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The horizontal transfer of antibiotic resistance genes among bacteria is a pressing global issue. The bacterial defense system clustered regularly interspaced short palindromic repeats (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 antiplasmid efficacy in an agricultural niche: manure. Analyzing 1,986 E. faecalis genomes from human and animal hosts, we show that the prevalence of CRISPR-Cas subtypes is similar between clinical and agricultural E. faecalis strains. Using plasmid conjugation assays, we found that CRISPR-Cas is a significant barrier against resistance plasmid transfer in manure. Finally, we used a CRISPR-based antimicrobial approach to cure resistant E. faecalis of erythromycin resistance, but this 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 One Health approach. [ABSTRACT FROM AUTHOR]

The emergence of drug-resistant bacteria, particularly methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), has increased the need to discover novel antimicrobial agents that are effective against these species. Here, we describe the identification and purification of the mutacin BHT-B-like gene locus and bacteriocin peptide from Streptococcus ursoris, which is closely related to Streptococcus ratti; hence, we named this bacteriocin ursoricin. Ursoricin is a cationic, chromosome-encoded peptide that has potent antimicrobial effects against Gram-positive pathogens, including MRSA and VRE, with minimum inhibitory concentrations in the micromolar range. Ursoricin also inhibits the biofilm formation of high biofilm-form ing S. aureus. Antibacterial activity was retained after treatment at 100°C for 60 min at a pH range of 3-9 and was partially reduced by treatment with proteinase K for 2 h (63% residual activity). The potent anti-MRSA, anti-VRE, and antibiofilm effects of ursoricin suggest that it is a possible candidate for the treatment of MRSA, VRE, and biofilm-associated infections. [ABSTRACT FROM AUTHOR]

ABSTRACT Insect gut microbes play important roles in digestion, metabolism, development, and environmental adaptation. Parasitoid wasps are one of the most important biological control agents in pest control, while the gut microbial species compositions and the associated functions have been poorly investigated. Two endoparasitoid wasps, Cotesia vestalis and Diadromus collaris, parasitize the larval stage and pupal stage of the diamondback moth, Plutella xylostella, respectively. Using whole-genome shotgun metagenomic sequencing, we characterized the gut microbial composition, diversity, and potential functional roles associated with the two parasitoid wasp larvae. The results reveal that Proteobacteria and Firmicutes are the dominant phyla in the gut of C. vestalis and D. collaris larvae, with Rhizobium and Enterococcus being the dominant genera. The putative microbial functions associated with the two parasitoid wasps might play a virtual role in assisting in consuming the host’s nutritional composition. The enriched CAZymes family genes are primarily involved in the degradation and synthesis of chitin. Despite the richness of microbial species and communities, the microbes species and the microbial community structure exhibit significant similarity between the two parasitoid wasps and between the parasitoid wasp and the host P. xylostella. Notably, the prevalence of the genus Enterococcus shared among them suggests a possible link of gut microbes between the host and their associated parasitoids. Our study offers insights into the gut microbe-based interactions between the host and parasitoid wasps for the first time, potentially paving the way for the development of an ecologically friendly biocontrol strategy against the pest P. xylostella.IMPORTANCEEndoparasitoid wasps spend the majority of their lifespan within their host and heavily rely on the host’s nutrition for survival. There is limited understanding regarding the composition and physiological impacts of gut microbial communities in parasitoid wasps, particularly during the larval stage, which is directly linked to the host. Based on a thorough characterization of the gut microbe and comprehensive comparative analysis, we found the microbial species of the larval parasitoid wasp Cotesia vestalis and the pupal parasitoid wasp Diadromus collaris were similar, sharing 159 genera and 277 species, as were the microbial community structure. Certain of the dominant microbial strains of the two parasitoid wasps were similar to that of their host Plutella xylostella larvae, revealing host insect may affect the microbial community of the parasitoid wasps. The putative microbial functions associated with the parasitoid wasp larvae play an important role in dietary consumption.

ABSTRACTObesity and overweight among companion animals are significant concerns, paralleling the issues observed in human populations. Recent research has highlighted the potential benefits of various probiotics in addressing weight-related changes, obesity, and associated pathologies. In this study, we delved into the beneficial probiotic mechanisms in high-fat-induced obese canines, revealing that Enterococcus faecium IDCC 2102 (IDCC 2102) and Bifidobacterium lactis IDCC 4301 (IDCC 4301) have the capacity to mitigate the increase in body weight and lipid accumulation in obese canines subjected to a high-fat diet and hyperlipidemic Caenorhabditis elegans (C. elegans) strain VS29. Both IDCC 2102 and IDCC 4301 demonstrated the ability to reduce systemic inflammation and hormonal disruptions induced by obesity. Notably, these probiotics induced modifications in the microbiota by promoting lactic acid bacteria, including Lactobacillaceae, Ruminococcaceae, and S24-7, with concomitant activation of pyruvate metabolism. IDCC 4301, through the generation of bacterial short-chain fatty acids and carboxylic acids, facilitated glycolysis and contributed to ATP synthesis. Meanwhile, IDCC 2102 produced bacterial metabolites such as acetic acid and butyric acid, exhibiting a particular ability to stimulate dopamine synthesis in a canine model. This stimulation led to the restoration of eating behavior and improvements in glucose and insulin tolerance. In summary, we propose novel probiotics for the treatment of obese animals based on the modifications induced by IDCC 2102 and IDCC 4301. These probiotics enhanced systemic energy utilization in response to high caloric intake, thereby preventing lipid accumulation and restoring stability to the fecal microbiota. Consequently, this intervention resulted in a reduction in systemic inflammation caused by the high-fat diet.IMPORTANCEProbiotic supplementation affected commensal bacterial proliferation, and administering probiotics increased glycolysis and activated pyruvate metabolism in the body, which is related to propanate metabolism as a result of pyruvate metabolism activation boosting bacterial fatty acid production via dopamine and carboxylic acid specialized pathways, hence contributing to increased ATP synthesis and energy metabolism activity.

ABSTRACTEnterococcus faecalis (Efs) and Enterococcus faecium (Efm) are major causes of multiresistant healthcare-associated or nosocomial infections. Efm has been traditionally divided into clades A (healthcare associated) and B (community associated) but clade B has been recently reassigned to Enterococcus lactis (Elc). However, identification techniques do not routinely differentiate Elc from Efm. As part of a longitudinal study to investigate the antimicrobial resistance of Enterococcus in dairy cattle, isolates initially identified as Efm were confirmed as Elc after Oxford-Nanopore long-fragment whole-genome sequencing and genome comparisons. An Efm-specific PCR assay was developed and used to identify isolates recovered from animal feces on five farms, resulting in 44 Efs, 23 Efm, and 59 Elc. Resistance, determined by broth microdilution, was more frequent in Efs than in Efm and Elc but all isolates were susceptible to ampicillin, daptomycin, teicoplanin, tigecycline, and vancomycin. Genome sequencing analysis of 32 isolates identified 23 antimicrobial resistance genes (ARGs, mostly plasmid-located) and 2 single nucleotide polymorphisms associated with resistance to 10 antimicrobial classes, showing high concordance with phenotypic resistance. Notably, linezolid resistance in Efm was encoded by the optrA gene, located in plasmids downstream of the fexA gene. Although most Elc lacked virulence factors and genetic determinants of resistance, one isolate carried a plasmid with eight ARGs. This study showed that Elc is more prevalent than Efm in dairy cattle but carries fewer ARGs and virulence genes. However, Elc can carry multi-drug-resistant plasmids like those harbored by Efm and could act as a donor of ARGs for other pathogenic enterococcal species.IMPORTANCEEnterococcus species identification is crucial due to differences in pathogenicity and antibiotic resistance profiles. The failure of traditional methods or whole-genome sequencing-based taxonomic classifiers to distinguish Enterococcus lactis (Elc) from Enterococcus faecium (Efm) results in a biased interpretation of Efm epidemiology. The Efm species-specific real-time PCR assay developed here will help to properly identify Efm (only the formerly known clade A) in future studies. Here, we showed that Elc is prevalent in dairy cattle, and although this species carries fewer genetic determinants of resistance (GDRs) than Enterococcus faecalis (Efs) and Efm, it can carry multi-drug-resistant (MDR) plasmids and could act as a donor of resistance genes for other pathogenic enterococcal species. Although all isolates (Efs, Efm, and Elc) were susceptible to critically or highly important antibiotics like daptomycin, teicoplanin, tigecycline, and vancomycin, the presence of GDRs in MDR-plasmids is a concern since antimicrobials commonly used in livestock could co-select and confer resistance to critically important antimicrobials not used in food-producing animals.

