Decoding the complexity of delayed wound healing following Enterococcus faecalis infection.

Wound infections are highly prevalent and can lead to delayed or failed healing, causing significant morbidity and adverse economic impacts. These infections occur in various contexts, including diabetic foot ulcers, burns, and surgical sites. Enterococcus faecalis is often found in persistent non-healing wounds, but its contribution to chronic wounds remains understudied. To address this, we employed single-cell RNA sequencing (scRNA-seq) on infected wounds in comparison to uninfected wounds in a mouse model. Examining over 23,000 cells, we created a comprehensive single-cell atlas that captures the cellular and transcriptomic landscape of these wounds. Our analysis revealed unique transcriptional and metabolic alterations in infected wounds, elucidating the distinct molecular changes associated with bacterial infection compared to the normal wound healing process. We identified dysregulated keratinocyte and fibroblast transcriptomes in response to infection, jointly contributing to an anti-inflammatory environment. Notably, E. faecalis infection prompted a premature, incomplete epithelial-mesenchymal transition in keratinocytes. Additionally, E. faecalis infection modulated M2-like macrophage polarization by inhibiting pro-inflammatory resolution in vitro, in vivo, and in our scRNA-seq atlas. Furthermore, we discovered macrophage crosstalk with neutrophils, which regulates chemokine signaling pathways, while promoting anti-inflammatory interactions with endothelial cells. Overall, our findings offer new insights into the immunosuppressive role of E. faecalis in wound infections.

If wounds get infected, they heal much more slowly, sometimes leading to skin damage and other complications, including disseminated infections or even amputation. Infections can happen in many types of wounds, ranging from ulcers in patients with diabetes to severe burns. If infections are not cleared quickly, the wounds can become 'chronic' and are unable to heal without intervention. Enterococcus faecalis is a type of bacteria that normally lives in the gut. Within that environment, in healthy people, it is not harmful. However, if it comes into contact with wounds ­ particularly diabetic ulcers or the site of a surgery ­ it can cause persistent infections and prevent healing. Although researchers are beginning to understand how E. faecalis initially colonises wounds, the biological mechanisms that transform these infections into chronic wounds are still largely unknown. Celik et al. therefore set out to investigate exactly how E. faecalis interferes with wound healing. To do this, Celik et al. looked at E. faecalis-infected wounds in mice and compared them to uninfected ones. Using a genetic technique called single-cell RNA sequencing, Celik et al. were able to determine which genes were switched on in individual skin and immune cells at the site of the wounds. This in turn allowed the researchers to determine how those cells were behaving in both infected and uninfected conditions. The experiments revealed that when E. faecalis was present in wounds, several important cell types in the wounds did not behave normally. For example, although the infected skin cells still underwent a change in behaviour required for healing (called an epithelial-mesenchymal transition), the change was both premature and incomplete. In other words, the skin cells in infected wounds started changing too early and did not finish the healing process properly. E. faecalis also changed the way macrophages and neutrophils worked within the wounds. These are cells in our immune system that normally promote inflammation, a process involved in both uninfected wounds or during infections and is a key part of wound healing when properly controlled. In the E. faecalis-infected wounds, these cells' inflammatory properties were suppressed, making them less helpful for healing. These results shed new light on how E. faecalis interacts with skin cells and the immune system to disrupt wound healing. Celik et al. hope that this knowledge will allow us to find new ways to target E. faecalis infections, and ultimately develop treatments to help chronic wounds heal better and faster.

Coaggregated E. faecalis with F. nucleatum regulated environmental stress responses and inflammatory effects.

