and effective in an agricultural setting, and has the potential to be utilized for depleting antibiotic-resistant populations. Our work has broad implications for tackling antibiotic resistance in the increasingly relevant agricultural setting, in line with a OneHealth approach.

Inter-species diversity and functional genomic analyses of closed genome assemblies of clinically isolated, megaplasmid-containing Enterococcus raffinosus Er676 and ATCC49464.

Enterococcus raffinosus is an understudied member of its genus possessing a characteristic megaplasmid contributing to a large genome size. Although less commonly associated with human infection compared to other enterococci, this species can cause disease and persist in diverse niches such as the gut, urinary tract, blood and environment. Few complete genome assemblies have been published to date for E. raffinosus . In this study, we report the complete assembly of the first clinical urinary E. raffinosus strain, Er676, isolated from a postmenopausal woman with history of recurrent urinary tract infection. We additionally completed the assembly of clinical type strain ATCC49464. Comparative genomic analyses reveal inter-species diversity driven by large accessory genomes. The presence of a conserved megaplasmid indicates it is a ubiquitous and vital genetic feature of E. raffinosus . We find that the E. raffinosus chromosome is enriched for DNA replication and protein biosynthesis genes while the megaplasmid is enriched for transcription and carbohydrate metabolism genes. Prophage analysis suggests that diversity in the chromosome and megaplasmid sequences arises, in part, from horizontal gene transfer. Er676 demonstrated the largest genome size reported to date for E. raffinosus and the highest probability of human pathogenicity. Er676 also possesses multiple antimicrobial resistance genes, of which all but one are encoded on the chromosome, and has the most complete prophage sequences. Complete assembly and comparative analyses of the Er676 and ATCC49464 genomes provide important insight into the inter-species diversity of E. raffinosus that gives it its ability to colonize and persist in the human body. Investigating genetic factors that contribute to the pathogenicity of this species will provide valuable tools to combat diseases caused by this opportunistic pathogen.

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile.

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term PnimBG) in the promoter of gene nimB, resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the PnimBG mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

Functional and genetic adaptations contributing to Enterococcus faecalis persistence in the female urinary tract.

Enterococcus faecalis is the leading Gram-positive bacterial species implicated in urinary tract infection (UTI). An opportunistic pathogen, E. faecalis is a commensal of the human gastrointestinal tract (GIT) and its presence in the GIT is a predisposing factor for UTI. The mechanisms by which E. faecalis colonizes and survives in the urinary tract (UT) are poorly understood, especially in uncomplicated or recurrent UTI. The UT is distinct from the GIT and is characterized by a sparse nutrient landscape and unique environmental stressors. In this study, we isolated and sequenced a collection of 37 clinical E. faecalis strains from the urine of primarily postmenopausal women. We generated 33 closed genome assemblies and four highly contiguous draft assemblies and conducted a comparative genomics to identify genetic features enriched in urinary E. faecalis with respect to E. faecalis isolated from the human GIT and blood. Phylogenetic analysis revealed high diversity among urinary strains and a closer relatedness between urine and gut isolates than blood isolates. Plasmid replicon (rep) typing further underscored possible UT-GIT interconnection identifying nine shared rep types between urine and gut E. faecalis . Both genotypic and phenotypic analysis of antimicrobial resistance among urinary E. faecalis revealed infrequent resistance to front-line UTI antibiotics nitrofurantoin and fluoroquinolones and no vancomycin resistance. Finally, we identified 19 candidate genes enriched among urinary strains that may play a role in adaptation to the UT. These genes are involved in the core processes of sugar transport, cobalamin import, glucose metabolism, and post-transcriptional regulation of gene expression. IMPORTANCE: Urinary tract infection (UTI) is a global health issue that imposes substantial burden on healthcare systems. Women are disproportionately affected by UTI with >60% of women experiencing at least one UTI in their lifetime. UTIs can recur, particularly in postmenopausal women, leading to diminished quality of life and potentially life-threatening complications. Understanding how pathogens colonize and survive in the urinary tract is necessary to identify new therapeutic targets that are urgently needed due to rising rates of antimicrobial resistance. How Enterococcus faecalis , a bacterium commonly associated with UTI, adapts to the urinary tract remains understudied. Here, we generated a collection of high-quality closed genome assemblies of clinical urinary E. faecalis isolated from the urine of postmenopausal women that we used alongside detailed clinical metadata to perform a robust comparative genomic investigation of genetic factors that may mediate urinary E. faecalis adaptation to the female urinary tract.

Targeted IS-element sequencing uncovers transposition dynamics during selective pressure in enterococci.

