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.

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.

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.

Vancomycin-resistant Enterococcus faecium in Japan, 2007-2015: a molecular epidemiology analysis focused on examining strain characteristics over time.

IMPORTANCE: Our study emphasizes the efficacy of whole-genome sequencing (WGS) in addressing outbreaks of vancomycin-resistant enterococci. WGS enables the identification and tracking of resistant bacterial strains, early detection and management of novel infectious disease outbreaks, and the appropriate selection and use of antibiotics. Furthermore, our approach deepens our understanding of how resistant bacteria transfer genes and adapt to their environments or hosts. For modern medicine, these insights have significant implications for controlling infections and effectively managing antibiotic use in the current era, where antibiotic resistance is progressing.

Epidemiology of vancomycin-resistant enterococci in the United Arab Emirates: a retrospective analysis of 12 years of national AMR surveillance data.

Introduction: Enterococci are usually low pathogenic, but can cause invasive disease under certain circumstances, including urinary tract infections, bacteremia, endocarditis, and meningitis, and are associated with peritonitis and intra-abdominal abscesses. Increasing resistance of enterococci to glycopeptides and fluoroquinolones, and high-level resistance to aminoglycosides is a concern. National antimicrobial resistance (AMR) surveillance data for enterococci from the Middle East and North Africa (MENA) and the Gulf region is scarce. Methods: A retrospective 12-year analysis of N = 37,909 non-duplicate diagnostic Enterococcus spp. isolates from the United Arab Emirates (UAE) was conducted. Data was generated by routine patient care during 2010-2021, collected by trained personnel and reported by participating surveillance sites to the UAE National AMR Surveillance program. Data analysis was conducted with WHONET. Results: Enterococcus faecalis was the most commonly reported species (81.5%), followed by Enterococcus faecium (8.5%), and other enterococci species (4.8%). Phenotypically vancomycin-resistant enterococci (VRE) were found in 1.8% of Enterococcus spp. isolates. Prevalence of VRE (%VRE) was highest for E. faecium (8.1%), followed by E. faecalis (0.9%). A significant level of resistance to glycopeptides (%VRE) for these two species has been observed in the majority of observed years [E. faecalis (0-2.2%), 2010: 0%, 2021: 0.6%] and E. faecium (0-14.2%, 2010: 0%, 2021: 5.8%). Resistance to fluoroquinolones was between 17 and 29% (E. faecalis) and was higher for E. faecium (between 42 and 83%). VRE were associated with higher patient mortality (RR: 2.97), admission to intensive care units (RR: 2.25), and increased length of stay (six excess inpatient days per VRE case), as compared to vancomycin-susceptible Enterococcus spp. Discussion: Published data on Enterococcus infections, in particular VRE-infections, in the UAE and MENA region is scarce. Our data demonstrates that VRE-enterococci are relatively rare in the UAE, however showing an increasing resistance trend for several clinically important antibiotic classes, causing a concern for the treatment of serious infections caused by enterococci. This study also demonstrates that VRE were associated with higher mortality, increased intensive care unit admission rates, and longer hospitalization, thus poorer clinical outcome and higher associated costs in the UAE. We recommend the expansion of current surveillance techniques (e.g., local VRE screening), stricter infection prevention and control strategies, and better stewardship interventions. Further studies on the molecular epidemiology of enterococci are needed.

Australian Group on Antimicrobial Resistance (AGAR) Australian Enterococcal Surveillance Outcome Program (AESOP) Bloodstream Infection Annual Report 2022.