To investigate the cell-cell interactions of intergeneric bacterial species, the study detected the survival of Enterococcus faecalis (Ef) under monospecies or coaggregation state with Fusobacterium nucleatum subsp. polymorphum (Fnp) in environmental stress. Ef and Fnp infected the human macrophages with different forms (Ef and Fnp monospecies, Ef-Fnp coaggregates, Ef + Fnp cocultures) for exploring the immunoregulatory effects and the relevant molecular mechanisms. Meanwhile, the transcriptomic profiles of coaggregated Ef and Fnp were analyzed. Ef was shown to coaggregate with Fnp strongly in CAB within 90 min by forming multiplexes clumps. Coaggregation with Fnp reinforced Ef resistance against unfavorable conditions including alkaline, hypertonic, nutrient-starvation, and antibiotic challenges. Compared with monospecies and coculture species, the coaggregation of Ef and Fnp significantly facilitates both species to invade dTHP-1 cells and aid Ef to survive within the cells. Compared with coculture species, dual-species interaction of Ef and Fnp significantly decreased the levels of pro-inflammatory cytokines IL-6, TNF-α, and chemokines MCP-1 secreted by dTHP-1 cells and lessened the phosphorylation of p38, JNK, and p65 signaling pathways. The transcriptome sequencing results showed that 111 genes were differentially expressed or Ef-Fnp coaggregated species compared to Ef monospecies; 651 genes were differentially expressed for Fnp when coaggregation with Ef. The analysis of KEGG pathway showed that Ef differentially expressed genes (DEGs) were enriched in quorum sensing and arginine biosynthesis pathway; Fnp DEGs were differentially concentrated in lipopolysaccharide (LPS) biosynthesis, biofilm formation, and lysine degradation pathway compared to monospecies. KEY POINTS: • Coaggregated with Fnp aids Ef's survival in environmental stress, especially in root canals after endodontic treatment. • The coaggregation of Ef and Fnp may weaken the pro-inflammatory response and facilitate Ef to evade killed by macrophages. • The coaggregation between Ef and Fnp altered interspecies transcriptional profiles.

Genomic epidemiology reveals multiple mechanisms of linezolid resistance in clinical enterococci in China.

BACKGROUND: Infections caused by linezolid-resistant enterococci (LRE) are clinically difficult to treat and threaten patient health. However, there is a lack of studies on long time-span LRE strains in China. For this reason, our study comprehensively revealed the resistance mechanisms of LRE strains collected in a Chinese tertiary care hospital from 2011 to 2022. METHODS: Enterococcal strains were screened and verified after retrospective analysis of microbial data. Subsequently, 65 LRE strains (61 Enterococcus faecalis and 4 Enterococcus faecium, MIC ≥ 8 µg/ml), 1 linezolid-intermediate Enterococcus faecium (MIC = 4 µg/ml) and 1 linezolid-susceptible Enterococcus faecium (MIC = 1.5 µg/ml) were submitted for whole-genome sequencing (WGS) analysis and bioinformatics analysis. RESULTS: The optrA gene was found to be the most common linezolid resistance mechanism in our study. We identified the wild-type OptrA and various OptrA variants in 98.5% of LRE strains (61 Enterococcus faecalis and 3 Enterococcus faecium). We also found one linezolid-resistant Enterococcus faecium strain carried both optrA and cfr(D) gene, while one linezolid-resistant Enterococcus faecium only harbored the poxtA gene. Most optrA genes (55/64) were located on plasmids, with impB-fexA-optrA, impB-fexA-optrA-erm(A), fexA-optrA-erm(A), and fexA-optrA segments. A minority of optrA genes (9/64) were found on chromosomes with the Tn6674-like platform. Besides, other possible linezolid resistance-associated mechanisms (mutations in the rplC and rplD genes) were also found in 26 enterococcal strains. CONCLUSIONS: Our study suggested that multiple mechanisms of linezolid resistance exist among clinical LRE strains in China.

Insights into the novel Enterococcus faecalis phage: A comprehensive genome analysis.

Enterococcus faecalis, a Gram-positive bacterium, poses a significant clinical challenge owing to its intrinsic resistance to a broad spectrum of antibiotics, warranting urgent exploration of innovative therapeutic strategies. This study investigated the viability of phage therapy as an alternative intervention for antibiotic-resistant E. faecalis, with a specific emphasis on the comprehensive genomic analysis of bacteriophage SAM-E.f 12. The investigation involved whole-genome sequencing of SAM-E.f 12 using Illumina technology, resulting in a robust dataset for detailed genomic characterization. Bioinformatics analyses were employed to predict genes and assign functional annotations. The bacteriophage SAM-E.f 12, which belongs to the Siphoviridae family, exhibited substantial potential, with a burst size of 5.7 PFU/infected cells and a latent period of 20 min. Host range determination experiments demonstrated its effectiveness against clinical E. faecalis strains, positioning SAM-E.f 12 as a precise therapeutic agent. Stability assays underscore resilience across diverse environmental conditions. This study provides a comprehensive understanding of SAM-E.f 12 genomic composition, lytic lifecycle parameters, and practical applications, particularly its efficacy in murine wound models. These results emphasize the promising role of phage therapy, specifically its targeted approach against antibiotic-resistant E. faecalis strains. The nuanced insights derived from this research will contribute to the ongoing pursuit of efficacious phage therapies and offer valuable implications for addressing the clinical challenges associated with E. faecalis infections.