ABSTRACTToxin production by Clostridioides difficile damages the colonic epithelium and leads to a robust inflammatory response. This disruption of the epithelial barrier markedly alters the nutritional landscape in the C. difficile-infected gut. The impact of toxin-mediated nutritional remodeling during C. difficile infection (CDI) on resident microbiota remains largely unexplored. One group of opportunistic pathogens, the enterococci, thrive during CDI, but it is unclear what strategies they employ to survive in this altered environment. Here, we demonstrate that Enterococcus faecalis, a heme auxotroph, takes advantage of C. difficile toxin-mediated damage to acquire host heme for enhanced fitness. Specifically, heme acquired from the C. difficile-infected gut is used by E. faecalis to populate a heme-dependent cytochrome and aerobically respire. This fitness advantage is specific to C. difficile toxin-mediated damage, as infection with a toxin-null strain of C. difficile does not provide E. faecalis with a fitness advantage. Finally, targeted disruption of the E. faecalis cytochrome (cydABDC) operon leads to a fitness defect in the C. difficile-infected gut. Together, this work demonstrates that C. difficile toxin remodels the gut ecosystem and improves the fitness of E. faecalis in a cydABDC-dependent manner. These data further highlight growing evidence of a cooperative partnership between C. difficile and enterococci that has implications on susceptibility to and severity of CDI.IMPORTANCEClostridioides difficile and Enterococcus faecalis are two pathogens of great public health importance. Both bacteria colonize the human gastrointestinal tract where they are known to interact in ways that worsen disease outcomes. We show that the damage associated with C. difficile infection (CDI) releases nutrients that benefit E. faecalis. One particular nutrient, heme, allows E. faecalis to use oxygen to generate energy and grow better in the gut. Understanding the mechanisms of these interspecies interactions could inform therapeutic strategies for CDI.

ABSTRACTEnterococcus 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. How E. faecalis colonizes and survives in the urinary tract (UT) is 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 4 highly contiguous draft assemblies and conducted a comparative genomics analysis study 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 analyses of antimicrobial resistance among urinary E. faecalis revealed infrequent resistance to the front-line UTI antibiotics nitrofurantoin and fluoroquinolones and no vancomycin resistance. Finally, comparing gene presence and absence among urinary and gut strains, we identified 19 candidate genes enriched among urinary strains. These genes are involved in the core processes of sugar transport, cobalamin import, glucose metabolism, and post-transcriptional regulation of gene expression.IMPORTANCEUrinary 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.

Enterococcus bacteria inhabit human and soil environments that show a wide range of pH values. Strains include commensals as well as antibiotic-resistant pathogens. We investigated the adaptation to pH stress in E. faecalis OG1RF by conducting experimental evolution under acidic (pH 4.8), neutral pH (pH 7.0), and basic (pH 9.0) conditions. A serial planktonic culture was performed for 500 generations and in a high-pH biofilm culture for 4 serial bead transfers. Nearly all of the mutations led to nonsynonomous codons, indicating adaptive selection. All of the acid-adapted clones from the planktonic culture showed a mutation in fusA (encoding elongation factor G). The acid-adapted fusA mutants had a trade-off of decreased resistance to fusidic acid (fusidate). All of the baseadapted clones from the planktonic cultures as well as some from the biofilm-adapted cultures showed mutations that affected the Pst phosphate ABC transporter (pstA, pstB, pstB2, pstC) and pyrR (pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). The biofilm cultures produced small-size colonies on brain heart infusion agar. These variants each contained a single mutation in pstB2, pstC, or pyrR. The pst and pyrR mutants outgrew the ancestral strain at pH 9.2, with a trade-off of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones that evolved at high pH (but not at low pH) include opp1BCDF (oligopeptide ABC transporter), ccpA (catabolite control protein A), and ftsZ (septation protein). Overall, the experimental evolution of E. faecalis showed a strong pH dependence, favoring the fusidate-sensitive elongation factor G modification at low pH and the loss of phosphate transport genes at high pH. IMPORTANCE E. faecalis bacteria are found in dental biofilms, where they experience low pH as a result of fermentative metabolism. Thus, the effect of pH on antibiotic resistance has clinical importance. The loss of fusidate resistance is notable for OG1RF strains in which fusidate resistance is assumed to be a stable genetic marker. In endodontal infections, enterococci can resist calcium hydroxide therapy that generates extremely high pH values. In other environments, such as the soil and plant rhizosphere, enterococci experience acidification that is associated with climate change. Thus, the pH modulation of natural selection in enterococci is important for human health as well as for understanding soil environments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACT Enterococcus faecium is a difficult-to-treat pathogen with emerging resistance to most clinically available antibiotics. Daptomycin (DAP) is the standard of care, but even high DAP doses (12 mg/kg body weight/day) failed to eradicate some vancomycin-resistant strains. Combination DAP-ceftaroline (CPT) may increase β-lactam affinity for target penicillin binding proteins (PBP); however, in a simulated endocardial vegetation (SEV) pharmacokinetic/pharmacodynamic (PK/PD) model, DAP-CPT did not achieve therapeutic efficacy against a DAP-nonsusceptible (DNS) vancomycin-resistant E. faecium (VRE) isolate. Phage-antibiotic combinations (PAC) have been proposed for resistant high-inoculum infections. We aimed to identify PAC with maximum bactericidal activity and prevention/reversal of phage and antibiotic resistance in an SEV PK/PD model against DNS isolate R497. Phage-antibiotic synergy (PAS) was evaluated with modified checkerboard MIC and 24-h time-kill analyses (TKA). Human-simulated antibiotic doses of DAP and CPT with phages NV-497 and NV-503-01 were then evaluated in 96-h SEV PK/PD models against R497. Synergistic and bactericidal activity was identified with the PAC of DAP-CPT combined with phage cocktail NV-497–NV-503-01, demonstrating a significant reduction in viability down to 3-log10 CFU/g (–Δ, 5.77-log10 CFU/g; P

ABSTRACT Vancomycin-resistant E. faecium (VREfm) is a significant public health concern because of limited treatment options. Genomic surveillance can be used to monitor VREfm transmission and evolution. Genomic analysis of VREfm has not been reported for the Dallas/Fort Worth/Arlington, TX, area, which is currently the 4th largest metropolitan area in the United States. Our study aimed to address this gap in knowledge by analyzing the genomes of 46 VREfm strains and 1 vancomycin-sensitive comparator collected during routine fecal surveillance of high-risk patients upon admission to a Dallas, TX, hospital system (August to October 2015). Thirty-one complete and 16 draft genome sequences were generated. The closed VREfm genomes possessed up to 12 extrachromosomal elements each. Overall, 251 closed putative plasmid sequences assigned to previously described and newly defined rep family types were obtained. Phylogenetic analysis identified 10 different sequence types (STs) among the isolates, with the most prevalent being ST17 and ST18. Strikingly, all but three of the VREfm isolates encoded vanA-type vancomycin resistance within Tn1546-like elements on a pRUM-like (rep17) plasmid backbone. Relative to a previously reported typing scheme for the vanA-carrying Tn1546, new variants of the Tn1546 were identified that harbored a combination of 7 insertion sequences (IS), including 3 novel IS elements reported here (ISEfa16, ISEfa17, and ISEfa18). We conclude that pRUM-like plasmids are important vectors for vancomycin resistance in the Dallas, TX, area and should be a focus of plasmid surveillance efforts. IMPORTANCE Vancomycin is an antibiotic used to treat infections caused by multidrug-resistant Gram-positive bacteria. Vancomycin resistance is common in clinical isolates of the Gram-positive pathogen Enterococcus faecium. Among E. faecium strains, vancomycin resistance genes can be disseminated by plasmids with different host ranges and transfer efficiencies. Surveillance of resistance plasmids is critical to understanding antibiotic resistance transmission. This study analyzed the genome sequences of VREfm isolates collected from the Dallas, TX, area, with particular focus on the mobile elements associated with vancomycin resistance genes. We found that a single plasmid family, the pRUM-like family, was associated with vancomycin resistance in the majority of isolates sampled. Our work suggests that the pRUM-like plasmids should continue to be studied to understand their mechanisms of maintenance, transmission, and evolution in VREfm.