From 1 January to 31 December 2022, fifty-five institutions across Australia participated in the Australian Enterococcal Surveillance Outcome Program (AESOP). The aim of AESOP 2022 was to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial resistant, and to characterise the molecular epidemiology of the Enterococcus faecium isolates. Of the 1,535 unique episodes of enterococcal bacteraemia investigated, 92.8% were caused by either E. faecalis (52.9%) or E. faecium (39.9%). Ampicillin and vancomycin resistance were not detected in E. faecalis but were detected in 95.4% and 46.9% of E. faecium respectively. One E. faecalis isolate, with a daptomycin minimum inhibitory concentration (MIC) of 8.0 mg/L, harboured the F478L GdpD mutation. One E. faecium with a daptomycin MIC of 24.0 mg/L harboured the A20D Cls mutation; both mutations are known to be associated with daptomycin resistance. Two E. faecium isolates, one with a linezolid MIC ≥ 256 mg/L and the other with a linezolid MIC of 16 mg/L, harboured the 23S rRNA G2576T mutation, a mutation associated with linezolid resistance in enterococci. Overall, 48.8% of E. faecium harboured either the vanA or the vanB gene, of which 28.0% harboured vanA and 72.0% harboured vanB. The percentage of vancomycin-resistant E. faecium bacteraemia isolates in Australia remains substantially higher than that recorded in most European countries. The E. faecium isolates consisted of 62 multi-locus sequence types (STs); 85.5% of isolates were classified into eight major STs each containing ten or more isolates. All major STs belonged to clonal complex (CC) 17, a major hospital-adapted polyclonal E. faecium cluster. The major STs (ST17, ST78, ST80, ST117, ST555, ST796, ST1421, and ST1424) were each found across most regions of Australia. The predominant ST was ST17, which was identified in all regions. Overall, 53.7% of isolates belonging to the eight major STs harboured the vanA or vanB gene. AESOP 2022 has shown that enterococcal bacteraemia episodes in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin resistant vanA- or vanB-positive E. faecium which have limited treatment options.

The Emergence of Antimicrobial Resistance and Virulence Characteristics in Enterococcus Species Isolated from Bovine Milk.

Enterococcus spp., including E. faecalis and E. faecium, pose risks to dairy farms as opportunistic pathogens. The study evaluates antimicrobial resistance (AMR) and virulence characteristics of Enterococcus spp. isolated from bovine milk. Bile esculin agar was used to assess 1471 milk samples, followed by colony identification, gram staining, catalase tests, and 45 °C incubation. PCR analysis targeted E. faecalis and E. faecium in characteristic Enterococcus spp. colonies, with MALDI-TOF used for negative samples. Multiple tests, including disk diffusion, chromogenic VRE agar for vancomycin resistance, Vancomycin Etest® for MIC determination, and PCR for virulence factors (cylA, esp, efaA, ace, asa1, gelE, and hyl genes), were performed. Out of 100 identified strains, E. durans (30.66%), E. faecium (26.28%), and E. faecalis (18.25%) were predominant. AMR in Enterococcus spp. varied, with the highest rates against rifampicin (27%), tetracycline (20%), and erythromycin (18%). Linezolid (5%), vancomycin, ciprofloxacin, and teicoplanin (3% each) had lower prevalence. E. faecium and E. faecalis showed high AMR to rifampicin, erythromycin, and tetracycline. Thirty-two strains (18.98%) grew on VRE Chromoselect agar, while 4 (2 E. faecalis and 2 E. faecium) showed vancomycin resistance by MIC values. E. faecalis carried gelE (45.5%) and asa1 (36%), and E. gallinarum had 9.1% with the asa1 gene. Detecting resistant Enterococcus in bovine milk supports control strategies for enterococci on dairy farms, highlighting AMR concerns in the food chain.

Corrigendum: Microbiological and clinical characteristics of bloodstream infections in general intensive care unit: A retrospective study.

[This corrects the article DOI: 10.3389/fmed.2022.876207.].

Infant food production environments: A potential reservoir for vancomycin-resistant enterococci non-nosocomial infections.