Insertion sequences (IS) are simple transposons implicated in the genome evolution of diverse pathogenic bacterial species. Enterococci have emerged as important human intestinal pathogens with newly adapted virulence potential and antibiotic resistance. These genetic features arose in tandem with large-scale genome evolution mediated by mobile elements. Pathoadaptation in enterococci is thought to be mediated in part by the IS element IS256 through gene inactivation and recombination events. However, the regulation of IS256 and the mechanisms controlling its activation are not well understood. Here, we adapt an IS256-specfic deep sequencing method to describe how chronic lytic phage infection drives widespread diversification of IS256 in E. faecalis and how antibiotic exposure is associated with IS256 diversification in E. faecium during a clinical human infection. We show through comparative genomics that IS256 is primarily found in hospital-adapted enterococcal isolates. Analyses of IS256 transposase gene levels reveal that IS256 mobility is regulated at the transcriptional level by multiple mechanisms in E. faecalis, indicating tight control of IS256 activation in the absence of selective pressure. Our findings reveal that stressors such as phages and antibiotic exposure drives rapid genome-scale transposition in the enterococci. IS256 diversification can therefore explain how selective pressures mediate evolution of the enterococcal genome, ultimately leading to the emergence of dominant nosocomial lineages that threaten human health.

Enterococcus faecalis Strains with Compromised CRISPR-Cas Defense Emerge under Antibiotic Selection for a CRISPR-Targeted Plasmid.

Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Non-MDR E. faecalis strains frequently possess CRISPR-Cas systems, which reduce the frequency of MGE acquisition. We demonstrated in previous studies that E. faecalis populations can transiently maintain both a functional CRISPR-Cas system and a CRISPR-Cas target. In this study, we used serial passage and deep sequencing to analyze these populations. In the presence of antibiotic selection for the plasmid, mutants with compromised CRISPR-Cas defense and enhanced ability to acquire a second antibiotic resistance plasmid emerged. Conversely, in the absence of selection, the plasmid was lost from wild-type E. faecalis populations but not E. faecalis populations that lacked the cas9 gene. Our results indicate that E. faecalis CRISPR-Cas can become compromised under antibiotic selection, generating populations with enhanced abilities to undergo horizontal gene transfer. IMPORTANCE Enterococcus faecalis is a leading cause of hospital-acquired infections and disseminator of antibiotic resistance plasmids among Gram-positive bacteria. We have previously shown that E. faecalis strains with an active CRISPR-Cas system can prevent plasmid acquisition and thus limit the transmission of antibiotic resistance determinants. However, CRISPR-Cas is not a perfect barrier. In this study, we observed populations of E. faecalis with transient coexistence of CRISPR-Cas and one of its plasmid targets. Our experimental data demonstrate that antibiotic selection results in compromised E. faecalis CRISPR-Cas function, thereby facilitating the acquisition of additional resistance plasmids by E. faecalis.

Human Serum Supplementation Promotes Streptococcus mitis Growth and Induces Specific Transcriptomic Responses.

Streptococcus mitis is a normal member of the human oral microbiota and a leading opportunistic pathogen causing infective endocarditis (IE). Despite the complex interactions between S. mitis and the human host, understanding of S. mitis physiology and its mechanisms of adaptation to host-associated environments is inadequate, especially compared with other IE bacterial pathogens. This study reports the growth-promoting effects of human serum on S. mitis and other pathogenic streptococci, including S. oralis, S. pneumoniae, and S. agalactiae. Using transcriptomic analyses, we identified that, with the addition of human serum, S. mitis downregulates uptake systems for metal ions and sugars, fatty acid biosynthetic genes, and genes involved in stress response and other processes related with growth and replication. S. mitis upregulates uptake systems for amino acids and short peptides in response to human serum. Zinc availability and environmental signals sensed by the induced short peptide binding proteins were not sufficient to confer the growth-promoting effects. More investigation is required to establish the mechanism for growth promotion. Overall, our study contributes to the fundamental understanding of S. mitis physiology under host-associated conditions. IMPORTANCE S. mitis is exposed to human serum components during commensalism in the human mouth and bloodstream pathogenesis. However, the physiological effects of serum components on this bacterium remain unclear. Using transcriptomic analyses, S. mitis biological processes that respond to the presence of human serum were revealed, improving the fundamental understanding of S. mitis physiology in human host conditions.

Comparative Genomics of Streptococcus oralis Identifies Large Scale Homologous Recombination and a Genetic Variant Associated with Infection.