Comparative analysis of proteomic adaptations in Enterococcus faecalis and Enterococcus faecium after long term bile acid exposure.

BACKGROUND: All gastrointestinal pathogens, including Enterococcus faecalis and Enterococcus faecium, undergo adaptation processes during colonization and infection. In this study, we investigated by data-independent acquisition mass spectrometry (DIA-MS) two crucial adaptations of these two Enterococcus species at the proteome level. Firstly, we examined the adjustments to cope with bile acid concentrations at 0.05% that the pathogens encounter during a potential gallbladder infection. Therefore, we chose the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA), as these are the most prominent bile acids. Secondly, we investigated the adaptations from an aerobic to a microaerophilic environment, as encountered after oral-fecal infection, in the absence and presence of deoxycholic acid (DCA). RESULTS: Our findings showed similarities, but also species-specific variations in the response to the different bile acids. Both Enterococcus species showed an IC50 in the range of 0.01- 0.023% for DCA and CDCA in growth experiments and both species were resistant towards 0.05% CA. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis (424 down-expressed proteins with DCA, 376 down-expressed proteins with CDCA) and in E. faecium (362 down-expressed proteins with DCA, 391 down-expressed proteins with CDCA). Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a "general bile acid response". Among these, various subunits of a V-type ATPase, different ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions instead of aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously. CONCLUSIONS: Overall, these findings provide a detailed insight into the proteomic stress response of two Enterococcus species and help to understand the resistance potential and the stress-coping mechanisms of these important gastrointestinal bacteria.

Optimization of the antifungal properties of the bacterial peptide EntV by variant analysis.

Fungal resistance to commonly used medicines is a growing public health threat, and there is a dire need to develop new classes of antifungals. We previously described a peptide produced by Enterococcus faecalis, EntV, that restricts Candida albicans to a benign form rather than having direct fungicidal activity. Moreover, we showed that one 12-amino acid (aa) alpha helix of this peptide retained full activity, with partial activity down to the 10aa alpha helix. Using these peptides as a starting point, the current investigation sought to identify the critical features necessary for antifungal activity and to screen for new variants with enhanced activity using both biofilm and C. elegans infection assays. First, the short peptides were screened for residues with critical activity by generating alanine substitutions. Based on this information, we used synthetic molecular evolution (SME) to rationally vary the specific residues of the 10aa variant in combination to generate a library that was screened to identify variants with more potent antifungal activity than the parent template. Five gain-of-function peptides were identified. Additionally, chemical modifications to the peptides to increase stability, including substitutions of D-amino acids and hydrocarbon stapling, were investigated. The most promising peptides were additionally tested in mouse models of oropharyngeal and systemic candidiasis where their efficacy in preventing infection was demonstrated. The expectation is that these discoveries will contribute to the development of new therapeutics in the fight against antimicrobial resistant fungi. IMPORTANCE: Since the early 1980s, the incidence of disseminated life-threatening fungal infections has been on the rise. Worldwide, Candida and Cryptococcus species are among the most common agents causing these infections. Simultaneously, with this rise of clinical incidence, there has also been an increased prevalence of antifungal resistance, making treatment of these infections very difficult. For example, there are now strains of Candida auris that are resistant to all three classes of currently used antifungal drugs. In this study, we report on a strategy that allows for the development of novel antifungal agents by using synthetic molecular evolution. These discoveries demonstrate that the enhancement of antifungal activity from naturally occurring peptides is possible and can result in clinically relevant agents that have efficacy in multiple in vivo models as well as the potential for broad-spectrum activity.

Fully Characterized Effective Bacteriophages Specific against Antibiotic-Resistant Enterococcus faecalis, the Causative Agent of Dental Abscess.