Enterococcus has been considered one of the most important nosocomial pathogens for human infections, and the hospital environment is an important reservoir for vancomycin-resistant enterococci (VRE) that leads to antimicrobial therapeutic failure. However, infant foods and their production environments could pose risks for the immature population, while this question remains unaddressed. This study conducted an extensive and thorough Enterococcus isolation, VRE risk assessment of the Chinese infant food production chains and additional online-marketing infant foods, including powdered infant formula (PIF) and infant complementary food (ICF). To investigate the prevalence of Enterococcus along infant food chains and commodities, a total of 482 strains of Enterococcus, including E. faecium (n = 363), E. faecalis (n = 84), E. casseliflavus (n = 13), E. mundtii (n = 12), E. gallinarum (n = 4), E. hirae (n = 4), and E. durans (n = 2) were recovered from 459 samples collected from infant food production chains (71/254) and food commodities (67/205). A decreasing trend for Enterococcus detection rate was found in the PIF production chain (PIF-PC), particularly during the preparation of the PIF base powder (From 100 % in raw milk to 8.70 % in end products), while an increasing trend was observed in the ICF production chain (ICF-PC) mainly during the initial processing of farm crops and the further processing of the product (20 % at farm crops increasing to 76.92 % at end products). The result indicated that the PIF-PC process effectively reduced Enterococcus contamination, while the ICF-PC showed the opposite trend. Importantly, eleven VRE isolates were recovered from the infant food production chain, including seven E. casseliflavus isolates carrying vanC2/C3 and four E. gallinarum isolates carrying vanC1. Ten VRE isolates were from food production environments. Collectively, our study demonstrated that infant food production environments represent potential reservoirs for VRE non-nosocomial infections in vulnerable populations.

A Review of Detection Methods for Vancomycin-Resistant Enterococci (VRE) Genes: From Conventional Approaches to Potentially Electrochemical DNA Biosensors.

Vancomycin-resistant Enterococci (VRE) genes are bacteria strains generated from Gram-positive bacteria and resistant to one of the glycopeptides antibiotics, commonly, vancomycin. VRE genes have been identified worldwide and exhibit considerable phenotypic and genotypic variations. There are six identified phenotypes of vancomycin-resistant genes: VanA, VanB, VanC, VanD, VanE, and VanG. The VanA and VanB strains are often found in the clinical laboratory because they are very resistant to vancomycin. VanA bacteria can pose significant issues for hospitalized patients due to their ability to spread to other Gram-positive infections, which changes their genetic material to increase their resistance to the antibiotics used during treatment. This review summarizes the established methods for detecting VRE strains utilizing traditional, immunoassay, and molecular approaches and then focuses on potential electrochemical DNA biosensors to be developed. However, from the literature search, no information was reported on developing electrochemical biosensors for detecting VRE genes; only the electrochemical detection of vancomycin-susceptible bacteria was reported. Thus, strategies to create robust, selective, and miniaturized electrochemical DNA biosensor platforms to detect VRE genes are also discussed.

Australian Group on Antimicrobial Resistance (AGAR) Australian Enterococcal Surveillance Outcome Program (AESOP).

Abstract: From 1 January to 31 December 2021, forty-eight institutions around Australia participated in the Australian Enterococcal Surveillance Outcome Programme (AESOP). The aim of AESOP 2021 was to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial resistant, and to characterise the molecular epidemiology of the Enterococcus faecium isolates. Of the 1,297 unique episodes of enterococcal bacteraemia investigated, 94.4% were caused by either E. faecalis (54.1%) or E. faecium (40.3%). Ampicillin resistance was detected in one E. faecalis isolate and in 89.3% of E. faecium isolates. Vancomycin non-susceptibility was not detected in E. faecalis but was detected in 37.9% of E. faecium. Overall, 39.6% of E. faecium harboured the vanA and/or vanB genes. For the vanA/vanB positive E. faecium isolates, 35.8% harboured the vanA gene and 64.2% the vanB gene. Although the percentage of vancomycin-resistant E. faecium bacteraemia isolates was significantly lower than that reported in the 2020 AESOP report (presumably due to the COVID-19 elective surgery restrictions placed on hospitals), it remains substantially higher than that recorded in most European countries. Isolates of E. faecium consisted of 73 multi-locus sequence types (STs); 77.2% of isolates were classified into seven major STs each containing more than ten isolates. All major STs belonged to clonal cluster (CC) 17, a major hospital-adapted polyclonal E. faecium cluster. The major STs (ST17, ST1424, ST796, ST78, ST80, ST1421 and ST555) were found across most regions of Australia. The predominant ST was ST17 which was identified in all regions except the Northern Territory. Overall, 46.5% of isolates belonging to the seven major STs harboured the vanA or vanB gene. The AESOP 2021 has shown that enterococcal bacteraemia episodes in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin resistant vanA- or vanB-positive E. faecium which have limited treatment options.