The viridans group streptococci (VGS) are a large consortium of commensal streptococci that colonize the human body. Many species within this group are opportunistic pathogens causing bacteremia and infective endocarditis (IE), yet little is known about why some strains cause invasive disease. Identification of virulence determinants is complicated by the difficulty of distinguishing between the closely related species of this group. Here, we analyzed genomic data from VGS that were isolated from blood cultures in patients with invasive infections and oral swabs of healthy volunteers and then determined the best-performing methods for species identification. Using whole-genome sequence data, we characterized the population structure of a diverse sample of Streptococcus oralis isolates and found evidence of frequent recombination. We used multiple genome-wide association study tools to identify candidate determinants of invasiveness. These tools gave consistent results, leading to the discovery of a single synonymous single nucleotide polymorphism (SNP) that was significantly associated with invasiveness. This SNP was within a previously undescribed gene that was conserved across the majority of VGS species. Using the growth in the presence of human serum and a simulated infective endocarditis vegetation model, we were unable to identify a phenotype for the enriched allele in laboratory assays, suggesting a phenotype may be specific to natural infection. These data highlighted the power of analyzing natural populations for gaining insight into pathogenicity, particularly for organisms with complex population structures like the VGS. IMPORTANCE The viridians group streptococci (VGS) are a large collection of closely related commensal streptococci, with many being opportunistic pathogens causing invasive diseases, such as bacteremia and infective endocarditis. Little is known about virulence determinants in these species, and there is a distinct lack of genomic information available for the VGS. In this study, we collected VGS isolates from invasive infections and healthy volunteers and performed whole-genome sequencing for a suite of downstream analyses. We focused on a diverse sample of Streptococcus oralis genomes and identified high rates of recombination in the population as well as a single genome variant highly enriched in invasive isolates. The variant lies within a previously uncharacterized gene, nrdM, which shared homology with the anaerobic ribonucleoside triphosphate reductase, nrdD, and was highly conserved among VGS. This work increased our knowledge of VGS genomics and indicated that differences in virulence potential among S. oralis isolates were, at least in part, genetically determined.

Recurrent urinary tract infection and estrogen shape the taxonomic ecology and function of the postmenopausal urogenital microbiome.

Postmenopausal women are severely affected by recurrent urinary tract infection (rUTI). The urogenital microbiome is a key component of the urinary environment. However, changes in the urogenital microbiome underlying rUTI susceptibility are unknown. Here, we perform shotgun metagenomics and advanced culture on urine from a controlled cohort of postmenopausal women to identify urogenital microbiome compositional and function changes linked to rUTI susceptibility. We identify candidate taxonomic biomarkers of rUTI susceptibility in postmenopausal women and an enrichment of lactobacilli in postmenopausal women taking estrogen hormone therapy. We find robust correlations between Bifidobacterium and Lactobacillus and urinary estrogens in women without urinary tract infection (UTI) history. Functional analyses reveal distinct metabolic and antimicrobial resistance gene (ARG) signatures associated with rUTI. Importantly, we find that ARGs are enriched in the urogenital microbiomes of women with rUTI history independent of current UTI status. Our data suggest that rUTI and estrogen shape the urogenital microbiome in postmenopausal women.

Genetically distant bacteriophages select for unique genomic changes in Enterococcus faecalis.

The human microbiota harbors diverse bacterial and bacteriophage (phage) communities. Bacteria evolve to overcome phage infection, thereby driving phage evolution to counter bacterial resistance. Understanding how phages select for genetic alterations in medically relevant bacteria is important as phages become established biologics for the treatment of multidrug-resistant (MDR) bacterial infections. Before phages can be widely used as standalone or combination antibacterial therapies, we must obtain a deep understanding of the molecular mechanisms of phage infection and how host bacteria alter their genomes to become resistant. We performed coevolution experiments using a single Enterococcus faecalis strain and two distantly related phages to determine how phage pressure impacts the evolution of the E. faecalis genome. Whole-genome sequencing of E. faecalis following continuous exposure to these two phages revealed mutations previously demonstrated to be essential for phage infection. We also identified mutations in genes previously unreported to be associated with phage infection in E. faecalis. Intriguingly, there was only one shared mutation in the E. faecalis genome that was selected by both phages tested, demonstrating that infection by two genetically distinct phages selects for diverse variants. This knowledge serves as the basis for the continued study of E. faecalis genome evolution during phage infection and can be used to inform the design of future therapeutics, such as phage cocktails, intended to target MDR E. faecalis.

Identification of a novel cationic glycolipid in Streptococcus agalactiae that contributes to brain entry and meningitis.