Background and Objectives: Enterococcus faecalis (E. faecalis) is a primary pathogen responsible for dental abscesses, which cause inflammation and pain when trapped between the crown and soft tissues of an erupted tooth. Therefore, this study aims to use specific phages as an alternative method instead of classical treatments based on antibiotics to destroy multidrug-resistant E. faecalis bacteria for treating dental issues. Materials and Methods: In the current study, twenty-five bacterial isolates were obtained from infected dental specimens; only five had the ability to grow on bile esculin agar, and among these five, only two were described to be extensive multidrug-resistant isolates. Results: Two bacterial isolates, Enterococcus faecalis A.R.A.01 [ON797462.1] and Enterococcus faecalis A.R.A.02, were identified biochemically and through 16S rDNA, which were used as hosts for isolating specific phages. Two isolated phages were characterized through TEM imaging, which indicated that E. faecalis_phage-01 had a long and flexible tail, belonging to the family Siphoviridae, while E. faecalis_phage-02 had a contractile tail, belonging to the family Myoviridae. Genetically, two phages were identified through the PCR amplification and sequencing of the RNA ligase of Enterococcus phage vB_EfaS_HEf13, through which our phages shared 97.2% similarity with Enterococcus phage vB-EfaS-HEf13 based on BLAST analysis. Furthermore, through in silico analysis and annotations of the two phages' genomes, it was determined that a total of 69 open reading frames (ORFs) were found to be involved in various functions related to integration excision, replication recombination, repair, stability, and defense. In phage optimization, the two isolated phages exhibited a high specific host range with Enterococcus faecalis among six different bacterial hosts, where E. faecalis_phage-01 had a latent period of 30 min with 115.76 PFU/mL, while E. faecalis_phage-02 had a latent period of 25 min with 80.6 PFU/mL. They were also characterized with stability at wide ranges of pH (4-11) and temperature (10-60 °C), with a low cytotoxic effect on the oral epithelial cell line at different concentrations (1000-31.25 PFU/mL). Conclusions: The findings highlight the promise of phage therapy in dental medicine, offering a novel approach to combating antibiotic resistance and enhancing patient outcomes. Further research and clinical trials will be essential to fully understand the therapeutic potential and safety profile of these bacteriophages in human populations.

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis.

Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.

Fibrinolytic-deficiencies predispose hosts to septicemia from a catheter-associated UTI.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis, a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI.

Reciprocal regulation of enterococcal cephalosporin resistance by products of the autoregulated yvcJ-glmR-yvcL operon enhances fitness during cephalosporin exposure.

Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal yvcJ-glmR-yvcL gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the yvcJ-glmR-yvcL operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in E. faecalis, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of Bacillus subtilis, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria.

Prevalence and virulence factors of haemolytic Enterococcus faecalis isolated from root filled teeth associated with periradicular lesions: A laboratory investigation in Thailand.

AIM: Previous endodontic research has provided limited understanding of the prevalence and roles of haemolytic and non-haemolytic Enterococcus faecalis strains in root filled teeth. This study aimed to determine the prevalence of these strains in root filled teeth with periradicular lesions and investigate their associated virulence factors. METHODOLOGY: A total of 36 root canal samples were collected from 36 subjects. The prevalence of E. faecalis was determined using culture and PCR methods. Antibiotic susceptibility of haemolytic and non-haemolytic E. faecalis strains was assessed using the broth dilution assay. The cytokine stimulation in periodontal ligament (PDL) cells and neutrophil migration were evaluated using real-time PCR and migration assay, respectively. Cell invasion ability of the strains was assessed using a cell culture model. Additionally, the virulence gene expression of the haemolytic and non-haemolytic strains was investigated using real-time PCR. The Mann-Whitney U and Spearman's ρ tests were used to examine the significant difference between the two strains and to analyse the correlation between phenotype and gene expression, respectively. RESULTS: Enterococcus faecalis was detected in 33.3% and 88.9% of samples by culture and real-time PCR, respectively. Haemolytic strains were found in 36.4% of subjects. Non-haemolytic strains exhibited susceptibility to erythromycin and varying susceptibility to tetracycline, while all haemolytic strains were resistant to both antibiotics. Haemolytic strains significantly upregulated the expression of IL-8, OPG and RANKL in PDL cells (p < .05). Notably, the fold increases in these genes were higher: IL-8 (556.1 ± 82.9 vs. 249.6 ± 81.8), OPG (2.2 ± 0.5 vs. 1.3 ± 0.2) and RANKL (1.8 ± 0.3 vs. 1.2 ± 0.1). Furthermore, haemolytic strains had a greater effect on neutrophil migration (68.7 ± 15.2% vs. 46.9 ± 11.4%) and demonstrated a higher level of internalization into oral keratinocyte cells (68.6 ± 0.4% vs. 33.8 ± 0.5%) (p < .05). They also showed enhanced expression of virulence genes associated with haemolysin, surface proteins, collagen-binding and aggregation substances. Gelatinase activity was only detectable in non-haemolytic strains. CONCLUSIONS: This study revealed that haemolytic strains E. faecalis possessed enhanced abilities in host invasion and a higher abundance of virulence factors, suggesting their potential contribution to more severe disease manifestations.

