Prevalence and Molecular Characteristics of Enterococci Isolated from Clinical Bovine Mastitis Cases in Ningxia.

Purpose: This study aimed to investigate the prevalence and genetic characterization of enterococcal isolates (Enterococcus faecalis, Enterococcus faecium and Enterococcus hirae) isolated from clinical bovine mastitis cases in Ningxia, China. Patients and Methods: The enterococci were identified by 16S rRNA amplification and sequencing. Antimicrobial resistance was determined by disc diffusion method. Virulence and antimicrobial resistance genes were detected by PCR assays. Results: Overall, 198 enterococcal isolates were identified from 2897 mastitis samples, including 137 (4.7%) E. faecalis, 50 (1.7%) E. faecium and 11 (0.4%) E. hirae. E. faecalis, E. faecium and E. hirae isolates showed high resistance to tetracycline (92.7%, 68.0%, 90.9%), followed by erythromycin (86.9%, 76.0%, 72.7%). The multidrug-resistant strains of E. faecalis and E. faecium were 29 (21.2%) and 13 (26.0%), respectively. The resistance of E. faecalis, E. faecium and E. hirae isolates to tetracycline is mainly attributed to the presence of tetL (alone or combined with tetM and/or tetK), the erythromycin resistance to ermB (alone or combined with ermC and/or ermA). Moreover, cpd (94.2%), gelE (77.4%), efaAfs (93.4%), and esp (79.6%) were the most common virulence genes in E. faecalis. In E. faecium, except for the gene efaAfs (82.0%), other virulence genes are rarely found. Only two strains of E. hirae carrying asa1 gene were detected. Conclusion: The results of this study can provide a reference for the prevention and treatment of bovine mastitis caused by enterococci.

Clinical Features and Genomic Epidemiology of Bloodstream Infections due to Enterococcal Species Other Than Enterococcus faecalis or E. faecium in Patients With Cancer.

Background: Non-Enterococcus faecium, non-E. faecalis (NFF) enterococci are a heterogeneous group of clinically pathogenic enterococci that include species with intrinsic low-level vancomycin resistance. Patients with cancer are at increased risk for bacteremia with NFF enterococci, but their clinical and molecular epidemiology have not been extensively described. Methods: We conducted a retrospective review of all patients (n = 70) with NFF bacteremia from 2016 to 2022 at a major cancer center. The main outcomes assessed were 30-day mortality, microbiological failure (positive blood cultures for ≥4 days), and recurrence of bacteremia (positive blood culture <14 days after clearance). Whole-genome sequencing was performed on all available NFF (n = 65). Results: Patients with hematological malignancies made up 56% of the cohort (77% had leukemia). The majority of solid malignancies (87%) were gastrointestinal in origin. The majority of infections (83%) originated from an intra-abdominal source. The most common NFF species were E. gallinarum (50%) and E. casseliflavus (30%). Most (61%) patients received combination therapy. Bacteremia recurred in 4.3% of patients, there was a 30-day mortality of 23%, and 4.3% had microbiological failure. E. gallinarum and E. casseliflavus isolates were genetically diverse with no spatiotemporal clustering to suggest a single strain. Frequencies of ampicillin resistance (4.3%) and daptomycin resistance (1.9%) were low. Patients with hematologic malignancy had infections with NFF enterococci that harbored more resistance genes than patients with solid malignancy (P = .005). Conclusions: NFF bacteremia is caused by a heterogeneous population of isolates and is associated with significant mortality. Hematological malignancy is an important risk factor for infection with NFF resistant to multiple antibiotics.

Activity-based protein profiling of serine hydrolases and penicillin-binding proteins in Enterococcus faecium.

Enterococcus faecium is a gut commensal bacterium which is gaining increasing relevance as an opportunistic, nosocomial pathogen. Its high level of intrinsic and acquired antimicrobial resistance is causing a lack of treatment options, particularly for infections with vancomycin-resistant strains, and prioritizes the identification and functional validation of novel druggable targets. Here, we use activity-based protein profiling (ABPP), a chemoproteomics approach using functionalized covalent inhibitors, to detect active serine hydrolases across 11 E. faecium and Enterococcus lactis strains. Serine hydrolases are a big and diverse enzyme family, that includes known drug targets such as penicillin-binding proteins (PBPs), whereas other subfamilies are underexplored. Comparative gel-based ABPP using Bocillin-FL revealed strain- and growth condition-dependent variations in PBP activities. Profiling with the broadly serine hydrolase-reactive fluorescent probe fluorophosphonate-TMR showed a high similarity across E. faecium clade A1 strains, but higher variation across A2 and E. lactis strains. To identify these serine hydrolases, we used a biotinylated probe analog allowing for enrichment and identification via liquid chromatography-mass spectrometry. We identified 11 largely uncharacterized targets (α,ß-hydrolases, SGNH-hydrolases, phospholipases, and amidases, peptidases) that are druggable and accessible in live vancomycin-resistant E. faecium E745 and may possess vital functions that are to be characterized in future studies.

Analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria.

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

The risk of viable but non-culturable (VBNC) enterococci and antibiotic resistance transmission during simulated municipal sludge composting.

Sludge composting is a sludge resource utilization method that can reduce pollutants, such as pathogens. Enterococci are regarded as more reliable and conservative indicators of pathogen inactivation than fecal coliforms, which are typically used as indicators of fecal pollution. Non-spore pathogenic bacteria may enter a viable but non-culturable (VBNC) state during composting, leading to residual risk. The VBNC status of bacteria is related to their survival during composting. However, the survival mechanisms of enterococci during sludge composting remain unclear. Therefore, this study aimed to investigate the VBNC state of enterococci in different phases of simulated sludge composting and the fate of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) during the composting process. This study is expected to provide a basis for subsequent exploration of possible methods to completely inactivate enterococci and reduce ARGs during sludge composting. Culturable enterococci were reduced in the thermophilic phase of sludge composting, but the proportion of VBNC subpopulation increased. It was reported for the first time that most VBNC enterococci were killed by extending the cooling phase of sludge compost, and by prolonging the cooling phase the types of ARG were reduced. However, there was a certain quantity (approximately 104/g dry weight) of culturable and VBNC enterococci in the compost products. In addition, MGEs and ARGs exist in both bacteria and compost products, leading to the risk of spreading antibiotic-resistant bacteria and antibiotic resistance when sludge compost products are used.

Spatio-Temporal Variation in the Exceedance of Enterococci in Lake Burley Griffin: An Analysis of 16 Years' Recreational Water Quality Monitoring Data.

Recreational waterbodies with high levels of faecal indicator bacteria (FIB) pose health risks and are an ongoing challenge for urban-lake managers. Lake Burley Griffin (LBG) in the Australian Capital city of Canberra is a popular site for water-based recreation, but analyses of seasonal and long-term patterns in enterococci that exceed alert levels (>200 CFU per 100 mL, leading to site closures) are lacking. This study analysed enterococci concentrations from seven recreational sites from 2001-2021 to examine spatial and temporal patterns in exceedances during the swimming season (October-April), when exposure is highest. The enterococci concentrations varied significantly across sites and in the summer months. The frequency of the exceedances was higher in the 2009-2015 period than in the 2001-2005 and 2015-2021 periods. The odds of alert-level concentrations were greater in November, December, and February compared to October. The odds of exceedance were higher at the Weston Park East site (swimming beach) and lower at the Ferry Terminal and Weston Park West site compared to the East Basin site. This preliminary examination highlights the need for site-specific assessments of environmental and management-related factors that may impact the public health risks of using the lake, such as inflows, turbidity, and climatic conditions. The insights from this study confirm the need for targeted monitoring efforts during high-risk months and at specific sites. The study also advocates for implementing measures to minimise faecal pollution at its sources.

Emergence of Virulent Extensively Drug-Resistant Vancomycin-Resistant Enterococci Among Diarrheic Pet Animals: A Possible Public Health Threat on the Move.