Bacterial membrane lipids are critical for membrane bilayer formation, cell division, protein localization, stress responses, and pathogenesis. Despite their critical roles, membrane lipids have not been fully elucidated for many pathogens. Here, we report the discovery of a novel cationic glycolipid, lysyl-glucosyl-diacylglycerol (Lys-Glc-DAG), which is synthesized in high abundance by the bacterium Streptococcus agalactiae (Group B Streptococcus, GBS). To our knowledge, Lys-Glc-DAG is more positively charged than any other known lipids. Lys-Glc-DAG carries 2 positive net charges per molecule, distinct from the widely described lysylated phospholipid lysyl-phosphatidylglycerol (Lys-PG) that carries one positive net charge due to the presence of a negatively charged phosphate moiety. We use normal phase liquid chromatography (NPLC) coupled with electrospray ionization (ESI) high-resolution tandem mass spectrometry (HRMS/MS) and genetic approaches to determine that Lys-Glc-DAG is synthesized by the enzyme MprF in GBS, which covalently modifies the neutral glycolipid Glc-DAG with the cationic amino acid lysine. GBS is a leading cause of neonatal meningitis, which requires traversal of the endothelial blood-brain barrier (BBB). We demonstrate that GBS strains lacking mprF exhibit a significant decrease in the ability to invade BBB endothelial cells. Further, mice challenged with a GBSΔmprF mutant developed bacteremia comparably to wild-type (WT) infected mice yet had less recovered bacteria from brain tissue and a lower incidence of meningitis. Thus, our data suggest that Lys-Glc-DAG may contribute to bacterial uptake into host cells and disease progression. Importantly, our discovery provides a platform for further study of cationic lipids at the host-pathogen interface.

Correction: CRISPR-based antimicrobials to obstruct antibiotic-resistant and pathogenic bacteria.

[This corrects the article DOI: 10.1371/journal.ppat.1009672.].

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies.

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data may include mobile genetic elements, such as plasmids and extrachromosomal phage, that contribute to the dissemination of antimicrobial resistance and further complicate treatment of urinary tract infection (UTI). In addition to providing fine resolution of genome structure, complete, closed genomes allow for the detailed comparative genomics and evolutionary analyses. The generation of complete genomes de novo has long been a challenging task due to limitations of available sequencing technology. Paired-end Next Generation Sequencing (NGS) produces high quality short reads often resulting in accurate but fragmented genome assemblies. On the contrary, Nanopore sequencing provides long reads of lower quality normally leading to error-prone complete assemblies. Such errors may hamper genome-wide association studies or provide misleading variant analysis results. Therefore, hybrid approaches combining both short and long reads have emerged as reliable methods to achieve highly accurate closed bacterial genomes. Reported herein is a comprehensive method for the culture of diverse urinary bacteria, species identification by 16S rRNA gene sequencing, extraction of genomic DNA (gDNA), and generation of short and long reads by NGS and Nanopore platforms, respectively. Additionally, this method describes a bioinformatic pipeline of quality control, assembly, and gene prediction algorithms for the generation of annotated complete genome sequences. Combination of bioinformatic tools enables the selection of high quality read data for hybrid genome assembly and downstream analysis. The streamlined approach for the hybrid de novo genome assembly described in this protocol may be adapted for the use in any culturable bacteria.

Characterization of presumptive vancomycin-resistant enterococci recovered during infection control surveillance in Dallas, Texas, USA.

Enterococcus faecalis and E. faecium are Gram-positive bacteria that normally inhabit the human gastrointestinal tract. They are also opportunistic pathogens and can cause nosocomial infection outbreaks. To prevent the spread of nosocomial infections, hospitals may rely on screening methods to identify patients colonized with multidrug-resistant organisms including vancomycin-resistant enterococci (VRE). Spectra VRE agar (Remel) contains vancomycin and other medium components that select for VRE and phenotypically differentiate between E. faecalis and E. faecium by colony colour. We obtained 66 de-identified rectal swab cultures on Spectra VRE agar that were obtained during routine patient admission surveillance at a hospital system in Dallas, Texas, USA. We analysed 90 presumptive VRE from 61 of the Spectra VRE agar cultures using molecular and culture methods. Using ddl typing, 55 were found to be E. faecium and 32 were found to be E. faecalis . While most of the E. faecium were positive for the vanA gene by PCR (52 of 55 strains), few of the E. faecalis were positive for either vanA or vanB (five of 32 strains). The 27 E. faecalis vanA- and vanB-negative strains could not be recultured on Spectra VRE agar. Overall, we found that Spectra VRE agar performed robustly for the identification of vancomycin-resistant E. faecium , but presumptive false positives were obtained for vancomycin-resistant E. faecalis .