Enterococcus faecalis is an important intestinal colonizing bacteria and can cause various tissue infections, including invasive blood infection (BI). The annual incidence of E. faecalis BI has been estimated to be ~4.5 per 100,000, with a fatality rate that can reach 20%. However, whether bacterial colonization or invasive infections are tissue based has not been thoroughly studied. In this study, we analyzed 537 clinical isolates from 7 different tissues to identify the key genomic elements that facilitate the colonization and invasive infection of E. faecalis. Comparative genomic analysis revealed that the BI E. faecalis isolates had the largest genome size but the lowest GC content, fsr quorum-sensing system genes were enriched in the BI E. faecalis, and the fsr gene cluster could enhance biofilm formation and serum resistance ability. Our findings also provide deep insight into the genomic differences between different tissue isolates, and the fsr quorum-sensing systems could be a key factor promoting E. faecalis invasion into the blood. [ABSTRACT FROM AUTHOR]

ABSTRACT Enterococcus faecalis is a normal commensal of the human gastrointestinal tract (GIT). However, upon disruption of gut homeostasis, this nonmotile bacterium can egress from its natural niche and spread to distal organs. While this translocation process can lead to life-threatening systemic infections, the underlying mechanisms remain largely unexplored. Our prior work showed that E. faecalis migration across diverse surfaces requires the formation of matrix-covered multicellular aggregates and the synthesis of exopolysaccharides, but how enterococcal cells are reprogrammed during this process is unknown. Whether surface penetration endows E. faecalis with adaptive advantages is also uncertain. Here, we report that surface penetration promotes the generation of a metabolically and phenotypically distinct E. faecalis population with an enhanced capacity to endure various forms of extracellular stress. Surface-invading enterococci demonstrated major ultrastructural alterations in their cell envelope characterized by increased membrane glycolipid content. These changes were accompanied by marked induction of specific transcriptional programs enhancing cell envelope biogenesis and glycolipid metabolism. Notably, the surface-invading population demonstrated superior tolerance to membrane-damaging antimicrobials, including daptomycin and β-defensins produced by epithelial cells. Genetic mutations impairing glycolipid biosynthesis sensitized E. faecalis to envelope stressors and reduced the ability of this bacterium to penetrate semisolid surfaces and translocate through human intestinal epithelial cell monolayers. Our study reveals that surface penetration induces distinct transcriptional, metabolic, and ultrastructural changes that equip E. faecalis with enhanced capacity to resist external stressors and thrive in its surrounding environment. IMPORTANCE Enterococcus faecalis inhabits the GIT of multiple organisms, where its establishment could be mediated by the formation of biofilm-like aggregates. In susceptible individuals, this bacterium can overgrow and breach intestinal barriers, a process that may lead to lethal systemic infections. While the formation of multicellular aggregates promotes E. faecalis migration across surfaces, little is known about the metabolic and physiological states of the enterococci encased in these surface-penetrating structures. The present study reveals that E. faecalis cells capable of migrating through semisolid surfaces genetically reprogram their metabolism toward increased cell envelope and glycolipid biogenesis, which confers superior tolerance to membrane-damaging agents. E. faecalis’s success as a pathobiont depends on its antimicrobial resistance, as well as on its rapid adaptability to overcome multiple environmental challenges. Thus, targeting adaptive genetic and/or metabolic pathways induced during E. faecalis surface penetration may be useful to better confront infections by this bacterium in the clinic.

Resistance to ampicillin and imipenem in Enterococcus faecalis is infrequent. However, the evolution of resistance can occur through prolonged antibiotic exposure during the treatment of chronic infections. In this study, we conducted a long-term evolution experiment using four genetically diverse strains of E. faecalis with varying susceptibilities to ampicillin and imipenem. Each strain was subjected to increasing concentrations of either ampicillin or imipenem over 200 days, with three independent replicates for each strain. Selective pressure from imipenem led to the rapid selection of highly resistant lineages across all genetic backgrounds, compared to ampicillin. In addition to high resistance, we describe, for the first time, the evolution of a β-lactam-dependent phenotype observed in lineages from all backgrounds. Whole-genome sequencing and bioinformatic analysis revealed mutations in three main functional classes: genes involved in cell wall synthesis and degradation, genes in the walK/R two-component system, and genes in the c-di-AMP pathway. Our analysis identified new mutations in genes known to be involved in resistance as well as novel genes potentially associated with resistance. Furthermore, the newly described β-lactam-dependent phenotype was correlated with the inactivation of c-di-AMP degradation, resulting in high levels of this second messenger. Together, these data highlight the diverse genetic mechanisms underlying resistance to ampicillin and imipenem in E. faecalis . The emergence of high resistance levels and β-lactam dependency underscores the importance of understanding evolutionary dynamics in the development of antibiotic resistance., Importance: Enterococcus faecalis is a major human pathogen, and treatment is frequently compromised by poor response to first-line antibiotics such as ampicillin. Understanding the factors that play a role in susceptibility/resistance to these drugs will help guide the development of much-needed treatments.

Enterococcus faecalis strain 13 (Ef#13) is a tongue isolate from a C57BL/6 mouse with experimental oropharyngeal candidiasis. Short-read sequencing revealed a genome of 2,767,598 bp with a GC content of 38.4%, and 2,649 genes were predicted., Competing Interests: The authors declare no conflict of interest.

We describe the genetic properties of two strains isolated from the elusive bile duct microbiome from solid organ transplant patients. Bacterial strains Enterococcus lactis (MS-STENT-08-E-001) and Klebsiella pneumoniae (MS-STENT-01-M-001) were isolated from the biofilms of bile duct catheters.

Enterococcus casseliflavus strains ASE2 and ASE4 were isolated from the soil of a lettuce farm in Salinas, California, in 2020. Their complete genome sequences were generated by the PacBio Sequel II platform. They both consist of a single circular chromosome without plasmids., Competing Interests: The authors declare no conflict of interest.

Our study presents the whole-genome sequences and annotation of five bacteria isolates, each demonstrating distinct antibiotic resistance. These isolates include Bacillus paranthracis RIT 841, Atlantibacter hermanii RIT 842, Pantoea leporis RIT 844, Enterococcus casseliflavus RIT 845, and Pseudomonas alkylphenolica RIT 846, underscoring the importance of understanding antimicrobial resistance., Competing Interests: The authors declare no conflict of interest.

SUMMARY Enterococcus faecalis and Enterococcus faecium are human pathobionts that exhibit a dual lifestyle as commensal and pathogenic bacteria. The pathogenic lifestyle is associated with specific conditions involving host susceptibility and intestinal overgrowth or the use of a medical device. Although the virulence of E. faecium appears to benefit from its antimicrobial resistance, E. faecalis is recognized for its higher pathogenic potential. E. faecalis has long been considered a predominantly extracellular pathogen; it adheres to and is taken up by a wide range of mammalian cells, albeit with less efficiency than classical intracellular enteropathogens. Carbohydrate structures, rather than proteinaceous moieties, are likely to be primarily involved in the adhesion of E. faecalis to epithelial cells. Consistently, few adhesins have been implicated in the adhesion of E. faecalis to epithelial cells. On the host side, very little is known about cognate receptors, except for the role of glycosaminoglycans during macrophage infection. Several lines of evidence indicate that E. faecalis internalization may involve a zipper-like mechanism as well as a macropinocytosis pathway. Conversely, E. faecalis can use several strategies to prevent engulfment in phagocytes. However, the bacterial and host mechanisms underlying cell infection by E. faecalis are still in their infancy. The most recent striking finding is the existence of an intracellular lifestyle where E. faecalis can replicate within a variety of host cells. In this review, we summarize and discuss the current knowledge of E. faecalis -host cell interactions and argue on the need for further mechanistic studies to prevent or reduce infections., Competing Interests: The authors declare no conflict of interest.

ABSTRACT Gut microbiota can have diverse impacts on hosts, the nature of which often depend on the circumstances. For insect gut microbes, the quality and nature of host diets can be a significant force in swinging the pendulum from inconsequential to functionally important. In our study, we addressed whether beneficial microbes in one species impart similar functions to related species under identical conditions. Using fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), and other noctuid hosts, we implemented an axenic rearing strategy and manipulated gut bacterial populations and dietary conditions. Our results revealed that some gut Enterococcus and Enterobacter isolates can facilitate utilization of a poor diet substrate by fall armyworm, but this was not the case for other more optimized diets. While Enterococcus provided benefits to fall armyworm, it was decidedly antagonistic to beet armyworm (Spodoptera exigua) under identical conditions. Unique isolates and bacterial introductions at early growth stages were critical to how both larval hosts performed. Our results provide robust evidence of the roles in which bacteria support lepidopteran larval growth, but also indicate that the directionality of these relationships can differ among congener hosts. IMPORTANCE Insects have intimate relationships with gut microbiota, where bacteria can contribute important functions to their invertebrate hosts. Lepidopterans are important insect pests, but how they engage with their gut bacteria and how that translates to impacts on the host are lacking. Here we demonstrate the facultative nature of gut microbiota in lepidopteran larvae and the importance of diet in driving mutualistic or antagonistic relationships. Using multiple lepidopteran species, we uncover that the same bacteria that can facilitate exploitation of a challenging diet in one host severely diminishes larval performance of another larval species. Additionally, we demonstrate the beneficial functions of gut microbiota on the hosts are not limited to one lineage, but rather multiple isolates can facilitate the exploitation of a suboptimal diet. Our results illuminate the context-dependent nature of the gut microbiomes in invertebrates, and how host-specific microbial engagement can produce dramatically different interactions.