Combined Action of Antibiotics and Bacteriocins against Vancomycin-Resistant Enterococci.

Antibiotics have been one of the most important discoveries in the area of applied medical microbiology; however, as a result of various factors, we are currently facing a dramatic and relatively dangerous increase in the number of cases of antibiotic resistance, and the need for new types of antimicrobials continues to grow. New approaches are needed to combat antibiotic-resistant pathogens. Bacteriocins, as part of the group of antimicrobial peptides, can be considered as alternatives and/or complements to known antibiotics. Their narrow spectra of activity can be explored for the control of various pathogens, such as vancomycin-resistant enterococci (VRE), as single therapies or in combination with known antibiotics. In the present study, we isolated bacteriocins from different lactic acid bacteria (LAB) strains, including Enterococcus and Pediococcus, and explored the possible synergistic inhibition of growth by bacteriocins and vancomycin. It was observed in the growth dynamics with previously selected VRE strains that the bacteriocins had a high specificity and a promising inhibitory effect against the VRE strains, and these results were validated by a propidium iodide viability test using flow cytometry. The data obtained indicate that the selected bacteriocins can be used to control VRE in the food industry or even as an alternative treatment to combat infections with antibiotic-resistant bacteria.

Microbiological and Clinical Characteristics of Bloodstream Infections in General Intensive Care Unit: A Retrospective Study.

Background: Bloodstream infections (BSI) are one of the common causes of morbidity and mortality in hospitals; however, the pathogenic spectrum and bacterial antibiotic resistance vary across the world. Therefore, identifying the pathogenic spectrum and changes in bacterial antibiotic resistance is critical in controlling BSI and preventing the irrational use of antibiotics. This study evaluated the microbiological and clinical data of BSI patients in the intensive care unit (ICU) of Tianjin Medical University General Hospital in Tianjin, China, to guide the selection of empirical antibiotic therapy. Methods: This study retrospectively analyzed the distribution and antibiotic resistance of pathogens based on the clinical data of BSI patients presented in the ICU of a tertiary teaching hospital from 2018 to 2020. Test performance for the prediction of pathogen species was assessed by receiver operating characteristic (ROC) analysis. Results: The analysis of the data of 382 BSI cases (10.40 cases per thousand patient day) revealed the most frequently isolated microorganisms to be Klebsiella pneumonia (11.52%), followed by Escherichia coli (9.95%), Staphylococcus epidermidis (9.95%), Candida parapsilosis (8.12%), and Enterococcus faecium (8.12%). Out of the isolated E. coli and K. pneumonia strains, 52.63, and 36.36%, respectively, were extended-spectrum ß-lactamase (ESBL) positive. The antibiotic-resistance rate of the ESBL-positive strains was 30.56% for piperacillin/tazobactam, 5.56% for imipenem, and 11.11% for tigecycline. In addition, most A. baumannii belonged to the group of multidrug-resistant (MDR) strains, with an antibiotic-resistance rate of 90.48% for meropenem and 16.00% for amikacin. However, polymyxin-resistant A. baumannii strains were not detected. Four strains of methicillin-resistant S. aureus (MRSA) (4/21, 19.05%) and one strain of vancomycin-resistant enterococci (VRE) were detected, with a resistance rate of 4.76 and 2.32%, respectively. Among the isolated 55 fungal strains, C. parapsilosis was the most common one (30/55, 56.36%), with an antibiotic-resistance rate of 5.77% for voriconazole, fluconazole, and itraconazole. The presence of amphotericin B-or flucytosine-resistant strains was not observed. Compared with the patients with Gram-positive and fungal pathogens, patients with Gram-negative bacteria exhibited the highest sequential organ failure assessment (SOFA) score (P < 0.001), lowest Glasgow Coma Scale (GCS) (P = 0.010), lowest platelet (PLT) value (P < 0.001), highest plasma creatinine (Cr) value (P = 0.016), and the highest procalcitonin (PCT) value (P < 0.001). The AUC in the ROC curve was 0.698 for the differentiation of Gram-negative BSI from Gram-positive BSI. A cutoff value of 8.47 ng/mL for PCT indicated a sensitivity of 56.9% and a specificity of 75.5%. The AUC in the ROC curve was 0.612 for the differentiation of bacteremia from fungemia. A cutoff value of 4.19 ng/mL for PCT indicated a sensitivity of 56.8% and a specificity of 62.7%. Conclusion: Among the bloodstream infection strains in ICU, Gram-negative bacteria have the highest drug resistance rate, and will cause more serious brain damage, renal function damage and thrombocytopenia. So clinician should pay more attention to the treatment of Gram-negative bacteria in patients with bloodstream infection in ICU. The test index of PCT can be used to distinguish Gram-negative bacteremia from Gram-positive and bacteremia from fungemia but not as an effective indicator, thereby indicating the need for further large-scale research.