The Integrity of Heme Is Essential for Reproducible Detection of Metronidazole-Resistant Clostridioides difficile by Agar Dilution Susceptibility Tests.

Metronidazole resistance in clinical Clostridioides difficile is often described as unstable, since resistant strains reportedly appear susceptible following freezer storage or brief passage. This has presented a conundrum for adopting susceptibility testing to accurately evaluate the connection between metronidazole resistance and decreased clinical efficacy of metronidazole in patients with C. difficile infections (CDIs). We discovered that supplementation of microbiological media with the metalloporphyrin heme is crucial for detection of metronidazole-resistant C. difficile using the agar dilution susceptibility testing method. Known metronidazole-resistant strains appeared susceptible to metronidazole in media lacking heme. Similarly, these resistant strains exhibited increased susceptibility to metronidazole when tested on heme-containing agars that were exposed to room light for more than 1 day, likely due to heme photodecomposition. In parallel experiments, resistance was reproducibly detected when heme-containing agars were either prepared and used on the same day or protected from light and then used on subsequent days. Notably, heme did not influence the susceptibilities of drug-susceptible strains that were of the same ribotype as the resistant strains. These findings firmly show that the consistent detection of metronidazole-resistant C. difficile is dependent upon heme and its protection from light. Studies are warranted to determine the extent to which this heme-associated metronidazole-resistant phenotype affects the clinical efficacy of metronidazole in CDI and the underlying genetic and biochemical mechanisms.

Streptococcus pneumoniae, S. pyogenes and S. agalactiae membrane phospholipid remodelling in response to human serum.

Streptococcus pneumoniae, S. pyogenes (Group A Streptococcus; GAS) and S. agalactiae (Group B Streptococcus; GBS) are major aetiological agents of diseases in humans. The cellular membrane, a crucial site in host-pathogen interactions, is poorly characterized in streptococci. Moreover, little is known about whether or how environmental conditions influence their lipid compositions. Using normal phase liquid chromatography coupled with electrospray ionization MS, we characterized the phospholipids and glycolipids of S. pneumoniae, GAS and GBS in routine undefined laboratory medium, streptococcal defined medium and, in order to mimic the host environment, defined medium supplemented with human serum. In human serum-supplemented medium, all three streptococcal species synthesize phosphatidylcholine (PC), a zwitterionic phospholipid commonly found in eukaryotes but relatively rare in bacteria. We previously reported that S. pneumoniae utilizes the glycerophosphocholine (GPC) biosynthetic pathway to synthesize PC. Through substrate tracing experiments, we confirm that GAS and GBS scavenge lysoPC, a major metabolite in human serum, thereby using an abbreviated GPC pathway for PC biosynthesis. Furthermore, we found that plasmanyl-PC is uniquely present in the GBS membrane during growth with human serum, suggesting GBS possesses unusual membrane biochemical or biophysical properties. In summary, we report cellular lipid remodelling by the major pathogenic streptococci in response to metabolites present in human serum.

ddcP, pstB, and excess D-lactate impact synergism between vancomycin and chlorhexidine against Enterococcus faecium 1,231,410.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens that cause life-threatening infections. To control hospital-associated infections, skin antisepsis and bathing utilizing chlorhexidine is recommended for VRE patients in acute care hospitals. Previously, we reported that exposure to inhibitory chlorhexidine levels induced the expression of vancomycin resistance genes in VanA-type Enterococcus faecium. However, vancomycin susceptibility actually increased for VanA-type E. faecium in the presence of chlorhexidine. Hence, a synergistic effect of the two antimicrobials was observed. In this study, we used multiple approaches to investigate the mechanism of synergism between chlorhexidine and vancomycin in the VanA-type VRE strain E. faecium 1,231,410. We generated clean deletions of 7 of 11 pbp, transpeptidase, and carboxypeptidase genes in this strain (ponA, pbpF, pbpZ, pbpA, ddcP, ldtfm, and vanY). Deletion of ddcP, encoding a membrane-bound carboxypeptidase, altered the synergism phenotype. Furthermore, using in vitro evolution, we isolated a spontaneous synergy escaper mutant and utilized whole genome sequencing to determine that a mutation in pstB, encoding an ATPase of phosphate-specific transporters, also altered synergism. Finally, addition of excess D-lactate, but not D-alanine, enhanced synergism to reduce vancomycin MIC levels. Overall, our work identified factors that alter chlorhexidine and vancomycin synergism in a model VanA-type VRE strain.