ABSTRACT Enterococcus faecalis is an opportunistic pathogen and a major cause of severe nosocomial infections. Treatment options against enterococcal infections are declining due to the resistance of enterococci to numerous antibiotics. A key risk factor for developing enterococcal infections is treatment with cephalosporin antibiotics, to which enterococci are intrinsically resistant. For susceptible organisms, cephalosporins inhibit bacterial growth by acylating the active site of penicillin-binding proteins (PBPs), key enzymes that catalyze peptidoglycan cross-linking. Two specific PBPs of enterococci, Pbp4(5) and PbpA(2b), exhibit low reactivity toward cephalosporins, allowing these PBPs to cross-link peptidoglycan in the presence of cephalosporins to drive resistance in enterococci, but the mechanisms by which these PBPs are regulated are poorly understood. The CroS/R two-component signal transduction system (TCS) is also required for cephalosporin resistance. Activation of CroS/R by cephalosporins leads to CroR-dependent changes in gene expression. However, the specific genes regulated by CroS/R that are responsible for cephalosporin resistance remain largely unknown. In this study, we characterized CroR-dependent transcriptome remodeling by RNA-seq, identifying pbp4(5) as a CroR regulon member in multiple, diverse lineages of E. faecalis. Through genetic analysis of the pbp4(5) and croR promoters, we uncovered a CroR-dependent regulatory motif. Mutations in this motif to disrupt CroR-dependent upregulation of pbp4(5) in the presence of cell wall stress resulted in a reduction of resistance to cephalosporins in E. faecalis, demonstrating that enhanced production of Pbp4(5) and likely other proteins involved in peptidoglycan biogenesis by the CroS/R system drives enterococcal cephalosporin resistance. IMPORTANCE Investigation into molecular mechanisms used by enterococci to subvert cephalosporin antibiotics is imperative for preventing and treating life-threatening infections. In this study, we used genetic means to investigate the functional output of the CroS/R TCS required for enterococcal resistance to cephalosporins. We found that enhanced production of the penicillin-binding protein Pbp4(5) upon exposure to cell wall stress was mediated by CroS/R and was critical for intrinsic cephalosporin resistance of E. faecalis.

ABSTRACT Enterococci are gram-positive, gastrointestinal (GI) tract commensal bacteria that have recently evolved into multidrug-resistant nosocomial pathogens. Enterococci are intrinsically hardy, meaning that they can thrive in challenging environments and outlast other commensal bacteria. Further adaptations enable enterococci to dominate the GI tracts of hospitalized patients, and this domination precedes invasive infection and facilitates transmission to other patients. A recent study by Boumasmoud et al. used whole genome sequencing (WGS) to characterize 69 vancomycin-resistant Enterococcus faecium (VREfm) isolates collected from a Swiss hospital. WGS uncovered a clone that was repeatedly sampled from dozens of patients over multiple years. This persistent clone accumulated mutations as well as a novel linear plasmid, which together likely increased its persistence in the GI tracts of infected patients. This study is one of several recent examples that highlight the genetic plasticity of VREfm as it adapts to the hospitalized gut and becomes a leading nosocomial pathogen.

A longitudinal study was conducted to assess the impact of different antimicrobial exposures of nursery-phase pigs on patterns of phenotypic antimicrobial resistance (AMR) in fecal indicator organisms throughout the growing phase. Based on practical approaches used to treat moderate to severe porcine reproductive and respiratory syndrome virus (PRRSV)-associated secondary bacterial infections, two antimicrobial protocols of differing intensities of exposure [44.1 and 181.5 animal-treatment days per 1000 animal days at risk (ATD)] were compared with a control group with minimal antimicrobial exposure (2.1 ATD). Litter-matched pigs (n = 108) with no prior antimicrobial exposure were assigned randomly to the treatment groups. Pen fecal samples were collected nine times during the wean-to-finish period and cultured for Escherichia coli and Enterococcus spp. Antimicrobial-susceptibility testing was conducted using NARMS Gram-negative and Gram-positive antibiotic panels. Despite up to 65-fold difference in ATD, few and modest differences were observed between groups and over time. Resistance patterns at marketing overall remained similar to those observed at weaning, prior to any antimicrobial exposures. Those differences observed could not readily be reconciled with the patterns of antimicrobial exposure. Resistance of E. coli to streptomycin was higher in the group exposed to 44.1 ATD, but no aminoglycosides were used. In all instances where resistances differed between time points, the higher resistance occurred early in the trial prior to any antimicrobial exposures. These minimal impacts on AMR despite substantially different antimicrobial exposures point to the lack of understanding of the drivers of AMR at the population level and the likely importance of factors other than antimicrobial exposure. [ABSTRACT FROM AUTHOR]

ABSTRACT Antimicrobial use in food-producing animals has come under increasing scrutiny due to its potential association with antimicrobial resistance (AMR). Monitoring of AMR in indicator microorganisms such as Enterococcus spp. in meat production facilities and retail meat products can provide important information on the dynamics and prevalence of AMR in these environments. In this study, swabs or samples were obtained from various locations in a commercial beef packing operation (n = 600) and from retail ground beef (n = 60) over a 19-month period. All samples/swabs were enriched for Enterococcus spp., and suspected enterococci isolates were identified using species-specific PCR primers. Enterococcus faecalis was the most frequently isolated species, followed by Enterococcus hirae, which was found mostly on post-hide removal carcasses and in ground beef. Enterococcus faecium (n = 9) and E. faecalis (n = 120) isolates were further characterized for AMR. Twenty-one unique AMR profiles were identified, with 90% of isolates resistant to at least two antimicrobials and two that were resistant to nine antimicrobials. Tetracycline resistance was observed most often in E. faecalis (28.8%) and was likely mediated by tet(M). Genomic analysis of selected E. faecalis and E. faecium isolates revealed that many of the isolates in this study clustered with other publicly available genomes from ground beef, suggesting that these strains are well adapted to the beef processing environment. IMPORTANCE Antimicrobial resistance (AMR) is a serious challenge facing the agricultural industry. Understanding the flow of antimicrobial-resistant bacteria through the beef fabrication process and into ground beef is an important step in identifying intervention points for reducing AMR. In this study, we used enterococci as indicator bacteria for monitoring AMR in a commercial beef packaging facility and in retail ground beef over a 19-month period. Although washing of carcasses post-hide removal reduced the isolation frequency of Enterococcus spp., a number of antimicrobial-resistant Enterococcus faecalis isolates were recovered from ground beef produced in the packaging plant. Genome analysis showed that several E. faecalis isolates were genetically similar to publicly available isolates recovered from retail ground beef in the United States.

ABSTRACT The rapid spread of antibiotic resistance among Enterococcus has prompted considerable interest in determining the dosage regimen of linezolid combined with fosfomycin. A checkerboard assay was employed to evaluate whether linezolid combined with fosfomycin had a synergistic effect on Enterococcus isolates from the hospital, including three drug-resistant strains (MIC of linezolid [MICLZD], ≥8 mg/L; MIC of fosfomycin [MICFOF], ≥256 mg/L). The in vitro static time-kill assay, dynamic pharmacokinetic (PK)/pharmacodynamic (PD) model, and semimechanistic PK/PD model were used to explore and predict effective combined dosage regimens. The checkerboard assay and in vitro static time-kill assay demonstrated that linezolid combined with fosfomycin has a synergistic effect on drug-resistant and sensitive Enterococcus. In the in vitro PK/PD model, the dosage regimen of linezolid (8 mg/L or 12 mg/L, steady-state concentration) combined with fosfomycin (6 g or 8 g) via a 0.5-h infusion every 8 h effectively suppressed bacterial growth at 24 h with a 3 log10 CFU/mL decrease compared with the initial inocula against two resistant and one sensitive Enterococcus isolates. The semimechanistic PK/PD model predicted that linezolid (more than 16 mg/L) combined with fosfomycin (6 g or 10 g) via a 0.5-h infusion every 8 h was required to achieve a 4 log10 CFU/mL decrease at 24 h against Enterococcus isolates (MICLZD ≥ 8 mg/L and MICFOF ≥ 256 mg/L). According to the prediction of the semimechanical PK/PD model, the effect of the combination was driven by linezolid, with fosfomycin enhancing the effect. Our study is the first to explore the synergistic effects of these two drugs from a qualitative and quantitative perspective and provides a simulation tool for future studies. IMPORTANCE In this study, we found that linezolid combined with fosfomycin could kill Enterococcus in vitro and that the administered dose was significantly lower after the combination treatment, which could reduce adverse effects and the development of drug resistance. The potential mechanism of the two-drug combination against Enterococcus was revealed from a quantitative perspective, which is an important step toward dose optimization in simulated humans. We hope that our research will help build a better relationship between clinicians and patients as we work together to address the challenges of antibiotic resistance in the 21st century.