Possibility of transfer and activation of 'silent' tetracycline resistance genes among Enterococcus faecalis under high-pressure processing.

In this study, the tetracycline resistance of Enterococcus faecalis strains isolated from food was determined and molecular analyses of the resistance background were performed by determining the frequency of selected tetracycline resistance genes. In addition, the effect of high-pressure stress (400 and 500 MPa) on the expression of selected genes encoding tetracycline resistance was determined, as well as changes in the frequency of transfer of these genes in isolates showing sensitivity to tetracyclines. In our study, we observed an increase in the expression of genes encoding tetracyclines, especially the tet(L) gene, mainly under 400 MPa pressure. The study confirmed the possibility of transferring genes encoding tetracyclines such as tet(M), tet(L), tet(K), tet(W) and tet(O) by horizontal gene transfer in both control strains and exposed to high-pressure. Exposure of the strains to 400 MPa pressure had a greater effect on the possibility of gene transfer and expression than the application of a higher-pressure. To our knowledge, this study for the first time determined the effect of high-pressure stress on the expression of selected genes encoding tetracycline resistance, as well as the possibility and changes in the frequency of transfer of these genes in Enterococcus faecalis isolates showing sensitivity to tetracyclines and possessing silent genes. Due to the observed possibility of increased expression of some of the genes encoding tetracycline resistance and the possibility of their spread by horizontal gene transfer to other microorganisms in the food environment, under the influence of high-pressure processing in strains phenotypically susceptible to this antibiotic, it becomes necessary to monitor this ability in isolates derived from foods.

Mapping the widespread distribution and transmission dynamics of linezolid resistance in humans, animals, and the environment.

BACKGROUND: The rise of linezolid resistance has been widely observed both in clinical and non-clinical settings. However, there were still data gaps regarding the comprehensive prevalence and interconnections of linezolid resistance genes across various niches. RESULTS: We screened for potential linezolid resistance gene reservoirs in the intestines of both humans and animals, in meat samples, as well as in water sources. A total of 796 bacteria strains out of 1538 non-duplicated samples were identified to be positive for at least one linezolid resistance gene, optrA, poxtA, cfr, and cfr(D). The prevalence of optrA reached 100% (95% CI 96.3-100%) in the intestines of pigs, followed by fish, ducks, and chicken at 77.5% (95% CI 67.2-85.3%), 62.0% (95% CI 52.2-70.9%), and 61.0% (95% CI 51.2-70.0%), respectively. The meat and water samples presented prevalences of 80.0% (95% CI 70.6-87.0%) and 38.0% (95% CI 25.9-51.9%), respectively. The unreported prevalence of the cfr(D) gene was also relatively higher at 13.0% (95% CI 7.8-21.0%) and 19.0% (95% CI 10.9-25.6%) for the feces samples of ducks and pigs, respectively. Enterococci were the predominant hosts for all genes, while several non-enterococcal species were also identified. Phylogenetic analysis revealed a significant genetic distance among linezolid resistance gene reservoirs, with polyclonal structures observed in strains within the same niche. Similar genetic arrays harboring assorted insertion sequences or transposons were shared by reservoirs displaying heterogeneous backgrounds, though large diversity in the genetic environment of linezolid resistance genes was also observed. CONCLUSIONS: The linezolid resistance genes were widespread among various niches. The horizontal transfer played a crucial role in driving the circulation of linezolid resistance reservoirs at the human-animal-environment interfaces. Video Abstract.