A Data-Driven Framework for Identifying Intensive Care Unit Admissions Colonized With Multidrug-Resistant Organisms.

Background: The rising prevalence of multi-drug resistant organisms (MDROs), such as Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE), and Carbapenem-resistant Enterobacteriaceae (CRE), is an increasing concern in healthcare settings. Materials and Methods: Leveraging data from electronic healthcare records and a unique MDRO universal screening program, we developed a data-driven modeling framework to predict MRSA, VRE, and CRE colonization upon intensive care unit (ICU) admission, and identified the associated socio-demographic and clinical factors using logistic regression (LR), random forest (RF), and XGBoost algorithms. We performed threshold optimization for converting predicted probabilities into binary predictions and identified the cut-off maximizing the sum of sensitivity and specificity. Results: Four thousand six hundred seventy ICU admissions (3,958 patients) were examined. MDRO colonization rate was 17.59% (13.03% VRE, 1.45% CRE, and 7.47% MRSA). Our study achieved the following sensitivity and specificity values with the best performing models, respectively: 80% and 66% for VRE with LR, 73% and 77% for CRE with XGBoost, 76% and 59% for MRSA with RF, and 82% and 83% for MDRO (i.e., VRE or CRE or MRSA) with RF. Further, we identified several predictors of MDRO colonization, including long-term care facility stay, current diagnosis of skin/subcutaneous tissue or infectious/parasitic disease, and recent isolation precaution procedures before ICU admission. Conclusion: Our data-driven modeling framework can be used as a clinical decision support tool for timely predictions, characterization and identification of high-risk patients, and selective and timely use of infection control measures in ICUs.

Australian Group on Antimicrobial Resistance (AGAR) Australian Enterococcal Sepsis Outcome Programme (AESOP) Annual Report 2020.

From 1 January to 31 December 2020, forty-nine institutions around Australia participated in the Australian Enterococcal Sepsis Outcome Programme (AESOP). The aims of AESOP 2020 were to determine the proportion of enterococcal bacteraemia isolates in Australia that were antimicrobial-resistant, and to characterise the molecular epidemiology of the E. faecium isolates. Of the 1,230 unique episodes of enterococcal bacteraemia investigated, 93.9% were caused by either E. faecalis (54.2%) or E. faecium (39.7%). Ampicillin resistance was not detected in E. faecalis but was detected in 88.2% of E. faecium . Vancomycin non-susceptibility was detected in 0.2% of E. faecalis and 32.6% of E. faecium . Overall, 35.2% of E. faecium harboured vanA and/or vanB genes. For the vanA/B positive E. faecium isolates, 38.8% harboured the vanA gene, 60.6% the vanB gene, and 0.6% harboured both vanA and vanB . Although the percentage of E. faecium bacteraemia isolates was significantly lower than that detected in the 2019 AESOP (presumably due to the COVID-19 elective surgery restrictions placed on hospitals), it remains substantially higher than that recorded in most European countries. The E. faecium isolates detected consisted of 71 multilocus sequence types (STs), with 81.7% of these isolates classified into eight major STs each containing ten or more isolates. All major STs belonged to clonal cluster 17 (CC17), a major hospital-adapted polyclonal E. faecium cluster. The major STs (ST17, ST1424, ST80, ST796, ST78, ST1421, ST555 and ST117) were found across most regions of Australia. The predominant clone was ST17, which was identified in all regions except the Northern Territory. Overall, 40.9% of isolates belonging to the eight major STs harboured the vanA or vanB gene. The AESOP 2020 has shown enterococcal bacteraemia episodes in Australia are frequently caused by polyclonal ampicillin-resistant high-level gentamicin-resistant vanA - or vanB -positive E. faecium which have limited treatment options.