Background: Vancomycin-resistant enterococci (VRE) have become an increasing public health concern in the past few decades, being associated with serious multidrug-resistant (MDR) infections. This study was conducted to investigate the role of diarrheic pet animals as potential reservoirs for virulent extensively drug-resistant (XDR) VRE and their threat on human health. Materials and Methods: Rectal swabs were collected from 153 diarrheic pet animals (80 dogs and 73 cats). The collected swabs were cultured on CHROMagarTMVRE for the isolation of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium, and then suspected colonies were identified as enterococci after Gram staining, conventional biochemical tests, and molecular techniques. VRE were basically identified using the disk diffusion method; however, molecular identification of vanA and vanB genes was carried out among confirmed VRE isolates. Moreover, three virulence genes (cytolysin A, cylA; enterococcal surface protein, esp; and hyaluronidase, hyl) were investigated in VRE isolates. Thereafter, VRE strains that harbored virulence genes were tested for antimicrobial susceptibility. Results: Eighteen out of 153 animals (11.8%) were positive for VRE, which were obtained from 15% and 8.2% of the examined dogs and cats, respectively. None of the obtained isolates carried the vanA gene, whereas the vanB gene was detected in E. faecalis (4/10) with a prevalence rate (40%). Of the obtained VRE isolates, five possessed esp and/or cylA, while all strains were negative for the hyl gene. Furthermore, four virulent VRE isolates exhibited an XDR pattern, and one isolate was MDR. Conclusion: Diarrheic pet animals could represent a potential zoonotic reservoir for virulent XDR vancomycin-resistant E. faecalis, which may have serious public health implications.

Epidemiology and genetic diversity of linezolid resistant enterococci clinical isolates in Belgium from 2013 to 2021.

OBJECTIVES: Linezolid resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are an emerging health threat as limited therapeutic options remain. The aim was to investigate the epidemiology, resistance mechanisms and genetic diversity of Belgian linezolid resistant enterococci (LRE) isolated between 2013 and 2021 received at the Belgian National Reference Centre (NRC) for Enterococci. METHODS: Linezolid susceptibility testing was performed upon request on 2458 submitted Enterococci strains. Whole genome sequencing was performed on all LRE strains. RESULTS: Seventy-eight LRE human isolates, of which 63 (81%) E. faecalis and 15 (19%) E. faecium strains, were submitted to the Belgian NRC for Enterococci. Of the linezolid resistant E. faecalis strains, 97% harboured the optrA (56% wild-type pE349) and 3% the poxtA gene. Of the linezolid resistant E. faecium strains, 54% harboured the G2576T point mutation in the V domain of the 23S rRNA genes, 23% the poxtA and 23% the optrA gene. Furthermore two E. faecium strains were identified with a combination of two resistance mechanisms ((i) optrA and poxtA and (ii) cfr(B) and G2576T point mutation respectively). Vancomycin resistance was observed in 15% (n=12) of the LRE. ST480 (n=42/63 typed strains, 67%) was the most frequently detected sequence type (STs) in linezolid resistant E. faecalis strains, while ST203 (n=5/15 typed strains, 33%) was the most frequently detected STs in linezolid resistant E. faecium strains. CONCLUSIONS: E. faecalis isolates harbouring optrA were the predominant LRE in Belgium with ST480 as the most prominent MLST. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.

The epidemiology of enterococci in a tertiary hospital and primary healthcare facilities in Fiji (2019-2022).

OBJECTIVES: We analysed 4 y of laboratory data to characterise the species and determine the antimicrobial susceptibility profiles of enterococci as human pathogens in Fiji. The study also investigated the molecular epidemiology amongst the subset of vancomycin-resistant enterococci (VRE). METHODS: This retrospective study reviewed bacteriological data from Colonial War Memorial Hospital (CWMH) and other healthcare facilities in the Central and Eastern divisions of Fiji. Phenotypic, antimicrobial susceptibility and vanA and vanB PCR testing were performed using locally approved protocols. The first clinical isolates per patient with antimicrobial susceptibility testing results in a single year were included in the analysis. Data was analysed using WHONET software and Microsoft Excel. RESULTS: A total of 1817 enterococcal isolates were reported, 1415 from CWMH and 402 from other healthcare facilities. The majority of isolates, 75% (n = 1362) were reported as undifferentiated Enterococcus spp., 17.8% (n = 324) were specifically identified as Enterococcus faecalis and 6.7% (n = 122) as E. faecium. Overall, 10% of the enterococci isolates were from blood cultures. Among isolates from CWMH, <15% of E. faecium were susceptible to ampicillin, and 17.2% were vancomycin resistant. Overall, 874 enterococcal isolates (including the undifferentiated species) were tested against vancomycin, of which 4.8% (n = 42) were resistance. All of the VRE isolates tested (n = 15) expressed vanA genes. CONCLUSIONS: This study demonstrates the clinical importance of VRE, particularly van A E. faecium in the national referral hospital in Fiji. Enhanced phenotypic and molecular surveillance data are needed to better understand enterococci epidemiology and help guide specific infection prevention and control measures and antibiotic prescribing guidelines.