Genome-wide analysis of acid tolerance genes of Enterococcus faecalis with RNA-seq and Tn-seq.

Enterococcus faecalis, a formidable nosocomial and community-acquired opportunistic pathogen, can persist a wide range of extreme environments, including low pH and nutrient deficiency. Clarifying the survival mechanism of E. faecalis in low-pH conditions is the key to combating the infectious diseases caused by E. faecalis. In this study, we combined transcriptome profiling (RNA-seq) and transposon insertion sequencing (TIS) to comprehensively understand the genes that confer these features on E. faecalis. The metadata showed that genes whose products are involved in cation transportation and amino acid biosynthesis were predominantly differentially expressed under acid conditions. The products of genes such as opp1C and copY reduced the hydrion concentration in the cell, whereas those of gldA2, gnd2, ubiD, and ubiD2 mainly participated in amino metabolism, increasing matters to neutralize excess acid. These, together with the folE and hexB genes, which are involved in mismatch repair, form a network of E. faecalis genes necessary for its survival under acid conditions.

IMPORTANCE: As a serious nosocomial pathogen, Enterococcus faecalis was considered responsible for large numbers of infections. Its ability to survive under stress conditions, such as acid condition and nutrient deficiency was indispensable for its growth and infection. Therefore, understanding how E. faecalis survives acid stress is necessary for the prevention and treatment of related diseases. RNA-seq and TIS provide us a way to analyze the changes in gene expression under such conditions.

VirulenceFinder for Enterococcus faecium and Enterococcus lactis: an enhanced database for detection of putative virulence markers by using whole-genome sequencing data.

Enterococcus faecium (Efm) is a leading cause of hospital-associated (HA) infections, often enriched in putative virulence markers (PVMs). Recently, the Efm clade B was assigned as Enterococcus lactis (Elts), which usually lack HA-Efm infection markers. Available databases for extracting PVM are incomplete and/or present an intermix of genes from Efm and Enterococcus faecalis, with distinct virulence profiles. In this study, we constructed a new database containing 27 PVMs [acm, scm, sgrA, ecbA, fnm, sagA, hylEfm, ptsD, orf1481, fms15, fms21-fms20 (pili gene cluster 1, PGC-1), fms14-fms17-fms13 (PGC-2), empA-empB-empC (PGC-3), fms11-fms19-fms16 (PGC-4), ccpA, bepA, gls20-glsB1, and gls33-glsB] from nine reference genomes (seven Efm + two Elts). The database was validated against these reference genomes and further evaluated using a collection of well-characterized Efm (n = 43) and Elts (n = 7) control strains, by assessing PVM presence/absence and its variants together with a genomic phylogeny constructed as single-nucleotide polymorphisms. We found a high concordance between the phylogeny and in silico findings of the PVM, with Elts clustering separately and mostly carrying Elts-specific PVM gene variants. Based on our validation results, we recommend using the database with raw reads instead of assemblies to avoid missing gene variants. This newly constructed database of 27 PVMs will enable a more comprehensive characterization of Efm and Elts based on WGS data. The developed database exhibits scalability and boasts a range of applications in public health, including diagnostics, outbreak investigations, and epidemiological studies. It can be further used in risk assessment for distinguishing between safe and unsafe enterococci.IMPORTANCEThe newly constructed database, consisting of 27 putative virulence markers, is highly scalable and serves as a valuable resource for the comprehensive characterization of these closely related species using WGS data. It holds significant potential for various public health applications, including hospital outbreak investigations, surveillance, and risk assessment for probiotics and feed additives.

Assessment of Zn and Cu in piglets' liver and kidney: impact in fecal Enterococcus spp.?

Zinc and copper have been used as growth promotors in alternative to antibiotics in pig's diet. The aim was the ascertainment of the Zn and Cu concentrations in piglets' liver and kidney and their impact in the reduced susceptibility to Zn, Cu, and antibiotics in enterococci, used as microbiota biomarker. Zn and Cu were determined in the livers and kidneys of 43 piglets slaughtered in Portugal, by flame atomic absorption spectrometry. Enterococci were isolated from feces for determining the identification of species (E. faecalis, E. faecium, and Enterococcus spp.); susceptibility to vancomycin, ciprofloxacin, linezolid, tigecycline, ampicillin, imipenem, and metals; and Cu tolerance genes. In piglets with Zn and Cu high or toxic levels, enterococci had reduced susceptibility to ions, reinforced by the presence of Cu tolerance genes and by resistance to antibiotics. The study relevance is to show the relationship between these metals' levels and decreased susceptibility to Cu, Zn, and antibiotics by enterococci. From the results, it could be supposed that the piglets were being fed with high doses of Zn and Cu which could select more resistant bacteria to both antibiotics and metals that could spread to environment and humans.