Reported here are the sequences for 11 Escherichia coli and four Enterococcus strains isolated from post-menopausal women with a recurrent urinary tract infection. Each of the Enterococcus strains were isolated along with an E. coli strain. This provides a resource of high-quality complete genomes from polymicrobial infections., Competing Interests: The authors declare no conflict of interest.

SUMMARYEnterococci are a diverse group of Gram-positive bacteria that are typically found as commensals in humans, animals, and the environment. Occasionally, they may cause clinically relevant diseases such as endocarditis, septicemia, urinary tract infections, and wound infections. The majority of clinical infections in humans are caused by two species: Enterococcus faecium and Enterococcus faecalis . However, there is an increasing number of clinical infections caused by non- faecium non- faecalis (NFF) enterococci. Although NFF enterococcal species are often overlooked, studies have shown that they may harbor antimicrobial resistance (AMR) genes and virulence factors that are found in E. faecium and E. faecalis . In this review, we present an overview of the NFF enterococci with a particular focus on human clinical manifestations, epidemiology, virulence genes, and AMR genes., Competing Interests: The authors declare no conflict of interest.

Enterococcus faecium is a ubiquitous Gram-positive bacterium that has been recovered from the environment, food, and microbiota of mammals. Commensal strains of E. faecium can confer beneficial effects on host physiology and immunity, but antibiotic usage has afforded antibiotic-resistant and pathogenic isolates from livestock and humans. However, the dissection of E. faecium functions and mechanisms has been restricted by inefficient gene-editing methods. To address these limitations, here, we report that the expression of E. faecium RecT recombinase significantly improves the efficiency of recombineering technologies in both commensal and antibiotic-resistant strains of E. faecium and other Enterococcus species such as E. durans and E. hirae. Notably, the expression of RecT in combination with clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 and guide RNAs (gRNAs) enabled highly efficient scarless single-stranded DNA recombineering to generate specific gene-editing mutants in E. faecium. Moreover, we demonstrate that E. faecium RecT expression facilitated chromosomal insertions of double-stranded DNA templates encoding antibiotic-selectable markers to generate gene deletion mutants. As a further proof of principle, we use CRISPR-Cas9-mediated recombineering to knock out both sortase A genes in E. faecium for downstream functional characterization. The general RecT-mediated recombineering methods described here should significantly enhance genetic studies of E. faecium and other closely related species for functional and mechanistic studies. [ABSTRACT FROM AUTHOR]

ABSTRACT Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyze the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus species. MRSE (n = 17) and Enterococcus species (Enterococcus faecalis n = 8 and Enterococcus faecium n = 10) strains were used. MIC of the mixture of DTAM, HTAM, and ENDX and that of vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and of vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays. MIC90 of tamoxifen metabolites was 1 mg/liter for MRSE strains and 2 mg/liter for E. faecalis and E. faecium strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 4× MIC for MRSE (SE385 and SE742) and at 2× MIC and 4× MIC for E. faecalis (EVR1 and EVR2) strains, respectively. SE385 and EVR2 strains treated with the tamoxifen metabolites mixture presented higher membrane permeabilization. Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drug treatments against these bacterial pathogens. IMPORTANCE The development of new antimicrobial therapeutic strategies requires immediate attention to avoid the tens of millions of deaths predicted to occur by 2050 as a result of MDR bacterial infections. In this study, we assessed the antibacterial activity of three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus spp. (E. faecalis and E. faecium). We found that the tamoxifen metabolites have antibacterial activity against MRSE, E. faecalis, and E. faecium strains by presenting MIC90 between 1 and 2 mg/liter and bactericidal activity over 24 h. In addition, this antibacterial activity is paralleled by an increased membrane permeability of these strains. Our results showed that tamoxifen metabolites might be potentially used as a therapeutic alternative when treating MRSE and E. faecalis strains in an animal model of infection.

Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis subsp. natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from chromosomally encoded CF10 (cCF10) without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30- to 50-kDa extracellular protease present in B. subtilis subsp. natto. Nattokinase of B. subtilis subsp. natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into LVTL plus VFV fragments. Moreover, the cleavage product LVTL (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis subsp. natto can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis subsp. natto and L peptide to mitigate E. faecalis antibiotic resistance transfer. [ABSTRACT FROM AUTHOR]

Enterococci are ubiquitous opportunistic pathogens that have become a major public health issue globally. The increasing prevalence of antimicrobial resistance in hospital-adapted enterococci had been thought to originate from livestock. However, this association between livestock and hospital-adapted enterococci is currently unclear. This study investigates the antimicrobial susceptibilities of enterococci isolated from pig cecal samples and compares the genomic characteristics of Enterococcus faecium from pigs to those of isolates from meat chickens and from human sepsis cases. From 200 cecal samples, antimicrobial susceptibility testing was performed for E. faecium (n = 84), E. hirae (n = 36), and E. faecalis (n = 17). Whole-genome sequencing was performed for all E. faecium isolates, and the sequences were compared to those of previously studied isolates from meat chickens and human sepsis cases through bioinformatics analysis. Resistance (non-wild type) to erythromycin, gentamicin, tetracycline, ampicillin, daptomycin, virginiamycin, and quinupristin-dalfopristin was identified. More importantly, except for a single isolate harboring the vanC operon, no resistance was observed in the three species to vancomycin, teicoplanin, and linezolid, which are critically important antimicrobials used to treat enterococcal infections in humans. The E. faecium isolates from chickens were genetically distinct from human and pig isolates, which were more closely related. Human strains that were closely related to pig strains were not typical "hospital-adapted strains" as previously identified. The results of this study show that enterococci from Australian finisher pigs are not a source of resistance to critically important antimicrobials and that E. faecium from pigs is not part of the current human hospitaladapted population. Resistance to the critically important antimicrobials vancomycin, teicoplanin, and linezolid is not found in enterococci collected from Australian finisher pigs. However, some antimicrobial resistance was observed. In particular, resistance to quinupristin-dalfopristin, a combination of two streptogramin class antimicrobials, was identified despite the absence of streptogramin use Australia-wide since 2005. Other observed resistance among enterococci from pigs include chloramphenicol, erythromycin, and tetracycline resistance. Genomic comparison of E. faecium from Australian pigs to isolates collected from previous studies on chickens and humans indicate that E. faecium from pigs are genetically more similar to those of humans than those from chickens. Despite the increased genetic similarities, E. faecium strains from pigs are phylogenetically distinct and did not belong to the dominant sequence types found in hospital-adapted strains causing sepsis in humans. Therefore, the results indicate that Australian finisher pigs are not a source of hospital-adapted E. faecium in Australia. [ABSTRACT FROM AUTHOR]

Linezolid is a last-resort antibiotic for the treatment of severe infections caused by multidrug-resistant Gram-positive organisms; although linezolid resistance remains uncommon, the number of linezolid-resistant enterococci has increased in recent years due to worldwide spread of acquired resistance genes (cfr, optrA, and poxtA) in clinical, animal, and environmental settings. In this study, we investigated the occurrence of linezolid-resistant enterococci in marine samples from two coastal areas in Italy. Isolates grown on florfenicol-supplemented Slanetz-Bartley agar plates were investigated for their carriage of optrA, poxtA, and cfr genes; optrA was found in one Enterococcus faecalis isolate, poxtA was found in three Enterococcus faecium isolates and two Enterococcus hirae isolates, and cfr was not found. Two of the three poxtA-carrying E. faecium isolates and the two E. hirae isolates showed related pulsed-field gel electrophoresis (PFGE) profiles. Two E. faecium isolates belonged to the new sequence type 1710, which clustered in clonal complex 94, encompassing nosocomial strains. S1 PFGE/hybridization assays showed a double (chromosome and plasmid) location of poxtA and a plasmid location of optrA. Whole-genome sequencing revealed that poxtA was contained in a Tn6657-like element carried by two plasmids (pEfm-EF3 and pEh-GE2) of similar size, found in different species, and that poxtA was flanked by two copies of IS1216 in both plasmids. In mating experiments, all but one strain (E. faecalis EN3) were able to transfer the poxtA gene to E. faecium 64/3. The occurrence of linezolid resistance genes in enterococci from marine samples is of great concern and highlights the need to improve practices aimed at limiting the transmission of linezolid-resistant strains to humans from environmental reservoirs. [ABSTRACT FROM AUTHOR]