Lusutrombopag as a Repurposing Drug in Combination with Aminoglycosides against Vancomycin-Resistant Enterococcus.

Due to the widespread abuse of antibiotics, drug resistance in Enterococcus has been increasing. However, the speed of antibiotic discovery cannot keep pace with the acquisition of bacterial resistance. Thus, drug repurposing is a proposed strategy to solve the crises. Lusutrombopag (LP) has been approved as a thrombopoietin receptor agonist by the Food and Drug Administration. This study demonstrated that LP exhibited significant antimicrobial activities against vancomycin-resistant Enterococcus in vitro with rare resistance occurrence. Further, LP combined with tobramycin exhibited synergistic antimicrobial effects in vitro and in vivo against Enterococcus. No in vitro or in vivo detectable toxicity was observed when using LP. Mechanism studies indicated that the disrupted proton motive force may account for LP's antimicrobial activity. In summary, these results demonstrate that LP has the previously undocumented potential to serve as an antibacterial agent against refractory infections caused by Enterococcus.

Comparison of efficacy and safety between daptomycin plus ß-lactam and daptomycin monotherapy for bloodstream infections due to gram-positive cocci: A systematic review and meta-analysis.

Objectives: We performed a comprehensive systematic review and meta-analysis to evaluate the clinical or microbiological outcomes and safety of a combination of daptomycin (DAP) and ß-lactams compared to DAP monotherapy in patients with blood stream infection (BSI) due to gram-positive cocci (GPC). Methods: We searched Scopus, PubMed, EMBASE, CINAHL, and Ityuushi databases up to January 30, 2023. Outcomes included all-cause mortality, clinical failure, and creatine phosphokinase (CPK) elevation. Results: Six cohorts or case-control studies fulfilled the inclusion criteria and were included in the final meta-analysis. Combination therapy of DAP and ß-lactams significantly reduced the mortality and clinical failure rate for all BSI due to GPC compared with the DAP monotherapy (mortality, odds ratio [OR] = 0.63, 95 % confidence interval [CI] = 0.41-0.98; clinical failure, OR = 0.42, 95 % CI = 0.22-0.81). In contrast, no significant difference was noted in the incidence of CPK elevation between the two groups (OR = 0.85, 95 % CI = 0.39-1.84). Conclusion: Altogether, combination therapy of DAP and ß-lactams can improve the prognosis for patients with BSI due to GPC compared with DAP alone. Therefore, it should be considered as an option for the empirical treatment of BSI caused by GPC.

Relationship Between COVID-19 and Linezolid-Resistant Enterococci: A Retrospective Single-Center Study.

AIM AND OBJECTIVES: To evaluate the correlation between whether the COVID-19 pandemic turned out to be a great premise for increasing the incidence of linezolid resistance infections. MATERIALS AND METHOD: The current retrospective study included data from March 2018 to March 2023 from a single center. The clinical records of the patients were reviewed to extract clinical data. Data gathered from medical records included demographic information, the type of specimen taken, the organism identified, and its sensitivity. Antibiotic susceptibility testing and bacterial identification are both done using the fully automated VITEK system. RESULTS: The total number of samples collected in all the groups, i.e., Group 1 (PRE-COVID), Group 2 (COVID), and Group 3 (POST-COVID), were 201, 127, and 1315, respectively. Out of a total of 201 samples in Group 1, i.e., from March 2018 to February 2020, 47 (23.38%) samples were collected from blood, 104 (51.74%) samples were collected from urine, and the rest of the samples were collected from other sources (pus, sputum, wound, stool, pleural fluid, etc.). In Group 2, i.e., from March 2020 to February 2021, the total number of samples collected was 127, of which 21 were collected from blood, 86 were from urine, and the remaining 20 samples were from other sources. A total of 1315 samples were collected between March 2021 and February 2023, i.e., in Group 3, 598 samples were collected from blood and 548 samples from urine. The most common isolates in the study were Enterococcus faecalis (35.7%) and Enterococcus faecium (61.0%). CONCLUSION: A new threat seems to be emerging in the era of COVID-19, the Enterococcus genus. Though the mechanism remains unidentified, the viral infection seems to cause changes in the bacterial flora, favoring Enterococcus and increasing gut permeability, which provides the perfect environment for Enterococcus bacteria to develop invasive infections. In our study, the prevalence of linezolid resistance was 18.2% for five years.