Factorial design and post-antibiotic sub-MIC effects of linezolid combined with fosfomycin against vancomycin-resistant enterococci.

Background: Antimicrobial drug resistance, including vancomycin-resistant enterococci (VRE), has long been an inescapable clinical problem. If vancomycin loose its therapeutic relevance, a regimen of linezolid combined with fosfomycin may provide an alternative option. Methods: In this study, the in vitro antimicrobial effect of linezolid combined with fosfomycin on several different types of VRE was investigated using a checkerboard method and time-kill assays. Based on the results of the 24 h time-kill assays, a 22 factorial design was then adopted. Finally, the post-antibiotic effect (PAE), post-antibiotic sub-minimum inhibitory concentration effect (PASME), and single sub-minimum inhibitory concentration effect (SME) of a combination of the two drugs on three selected strains was examined. Results: The checkerboard method and factorial design analysis showed that linezolid combined with fosfomycin not only had synergistic and additive effects but also had an interactive effect on VREs. The time-kill assays showed that 1× minimum inhibitory concentration (MIC) of linezolid combined with 1× MIC or 1/4× MIC of fosfomycin had no statistically significant difference in the bactericidal effect against VRE at 24 h (P>0.05). The combination of the two drugs did not significantly extend the PAE or the SME; however, in relation to 1/4× MIC, the combination of the two drugs significantly prolonged the duration of the PASME compared to that of a single drug on VREs (P<0.05; with values of 1.97±0.01, 4.32±0.18, and 1.74±0.13 h, respectively). Conclusions: Our results showed that when linezolid is selected for the treatment of VRE infections, sub-inhibitory concentrations of fosfomycin can be administered at multiple intervals to improve the therapeutic effect.

Genomic Analysis of Multidrug-Resistant Enterococcus faecium Harboring vanA Gene from Wastewater Treatment Plants.

The emergence of vancomycin-resistant Enterococcus faecium (Efm) harboring vanA gene and multidrug-resistant determinants is a relevant public health concern. It is an opportunistic pathogen responsible for nosocomial infections widely distributed in the environment, including wastewater treatment plants (WWTPs). Our study addresses a genomic investigation of vanA-carrying Efm from WWTPs in Brazil. Samples from five WWTPs supplied with sewage from different sources were evaluated. Here we present whole-genome sequencing of eight vanA-Efm isolates performed on Illumina MiSeq platform. All these isolates presented multidrug-resistant profile, and five strains were from treated wastewater. Multiple antimicrobial resistance genes (ARGs) were found, such as aph(3')-IIIa, ant(6')-Ia, erm(B), and msrC, some of them being allocated in plasmids. The virulence profile was predominantly constituted by efaAfm and acm genes and all isolates, except for one, were predicted as human pathogens. Multilocus sequence typing analysis revealed a new allele and five different STs, three previously described (ST32, ST168, and ST253) and two novel ones (ST1893 and ST1894). Six strains belonged to CC17, often associated with hospital outbreaks. As far as our knowledge, no genomic studies of vanA-Efm recovered from WWTPs revealed isolates belonging to CC17 in Brazil. Therefore, our findings point to the environmental spread of Efm carrying multiple ARGs.

Prevalence of Vancomycin-Resistant Enterococci and Antimicrobial Residues in Wastewater and Surface Water.