Enterococcal plasmid-encoded bacteriolysin Bac41 is a selective antimicrobial system that is considered to provide a competitive advantage to Enterococcus faecalis cells that carry the Bac41-coding plasmid. The Bac41 effector consists of the secreted proteins BacL1 and BacA, which attack the cell wall of the target E. faecalis cell to induce bacteriolysis. Here, we demonstrated that galU, which encodes UTP-glucose- 1-phosphate uridylyltransferase, is involved in susceptibility to the Bac41 system in E. faecalis. Spontaneous mutants that developed resistance to the antimicrobial effects of BacL1 and BacA were revealed to carry a truncation deletion of the C-terminal amino acid (aa) region 288 to 298 of the translated GalU protein. This truncation resulted in the depletion of UDP-glucose, leading to a failure to utilize galactose and produce the enterococcal polysaccharide antigen (EPA), which is expressed abundantly on the cell surface of E. faecalis. This cell surface composition defect that resulted from galU or EPA-specific genes caused an abnormal cell morphology, with impaired polarity during cell division and alterations of the limited localization of BacL1. Interestingly, these mutants had reduced susceptibility to beta-lactams besides Bac41, despite their increased susceptibility to other bacteriostatic antimicrobial agents and chemical detergents. These data suggest that a complex mechanism of action underlies lytic killing, as exogenous bacteriolysis induced by lytic bacteriocins or beta-lactams requires an intact cell physiology in E. faecalis. [ABSTRACT FROM AUTHOR]

ABSTRACT To cause infection, Staphylococcus aureus must withstand damage caused by host immune defenses. However, the mechanisms by which staphylococcal DNA is damaged and repaired during infection are poorly understood. Using a panel of transposon mutants, we identified the rexBA operon as being important for the survival of Staphylococcus aureus in whole human blood. Mutants lacking rexB were also attenuated for virulence in murine models of both systemic and skin infections. We then demonstrated that RexAB is a member of the AddAB family of helicase/nuclease complexes responsible for initiating the repair of DNA double-strand breaks. Using a fluorescent reporter system, we were able to show that neutrophils cause staphylococcal DNA double-strand breaks through reactive oxygen species (ROS) generated by the respiratory burst, which are repaired by RexAB, leading to the induction of the mutagenic SOS response. We found that RexAB homologues in Enterococcus faecalis and Streptococcus gordonii also promoted the survival of these pathogens in human blood, suggesting that DNA double-strand break repair is required for Gram-positive bacteria to survive in host tissues. Together, these data demonstrate that DNA is a target of host immune cells, leading to double-strand breaks, and that the repair of this damage by an AddAB-family enzyme enables the survival of Gram-positive pathogens during infection. IMPORTANCE To cause infection, bacteria must survive attack by the host immune system. For many bacteria, including the major human pathogen Staphylococcus aureus, the greatest threat is posed by neutrophils. These immune cells ingest the invading organisms and try to kill them with a cocktail of chemicals that includes reactive oxygen species (ROS). The ability of S. aureus to survive this attack is crucial for the progression of infection. However, it was not clear how the ROS damaged S. aureus and how the bacterium repaired this damage. In this work, we show that ROS cause breaks in the staphylococcal DNA, which must be repaired by a two-protein complex known as RexAB; otherwise, the bacterium is killed, and it cannot sustain infection. This provides information on the type of damage that neutrophils cause S. aureus and the mechanism by which this damage is repaired, enabling infection.

Bacteria can survive antibiotic treatment both by acquiring antibiotic resistance genes and through mechanisms of tolerance that are based on phenotypic changes and the formation of metabolically inactive cells. Here, we report an Enterococcus faecalis strain (E. faecalis UM001B) that was isolated from a cystic fibrosis patient and had no increase in resistance but extremely high-level tolerance to ampicillin, vancomycin, and tetracycline. Specifically, the percentages of cells that survived 3.5-h antibiotic treatment (at 100 μg · mlμ1) were 25.4% ± 4.3% and 51.9%±4.0% for ampicillin and tetracycline, respectively; vancomycin did not exhibit any significant killing. Consistent with the changes in antibiotic susceptibility, UM001B was found to have reduced penetration of ampicillin and vancomycin and accumulation of tetracycline compared to the reference strain ATCC 29212. Based on whole-genome sequencing, four amino acid substitutions were identified in one of the tetracycline efflux pump repressors (TetRs), compared to ATCC 29212. Results of molecular simulations and experimental assays revealed that these mutations could lead to higher levels of tetracycline efflux activity. Consistently, replicating these mutations in Escherichia coli MG1655 increased its tolerance to tetracycline. Overall, these findings provide new insights into the development of multidrug tolerance in E. faecalis, which can facilitate future studies to better control enterococcal infections. [ABSTRACT FROM AUTHOR]

Enterococcus faecium is a member of the human gastrointestinal (GI) microbiota but can also cause invasive infections, especially in immunocompromised hosts. Enterococci display intrinsic resistance to many antibiotics, and most clinical E. faecium isolates have acquired vancomycin resistance, leaving clinicians with a limited repertoire of effective antibiotics. As such, vancomycin-resistant E. faecium (VREfm) has become an increasingly difficult to treat nosocomial pathogen that is often associated with treatment failure and recurrent infections. We followed a patient with recurrent E. faecium bloodstream infections (BSIs) of increasing severity, which ultimately became unresponsive to antibiotic combination therapy over the course of 7 years. Whole-genome sequencing (WGS) showed that the patient was colonized with closely related E. faecium strains for at least 2 years and that invasive isolates likely emerged from a large E. faecium population in the patient's gastrointestinal (GI) tract. The addition of bacteriophage (phage) therapy to the patient's antimicrobial regimen was associated with several months of clinical improvement and reduced intestinal burden of VRE and E. faecium. In vitro analysis showed that antibiotic and phage combination therapy improved bacterial growth suppression compared to therapy with either alone. Eventual E. faecium BSI recurrence was not associated with the development of antibiotic or phage resistance in post-treatment isolates. However, an anti-phage-neutralizing antibody response occurred that coincided with an increased relative abundance of VRE in the GI tract, both of which may have contributed to clinical failure. Taken together, these findings highlight the potential utility and limitations of phage therapy to treat antibiotic-resistant enterococcal infections., Importance: Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit., Competing Interests: G.H. is a recipient of research grants from Allovir, Karius, and AstraZeneca. G.H. also serves on the scientific advisory boards of Karius and AstraZeneca and has received honoraria from MDOutlook.

Here, we report the annotated genome of enterococcal phage G01. The G01 genome is 41,189 bp in length and contains 67 predicted open reading frames. Host range analysis revealed G01 can infect 28.6% (6/21) of Enterococcus faecalis strains tested and appears to not require the enterococcal phage infection protein PIP EF ., Competing Interests: The authors declare no conflict of interest.

ABSTRACT Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy. IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

ABSTRACT Metabolic diseases, including type 2 diabetes and obesity, have become increasingly prevalent global health concerns. Studies over the past decade have established connections between the gastrointestinal microbiota and host metabolism, but the mechanisms behind these connections are only beginning to be understood. We were interested in identifying microbes that have the ability to modulate the levels of the incretin hormone glucagon-like peptide-1 (GLP-1). Using a human-derived cell line that is capable of secreting GLP-1 in response to stimulatory ligands (NCI-H716), we identified supernatants from several bacterial isolates that were capable of decreasing GLP-1 levels, including several strains of Enterococcus faecalis. We further identified the secreted protease GelE, an established virulence factor from E. faecalis, as being responsible for GLP-1 inhibition via direct cleavage of GLP-1 by GelE. Finally, we demonstrated that E. faecalis supernatants can disrupt a colonic epithelial monolayer and cleave GLP-1 in a gelE-dependent manner. This work suggests that a secreted factor from an intestinal microbe can traverse the epithelial barrier and impact levels of an important intestinal hormone. IMPORTANCE Humans have a complex and interconnected relationship with their gastrointestinal microbiomes, yet our interest in the microbiome tends to focus on overt pathogenic or probiotic activities, leaving the roles that commensal species may have on host physiology and metabolic processes largely unexplored. Commensal organisms in the microbiome produce and secrete many factors that have an opportunity to interact with the gastrointestinal tract and host biology. Here, we show that a secreted protease from E. faecalis, GelE, is able to degrade the gastrointestinal hormone GLP-1, which is responsible for regulating glucose homeostasis and appetite in the body. The disruption of natural GLP-1 signaling by GelE may have significant consequences for maintaining healthy blood glucose levels and in the development of metabolic disease. Furthermore, this work deepens our understanding of specific host-microbiome interactions.