The virtue of training: extending phage host spectra against vancomycin-resistant Enterococcus faecium strains using the Appelmans method.

Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra.

Antimicrobial resistance patterns, virulence genes, and biofilm formation in enterococci strains collected from different sources.

BACKGROUND: Currently, antibiotic-resistant strains of Enterococcus are considered to be one of the critical health challenges globally. This study aimed to investigate the antibiotic susceptibility pattern, biofilm formation capacity, and virulence genes of enterococci isolated from different sources. METHODS: In this cross-sectional study, environmental and fecal samples were collected from the hospital environment, volunteers, and hospital staff from October 2018 to August 2019. The isolates were identified by morphological and biochemical tests (gram staining, catalase, bile resistance, esculin hydrolysis, carbohydrate fermentation, growth in 6.5% NaCl, Pyrrolidonyl arylamidase, arginine dehydrolase), and PCR for ddl gene. An antimicrobial susceptibility test was performed by the standard disk agar diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Quantitative microplate assays were used to assess biofilm production. The bacterial DNAs were extracted by alkaline lysis method and polymerase chain reaction technique was used detect the esp, ace, and efaA virulence genes. RESULTS: Out of 145 isolates, 84 (57.9%) were identified as E. faecalis and 61 (42.1%) as E. faecium. Resistance to kanamycin and quinupristin-dalfopristin was 82.1% (69/84) and 85.7% (72/84), respectively, in E. faecalis isolates. Out of 61 E. faecalis isolates, 38 (62.4%) were resistant to kanamycin. Among the E. faecalis isolates, esp was the most dominant virulence gene (73.80%), followed by efaA, and ace, which were detected in 60.71%, and 30.95% isolates, respectively. In total, 68.27% of the strains were biofilm producers. Further, esp and efaA genes were more frequently found among E. faecalis strains with moderate and strong biofilm biomass. CONCLUSIONS: According to the findings of our study, enterococci strains isolated from different samples possess distinctive patterns of virulence genes. The esp, ace, and efaA genes were more prevalent among E. faecalis than E. faecium. Besides, the high level antibiotic resistance of normal flora and environmental enterococci strains is alarming the researchers.

A predictive score for the result of carbapenem-resistant Enterobacterales and vancomycin-resistant enterococci screening.

BACKGROUND: The duration of extensively drug-resistant bacteria (XDR) carriage depends on several factors for which the information can be difficult to recover. AIM: To determine whether past screening and clinical results of patients can predict the results of subsequent screening. METHODS: In total, 256 patients were retrospectively included from 10 healthcare centres in France from January 2014 to January 2022. We created a predictive clearance score, ranging from -5 to +7, that included the number of XDR species and the type of resistance detected in the sample, as well as the time from the last positive sample, the number of previous consecutive negative samples, and obtaining at least one negative PCR result in the collection. This score could be used for the upcoming rectal screening of a patient carrying an XDR as soon as the last screening sample was negative. FINDINGS: The negative predictive value was >99% for score ≤0. The median time to achieve XDR clearance was significantly shorter for a score of 0 (443 days (259-705)) than that based on previously published criteria. CONCLUSION: This predictive score shows high performance for the assessment of XDR clearance. Relative to previous guidelines, it could help to lift specific infection prevention and control measures earlier. Nevertheless, the decision should be made according to other factors, such as antimicrobial use and adherence to hand hygiene.

Repurposing fusidic acid as an antimicrobial against enterococci with a low probability of resistance development.

Drug repurposing constitutes a strategy to combat antimicrobial resistance, by using agents with known safety, pharmacokinetics, and pharmacodynamics. Previous studies have implemented new fusidic acid (FA) front-loading-dose regimens, allowing higher serum levels than those achievable with ordinary doses. As susceptibility breakpoints are affected by serum level, we evaluated the repurposing of FA as an antimicrobial product against enterococci. FA minimum inhibitory concentrations (MICs) against standard enterococci strains; Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 27270 were 2 and 4 µg/mL, respectively. The MIC against 98 enterococcal clinical isolates was ≤ 8 µg/mL; all would be susceptible if categorized according to recalculated breakpoints (≥ 16 µg/mL), based on the serum level achieved using the front-loading regimen. FA administration in vivo, using the BALB/c mouse infection model, significantly reduced bacterial burden by two to three log10 units in the liver and spleen of mice infected with vancomycin-susceptible and -resistant strains. Exposure of the standard enterococcal strains to increasing, but not fixed, FA concentrations resulted in resistant strains (MIC = 128 µg/mL), with thicker cell walls and slower growth rates. Only one mutation (M651I) was detected in the fusA gene of the resistant strain derived from serial passage of E. faecium ATCC 27270, which was retained in the revertant strain after passage in the FA-free medium. In conclusion, FA can be repurposed as an antimicrobial drug against enterococci with a low probability of mutational resistance development, and can be employed for treatment of infections attributable to vancomycin-resistant enterococci.