Global diversity of enterococci and description of 18 previously unknown species.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

Distribution of Virulence Genes among Enterococcus species Isolated from Patients of Urinary Tract Infection Attended in a Tertiary Care Hospital of Bangladesh.

Enterococcus species was frequently considered to be commensal organisms but last few decades it has emerged as an important cause of health care associated infections. The presence of virulent genes is one of a key factor for which Enterococcus spp. is gaining attention. In this study, we aim to determine the frequency of virulence genes in uropathogenic Enterococcus species. A total of 46 Enterococcus strains isolated from January 2017 to December 2017. Urine samples were collected from adult clinically suspected urinary tract infected patients from the inpatient and outpatient department of Dhaka Medical College Hospital, Bangladesh irrespective of sex and antibiotic intake. Potential virulence genes such as asa, esp, ace, ebp, cyl, gelE, pilA, pilB, sprE, scm, fms8, ecbA and hyl were detected by PCR using specific primers. Among 46 culture positive Enterococcus, 33(71.74%) were E. faecalis, 11(23.91%) were E. faecium, 2(4.35%) were unidentified. Of the 44 identified Enterococci (33 E. faecalis and 11 E. faecium), 43(97.73%) were positive for pilB, 41(93.18%) for both scm and fms8, 39(88.64%) were positive for ebp, 34(77.27%) for gelE, 32(72.78%) for esp, 31(70.45%) for ecbA, 30(68.18%) for sprE, 28(63.67%) for pilA, 25(56.82%) for ace, 21(47.73%) for cyl, 20(45.45%) for asa and 3(6.82%) for hyl gene. Different virulence factors could be associated with the pathogenicity of E. faecalis and E. faecium and these genes are extensively available among the Enterococcus species.

IS6 family insertion sequences promote optrA dissemination between plasmids varying in transfer abilities.

Plasmids are the primary vectors for intercellular transfer of the oxazolidinone and phenicol cross-resistance gene optrA, while insertion sequences (ISs) are mobile genetic elements that can mobilize plasmid-borne optrA intracellularly. However, little is known about how the IS-mediated intracellular mobility facilitates the dissemination of the optrA gene between plasmid categories that vary in transfer abilities, including non-mobilizable, mobilizable, and conjugative plasmids. Here, we performed a holistic genomic study of 52 optrA-carrying plasmids obtained from searches guided by the Comprehensive Antibiotic Resistance Database. Among the 132 ISs identified within 10 kbp from the optrA gene in the plasmids, IS6 family genes were the most prevalent (86/132). Homologous gene arrays containing IS6 family genes were shared between different plasmids, especially between mobilizable and conjugative plasmids. All these indicated the central role of IS6 family genes in disseminating plasmid-borne optrA. Thirty-three of the 52 plasmids were harbored by Enterococcus faecalis found mainly in humans and animals. By Nanopore sequencing and inverse PCR, the potential of the enterococcal optrA to be transmitted from a mobilizable plasmid to a conjugative plasmid mediated by IS6 family genes was further confirmed in Enterococcus faecalis strains recovered from the effluents of anaerobic digestion systems for treating chicken manure. Our findings highlight the increased intercellular transfer abilities and dissemination risk of plasmid-borne optrA gene caused by IS-mediated intracellular mobility, and underscore the importance of routinely monitoring the dynamic genetic contexts of clinically important antibiotic resistance genes to effectively control this critical public health threat. KEY POINTS: • IS6 was prevalent in optrA-plasmids varying in intercellular transfer abilities. • Enterococcal optrA-plasmids were widespread among human, animal, and the environment. • IS6 elevated the dissemination risk of enterococcal optrA-plasmids.

Whole-genome long-read sequencing to unveil Enterococcus antimicrobial resistance in dairy cattle farms exposed a widespread occurrence of Enterococcus lactis.

Enterococcus 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.