Class IIa bacteriocin antimicrobial peptides (AMPs) are a compelling alternative to current antimicrobials because of potential specific activity towards antibiotic-resistant bacteria, including vancomycin-resistant enterococci. Engineering of these molecules would be enhanced by a better understanding of AMP sequence-activity relationships to improve efficacy in vivo and limit effects of off-target activity. Towards this goal, we experimentally evaluated 210 natural and variant class IIa bacteriocins for antimicrobial activity against six strains of enterococci. Inhibitory activity was ridge regressed to AMP sequence to predict performance, achieving an area under the curve of 0.70 and demonstrating the potential of statistical models for identifying and designing AMPs. Active AMPs were individually produced and evaluated against eight enterococci strains and four Listeria strains to elucidate trends in susceptibility. It was determined that the mannose phosphotransferase system sequence is informative of susceptibility to class IIa bacteriocins, yet other factors, such as membrane composition, also contribute strongly to susceptibility. A broadly potent bacteriocin variant (lactocin DT1) from a Lactobacillus ruminis genome was identified as the only variant with inhibitory activity towards all tested strains, while a novel enterocin variant (DT2) from an E. faecium genome demonstrated specificity towards Listeria strains. Eight AMPs were evaluated for proteolytic stability to trypsin, chymotrypsin, and pepsin, and three C-terminal disulfide-containing variants, including divercin V41, were identified as compelling for future in vivo studies given high potency and proteolytic stability. [ABSTRACT FROM AUTHOR]

As climate change continues to stress freshwater resources, we have a pressing need to identify alternative (non-traditional) sources of microbially safe water for irrigation of fresh produce. This study is part of the center CONSERVE, which aims to facilitate the adoption of adequate agricultural water sources. A 26-month longitudinal study was conducted at 11 sites to assess the prevalence of bacteria indicating water quality, fecal contamination and crop contamination risk (Escherichia coli, total coliforms (TC), Enterococcus, Aeromonas). Sites included non-tidal freshwater rivers/creeks (NF), a tidal brackish river (TB), irrigation ponds (PW) and reclaimed water sites (RW). Water samples were filtered for bacterial quantification. E. coli, TC and enterococci (~86%, 98% and 90% positive, respectively; n=333), and Aeromonas (~98% positive; n=133) were widespread in water samples tested. Highest E. coli counts were in rivers, TC counts in TB rivers and enterococci in rivers and ponds (all p<0.001), compared to other water types. Aeromonas counts were consistent across sites. Seasonal dynamics were detected in NF and PW samples only. E. coli counts were higher in the vegetable crop-growing (May- October) than non-growing (November-April) season in all water types (p<0.05). Only one RW and both PW sites met the US Food Safety Modernization Act water standards. However, implementation of recommended mitigation measures of allowing time for microbial die-off between irrigation and harvest would bring all other sites into compliance within two days. This study provides comprehensive microbial data on alternative irrigation water and serves as an important resource for food safety planning and policy-setting. [ABSTRACT FROM AUTHOR]

Enterococci are commensals that proliferated as animals crawled ashore hundreds of millions of years ago. They are also leading causes of multidrug-resistant hospital-acquired infections. While most studies are driven by clinical interest, comparatively little is known about enterococci in the wild or the effect of human activity on them. Pharmaceutical pollution and runoff from other human activities are encroaching widely into natural habitats. To assess their reach into remote habitats, we investigated the identity, genetic relatedness, and presence of specific traits among 172 enterococcal isolates from wild Magellanic penguins. Four enterococcal species, 18 lineage groups, and different colonization patterns were identified. One Enterococcus faecalis lineage, sequence type 475 (ST475), was isolated from three different penguins, making it of special interest. Its genome was compared to those of other E. faecalis sequence types (ST116 and ST242) recovered from Magellanic penguins, as well as to an existing phylogeny of E. faecalis isolated from diverse origins over the past 100 years. No penguin-derived E. faecalis strains were closely related to dominant clinical lineages. Most possessed intact CRISPR defenses, few mobile elements, and antibiotic resistances limited to those intrinsic to the species and lacked pathogenic features conveyed by mobile elements. Interestingly, plasmids were identified in penguin isolates that also had been reported for other marine mammals. Enterococci isolated from penguins showed limited anthropogenic impact, indicating that they are likely representative of those naturally circulating in the ecosystem inhabited by the penguins. These findings establish an important baseline for detecting the encroachment of human activity into remote planetary environments. [ABSTRACT FROM AUTHOR]

Resistance to the "last-resort" antibiotics, such as carbapenems, has led to very few antibiotics being left to treat infections by multidrug-resistant bacteria. Spread of carbapenem resistance (CR) has been well characterized for the clinical environment. However, there is a lack of information about its environmental distribution. Our study reveals that CR is present in a wide range of Gram-negative bacteria in the coastal seawater environment, including four phyla, eight classes, and 30 genera. These bacteria were likely introduced into seawater via stormwater flows. Some CR isolates found here, such as Acinetobacter junii, Acinetobacter johnsonii, Brevundimonas vesicularis, Enterococcus durans, Pseudomonas monteilii, Pseudomonas fulva, and Stenotrophomonas maltophilia, are further relevant to human health. We also describe a novel metallo-γ-lactamase (MBL) for marine Rheinheimera isolates with CR, which has likely been horizontally transferred to Citrobacter freundii or Enterobacter cloacae. In contrast, another MBL of the New Delhi type was likely acquired by environmental Variovorax isolates from Escherichia coli, Klebsiella pneumoniae, or Acinetobacter baumannii utilizing a plasmid. Our findings add to the growing body of evidence that the aquatic environment is both a reservoir and a vector for novel CR genes. Resistance against the "last-resort" antibiotics of the carbapenem family is often based on the production of carbapenemases, and this has been frequently observed in clinical samples. However, the dissemination of carbapenem resistance (CR) in the environment has been less well explored. Our study shows that CR is commonly found in a range of bacterial taxa in the coastal aquatic environment and can involve the exchange of novel metallo-γ-lactamases from typical environmental bacteria to potential human pathogens or vice versa. The outcomes of this study contribute to a better understanding of how aquatic and marine bacteria can act as reservoirs and vectors for CR outside the clinical setting. [ABSTRACT FROM AUTHOR]

Cultivated fecal indicator bacteria such as Escherichia coli and enterococci are typically used to assess the sanitary quality of recreational waters. However, these indicators suffer from several limitations, such as the length of time needed to obtain results and the fact that they are commensal inhabitants of the gastrointestinal tract of many animals and have fate and transport characteristics dissimilar to pathogenic viruses. Numerous emerging technologies that offer same-day water quality results or pollution source information or that more closely mimic persistence patterns of disease-causing pathogens that may improve water quality management are now available, but data detailing geospatial trends in wastewater across the United States are sparse. We report geospatial trends of cultivated bacteriophage (somatic, F, and total coliphages and GB-124 phage), as well as genetic markers targeting polyomavirus, enterococci, E. coli, Bacteroidetes, and human-associated Bacteroides spp. (HF183/BacR287 and HumM2) in 49 primary influent sewage samples collected from facilities across the contiguous United States. Samples were selected from rural and urban facilities spanning broad latitude, longitude, elevation, and air temperature gradients by using a geographic information system stratified random site selection procedure. Most indicators in sewage demonstrated a remarkable similarity in concentration regardless of location. However, some exhibited predictable shifts in concentration based on either facility elevation or local air temperature. Geospatial patterns identified in this study, or the absence of such patterns, may have several impacts on the direction of future water quality management research, as well as the selection of alternative metrics to estimate sewage pollution on a national scale. [ABSTRACT FROM AUTHOR]

Quantitative PCR (qPCR) assays for human/sewage marker genes have demonstrated sporadic positive results in animal feces despite their high specificities to sewage and human feces. It is unclear whether these positive reactions are caused by true occurrences of microorganisms containing the marker gene (i.e., indicator organisms), or nonspecific amplification (false positive). The distribution patterns of human/sewage indicator organisms in animals have not been explored in depth, which is crucial for evaluating a marker gene's true or false positive reactions. Here we analyzed V6 region 16S ribosomal RNA (rRNA) gene sequences from 257 animal fecal samples and tested a subset of 184 using qPCR for human/sewage marker genes. Overall specificities of human/sewage marker genes within sequencing data were BacV6-21 (99.6%) > Lachno3 (96.9%) HF183 (96.1%, indexed by its inferred V6 sequence). Occurrence of some true cross reactions were associated with atypical compositions of organisms within the genus Blautia or Bacteroides. For human/sewage marker qPCR assays, specificities were HF183/Bac287R (96.7%) > BacV6-21 (96.2%) > Human Bacteroides (HB) (95.6%) > Lachno3 (94.0%). Select assays duplexed with either Escherichia coli (E. coli) or Enterococcus spp. were also validated. Most of the positive qPCR results in animals were low-level and on average two orders of magnitude lower than the copy numbers of E. coli and Enterococcus spp. The lower specificity in qPCR assays compared to sequencing data were mainly caused by amplification of sequences highly similar to the marker gene and not occurrence of the exact marker sequence in animal fecal samples. [ABSTRACT FROM AUTHOR]