Due to the extensive use of antimicrobial agents in human and veterinary medicine, residues of various antimicrobials get into wastewater and, subsequently, surface water. On the one hand, a combination of processes in wastewater treatment plants aims to eliminate chemical and biological pollutants; on the other hand, this environment may create conditions suitable for the horizontal transfer of resistance genes and potential selection of antibiotic-resistant bacteria. Wastewater and surface water samples (Morava River) were analyzed to determine the concentrations of 10 antibiotics and identify those exceeding so-called predicted no-effect environmental concentrations (PNECs). This study revealed that residues of five of the tested antimicrobials, namely ampicillin, clindamycin, tetracycline, tigecycline and vancomycin, in wastewater samples exceeded the PNEC. Vancomycin concentrations were analyzed with respect to the detected strains of vancomycin-resistant enterococci (VRE), in which the presence of resistance genes, virulence factors and potential relationship were analyzed. VRE were detected in 16 wastewater samples (11%) and two surface water samples (6%). The PNEC of vancomycin was exceed in 16% of the samples. Since the detected VRE did not correlate with the vancomycin concentrations, no direct relationship was confirmed between the residues of this antimicrobials and the presence of the resistant strains.

Screening patients at admission to Copenhagen hospitals for carriage of resistant bacteria after contact with healthcare systems abroad, 2016-2019.

OBJECTIVES: Patients having previous contact with healthcare systems abroad are routinely screened for resistant bacteria on admission to hospitals in Copenhagen. This study aimed to present carriage prevalence and geographical risk stratification, as well as phenotypic and genotypic characterisation of resistant isolates. METHODS: This study included screening samples analysed at one department of clinical microbiology in Copenhagen from 2016-2019. Patients who had previous contact with healthcare systems abroad within 6 months were screened at admission for methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) and carbapenemase-producing organisms (CPO). Isolates were characterised phenotypically and by whole-genome sequencing. The relative frequency of positive findings stratified by geographical regions correlated with relative frequency of Danish residents' travel destinations. RESULTS: Of 2849 screening sets included in the study, 103 (3.6%) were positive. A total of 120 resistant isolates were detected (36 MRSA, 31 VRE and 53 CPO). The carrier prevalence for MRSA was 1.3%, 1.1% for VRE and 1.5% for CPO. Southern and Western Asia were overrepresented travel destinations in positive screening sets (41%). For VRE, 40% were related to Southern Europe, which also represented 35% of travel destinations. Genotypic characterisation confirmed a heterogenous genomic background reflecting global distribution of resistant clones. CONCLUSIONS: Exposure targeted screening identified a substantial number of asymptomatic carriers of MRSA, VRE and CPO with heterogenous genetic backgrounds. Although some geographical regions were overrepresented, the complex epidemiology of the different pathogens did not allow a restriction of the screening strategy to certain geographical regions.

Anti-enterococcal and anti-oxidative potential of a thermophilic cyanobacterium, Leptolyngbya sp. HNBGU 003.

Enterococci, the opportunistic pathogens, pose several serious and life-threatening infections such as urinary tract infections, sepsis, and endocarditis. The situation is worsening due to the development of drug resistance in these pathogens against several antibiotics. The addition of anti-enterococcal compounds with antioxidant activity in fermented and packaged food may help prevent the transmission of food-borne enterococcal infections. Scientists are in continuous search of such compounds from various sources. Hence, the present study has tested the diethyl ether extracts of thermophilic cyanobacteria, selected based on a previous study, against the multidrug-resistant and -sensitive strains of Enterococcus faecium. Out of the eleven tested extracts, 72% have shown anti-enterococcal activity against both strains. Among the extracts with anti-enterococcal activity, the diethyl ether extract of Leptolyngbya sp. (DEEL-3) inhibited the growth of VRE in a dose-dependent manner with a minimum inhibitory concentration of 2.0 mg mL-1. The DEEL-3 has also shown its antioxidant potential in terms of DPPH scavenging with an IC50 of 3.16 mg mL-1. The organism was named Leptolyngbya sp. HNBGU 003 based on 16SrRNA sequence homology analysis and morphological features. Further, the GC-MS analysis of the DEEL-3 has revealed the predominance of two phenolic compounds, phenol, 2,4-bis(1,1-dimethylethyl)-, phosphite (3:1) and tris(2,4-di-tert-butylphenyl) phosphate, in it. Thus, the anti-enterococcal and antioxidant activity of DEEL-3 may be attributed to these phenolics, which may be isolated and developed as food additives.