Exploring the feasibility of bacteriocins EntK1 and EntEJ97s in treatment of systemic vancomycin resistant enterococci infections in mice.

AIMS: Enterocins K1 and EJ97 have specific antimicrobial activity against Enterococcus faecium and Enterococcus faecalis, respectively. The aim of this study was to investigate the utility of these enterocins for in vivo treatment of systemic enterococcal infections. METHODS AND RESULTS: The antimicrobial effect in blood was analysed and compared against the effect in saline. Colony forming unit counts revealed that the enterocins killed all the bacteria within 1 hour. Additionally, the bactericidal effect against E. faecalis was more rapid in blood, indicating a possible synergy between EntEJ97 and blood. Importantly, no enterocin resistant mutants emerged in these experiments. Injecting the enterocins intraperitoneally in an in vivo mouse model and using fluorescence and minimum inhibitory concentration determination to estimate concentrations of the peptides in plasma, indicate that the enterocins exist in circulation in therapeutic concentrations. Alanine aminotransferase detection, and haemolysis analysis indicates that there is no detectable liver damage or haemolytic effect after injection. CONCLUSIONS: The study revealed that EntK1 and EntEJ97 are able to kill all bacteria ex vivo in the presence of blood. In vivo experiments determine that the enterocins exist in circulation in therapeutic concentrations without causing liver damage or haemolysis. Future experiments should test these peptides for treatment of infection in a relevant in vivo model.

Rapid detection of vanB vancomycin-resistant enterococci by laboratory-developed PCR on enrichment broth.

Diagnostic accuracy of laboratory-developed PCR after overnight enrichment for the detection of vanB vancomycin-resistant enterococci was evaluated on 537 rectal swabs. Defining Ct-values of 27-34 (40 samples, 7 % inconclusive), we found an excellent sensitivity of 98,3 % and specificity of 99,7 % for the remaining 497 samples.

Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Discovery of Gambogic acid as an antibacterial adjuvant against vancomycin-resistant enterococci in vitro and in vivo.

BACKGROUND: The emergence and spread of vancomycin-resistant enterococci (VRE) have posed a significant challenge to clinical treatment, underscoring the need to develop novel strategies. As therapeutic options for VRE are limited, discovering vancomycin enhancer is a feasible way of combating VRE. Gambogic acid (GA) is a natural product derived from the resin of Garcinia hanburyi Hook.f. (Clusiaceae), which possesses antibacterial activity. PURPOSE: This study aimed to investigate the potential of GA as an adjuvant to restore the susceptibility of VRE to vancomycin. METHODS: In vitro antibacterial and synergistic activities were evaluated against vancomycin-susceptible and resistant strains by the broth microdilution method for the Minimal Inhibitory Concentrations (MICs) determination, and checkerboard assay and time-kill curve analysis for synergy evaluation. In vivo study was conducted on a mouse multi-organ infection model. The underlying antibacterial mechanism of GA was also explored. RESULTS: GA showed a potent in vitro activity against all tested strains, with MICs ranging from 2 to 4 µg/ml. The combination of GA and vancomycin exhibited a synergistic effect against 18 out of 23 tested VRE strains, with a median fractional inhibitory concentration index (FICI) of 0.254, and demonstrated a synergistic effect in the time-kill assay. The combination therapy exhibited a significant reduction in tissue bacterial load compared with either compound used alone. GA strongly binds to the ParE subunit of topoisomerase IV, a bacterial type II DNA topoisomerase, and suppresses its activity. CONCLUSIONS: The study suggests that GA has a significant antibacterial activity against enterococci, and sub-MIC concentrations of GA can restore the activity of vancomycin against VRE in vitro and in vivo. These findings indicate that GA has the potential to be a new antibacterial adjuvant to vancomycin in the treatment of infections caused by VRE.