Bacteriophage-derived lysin proteins are potentially effective antimicrobials that would benefit from engineered improvements to their bioavailability and specific activity. Here, the catalytic domain of LysEFm5, a lysin with activity against vancomycin-resistant Enterococcus faecium (VRE), was subjected to site-saturation mutagenesis at positions whose selection was guided by sequence and structural information from homologous proteins. A second-order Potts model with parameters inferred from large sets of homologous sequence information was used to predict the average change in the statistical fitness for mutant libraries with diversity at pairs of sites within the secondary catalytic shell. Guided by the statistical fitness, nine double mutant saturation libraries were created and plated on agar containing autoclaved VRE to quickly identify and segregate catalytically active (halo-forming) and inactive (non-halo-forming) variants. High-throughput DNA sequencing of 873 unique variants showed that the statistical fitness was predictive of the retention or loss of catalytic activity (area under the curve [AUC], 0.840 to 0.894), with the inclusion of more diverse sequences in the starting multiple-sequence alignment improving the classification accuracy when pairwise amino acid couplings (epistasis) were considered. Of eight random halo-forming variants selected for more sensitive testing, one showed a 1.8 (0.4)-fold improvement in specific activity and an 11.5 0.8°C increase in melting temperature compared to those of the wild type. Our results demonstrate that a computationally informed approach employing homologous protein information coupled with a mid-throughput screening assay allows for the expedited discovery of lysin variants with improved properties. IMPORTANCE Broad-spectrum antibiotics can indiscriminately kill most bacteria, including commensal species that are a part of the normal human flora. This can potentially lead to the proliferation of drug-resistant bacteria upon elimination of competing species and to unwanted autoimmune effects in patients. Bacteriophagederived lysin proteins are an alternative to conventional antibiotics that have coevolved alongside specific bacterial hosts. Lysins are capable of targeting conserved substrates in the bacterial cell wall essential for its viability. To engineer these proteins to exhibit improved therapeutically relevant properties, homology-guided statistical approaches can be used to identify compelling sites for mutation and to quantify the functional constraints acting on these sites to direct mutagenic library creation. The platform described herein couples this informed approach with a visual plate assay that can be used to simultaneously screen hundreds of mutants for catalytic activity, allowing for the streamlined identification of improved lysin variants. [ABSTRACT FROM AUTHOR]

ABSTRACT In Firmicutes, the nutrient-sensing regulators (p)ppGpp, the effector molecule of the stringent response, and CodY work in tandem to maintain bacterial fitness during infection. Here, we tested (p)ppGpp and codY mutant strains of Enterococcus faecalis in a catheter-associated urinary tract infection (CAUTI) mouse model and used global transcriptional analysis to investigate the relationship of (p)ppGpp and CodY. The absence of (p)ppGpp or single inactivation of codY led to lower bacterial loads in catheterized bladders and diminished biofilm formation on fibrinogen-coated surfaces under in vitro and in vivo conditions. Single inactivation of the bifunctional (p)ppGpp synthetase/hydrolase rel did not affect virulence, supporting previous evidence that the association of (p)ppGpp with enterococcal virulence is not dependent on the activation of the stringent response. Inactivation of codY in the (p)ppGpp0 strain restored E. faecalis virulence in the CAUTI model as well as the ability to form biofilms in vitro. Transcriptome analysis revealed that inactivation of codY restores, for the most part, the dysregulated metabolism of (p)ppGpp0 cells. While a clear linkage between (p)ppGpp and CodY with expression of virulence factors could not be established, targeted transcriptional analysis indicates that a possible association between (p)ppGpp and c-di-AMP signaling pathways in response to the conditions found in the bladder may play a role in enterococcal CAUTI. Collectively, data from this study identify the (p)ppGpp-CodY network as an important contributor to enterococcal virulence in catheterized mouse bladder and support that basal (p)ppGpp pools and CodY promote virulence through maintenance of a balanced metabolism under adverse conditions. IMPORTANCE Catheter-associated urinary tract infections (CAUTIs) are one of the most frequent types of infection found in the hospital setting that can develop into serious and potentially fatal bloodstream infections. One of the infectious agents that frequently causes complicated CAUTI is the bacterium Enterococcus faecalis, a leading cause of hospital-acquired infections that are often difficult to treat due to the exceptional multidrug resistance of some isolates. Understanding the mechanisms by which E. faecalis causes CAUTI will aid in the discovery of new druggable targets to treat these infections. In this study, we report the importance of two nutrient-sensing bacterial regulators, named (p)ppGpp and CodY, for the ability of E. faecalis to infect the catheterized bladder of mice.

ABSTRACT Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, are common constituents of the microbiome as well as increasingly problematic causes of infections. Interestingly, we previously showed that these two species antagonize each other’s virulence and that E. faecalis inhibition of C. albicans was specifically mediated by EntV. EntV is a bacteriocin encoded by the entV (ef1097) locus that reduces C. albicans virulence and biofilm formation by inhibiting hyphal morphogenesis. In this report, we studied the posttranslational modifications necessary for EntV antifungal activity. First, we show that the E. faecalis secreted enzyme gelatinase (GelE) is responsible for cleaving EntV into its 68-amino-acid, active form and that this process does not require the serine protease SprE. Furthermore, we demonstrate that a disulfide bond that forms within EntV is necessary for antifungal activity. Abrogating this bond by chemical treatment or genetic modification rendered EntV inactive against C. albicans. Moreover, we identified the likely catalyst of this disulfide bond, a previously uncharacterized thioredoxin within the E. faecalis genome called DsbA. Loss of DsbA, or disruption of its redox-active cysteines, resulted in loss of EntV antifungal activity. Finally, we show that disulfide bond formation is not a prerequisite for cleavage; EntV cleavage proceeded normally in the absence of DsbA. In conclusion, we present a model in which following secretion, EntV undergoes disulfide bond formation by DsbA and cleavage by GelE in order to generate a peptide capable of inhibiting C. albicans. IMPORTANCE Enterococcus faecalis and Candida albicans are among the most important and problematic pathobionts, organisms that normally are harmless commensals but can cause dangerous infections in immunocompromised hosts. In fact, both organisms are listed by the Centers for Disease Control and Prevention as serious global public health threats stemming from the increased prevalence of antimicrobial resistance. The rise in antifungal resistance is of particular concern considering the small arsenal of currently available therapeutics. EntV is a peptide with antifungal properties, and it, or a similar compound, could be developed into a therapeutic alternative, either alone or in combination with existing agents. However, to do so requires understanding what properties of EntV are necessary for its antifungal activity. In this work, we studied the posttranslational processing of EntV and what modifications are necessary for inhibition of C. albicans in order to fill this gap in knowledge.

ABSTRACT Teixobactin is a new antimicrobial of significant interest. It is active against a number of multidrug-resistant pathogens, including Staphylococcus aureus and Enterococcus faecalis, with no reported mechanisms of teixobactin resistance. However, historically, mechanisms of resistance always exist and arise upon introduction of a new antimicrobial into a clinical setting. Therefore, for teixobactin to remain effective long term, we need to understand how mechanisms of resistance could develop. Here we demonstrate that E. faecalis shows a remarkable intrinsic tolerance to high concentrations of teixobactin. This is of critical importance, as antimicrobial tolerance has been shown to precede the development of antimicrobial resistance. To identify potential pathways responsible for this tolerance, we determined the genomewide expression profile of E. faecalis strain JH2-2 in response to teixobactin using RNA sequencing. A total of 573 genes were differentially expressed (2.0-fold log2 change in expression) in response to teixobactin, with genes involved in cell wall biogenesis and division and transport/binding being among those that were the most upregulated. Comparative analyses of E. faecalis cell wall-targeting antimicrobial transcriptomes identified CroRS, LiaRS, and YclRK to be important two-component regulators of antimicrobial-mediated stress. Further investigation of CroRS demonstrated that deletion of croRS abolished tolerance to teixobactin and to other cell wall-targeting antimicrobials. This highlights the crucial role of CroRS in controlling the molecular response to teixobactin. IMPORTANCE Teixobactin is a new antimicrobial with no known mechanisms of resistance. Understanding how resistance could develop will be crucial to the success and longevity of teixobactin as a new potent antimicrobial. Antimicrobial tolerance has been shown to facilitate the development of resistance, and we show that E. faecalis is intrinsically tolerant to teixobactin at high concentrations. We subsequently chose E. faecalis as a model to elucidate the molecular mechanism underpinning teixobactin tolerance and how this may contribute to the development of teixobactin resistance.

In this study, we present the draft genome sequence of the Enterococcus sp. strain SB12, which was isolated from artisanal cheese of the Carpathian region (Ukraine). The de novo assembly produced 64 contigs, with a total length of 2,514,601 bp. Phylogenetic analysis revealed its proximity to the Enterococcus faecium strains., Competing Interests: The authors declare no conflict of interest.