Vancomycin-resistant Enterococcus Faecium Infections Research Articles

LiaX is a surrogate marker for cell envelope stress and daptomycin non-susceptibility in Enterococcus faecium.

Daptomycin (DAP) is often used as a first-line therapy to treat vancomycin-resistant Enterococcus faecium infections, but emergence of DAP non-susceptibility threatens the effectiveness of this antibiotic. Moreover, current methods to determine DAP minimum inhibitory concentrations (MICs) have poor reproducibility and accuracy. In enterococci, DAP resistance is mediated by the LiaFSR cell membrane stress response system, and deletion of liaR encoding the response regulator results in hypersusceptibility to DAP and antimicrobial peptides. The main genes regulated by LiaR are a cluster of three genes, designated liaXYZ. In Enterococcus faecalis, LiaX is surface-exposed with a C-terminus that functions as a negative regulator of cell membrane remodeling and an N-terminal domain that is released to the extracellular medium where it binds DAP. Thus, in E. faecalis, LiaX functions as a sentinel molecule recognizing DAP and controlling the cell membrane response, but less is known about LiaX in E. faecium. Here, we found that liaX is essential in E. faecium with an activated LiaFSR system. Unlike E. faecalis, E. faecium LiaX is not detected in the extracellular milieu and does not appear to alter phospholipid architecture. We further postulated that LiaX could be used as a surrogate marker for cell envelope activation and non-susceptibility to DAP. For this purpose, we developed and optimized a LiaX enzyme-linked immunosorbent assay (ELISA). We then assessed 86 clinical E. faecium bloodstream isolates for DAP MICs and used whole genome sequencing to assess for substitutions in LiaX. All DAP-resistant clinical strains of E. faecium exhibited elevated LiaX levels. Strikingly, 73% of DAP-susceptible isolates by standard MIC determination also had elevated LiaX ELISAs compared to a well-characterized DAP-susceptible strain. Phylogenetic analyses of predicted amino acid substitutions showed 12 different variants of LiaX without a specific association with DAP MIC or LiaX ELISA values. Our findings also suggest that many E. faecium isolates that test DAP susceptible by standard MIC determination are likely to have an activated cell stress response that may predispose to DAP failure. As LiaX appears to be essential for the cell envelope response to DAP, its detection could prove useful to improve the accuracy of susceptibility testing by anticipating therapeutic failure.

Protective effect of microbisporicin (NAI-107) against vancomycin resistant Enterococcus faecium infection in a Galleria mellonella model.

Increasing antimicrobial resistance in Enterococcus faecium necessitates the search for novel treatment agents, such as bacteriocins. In this study, we conducted an in vivo assessment of five bacteriocins, namely Lacticin Z, Lacticin Q, Garvicin KS (ABC), Aureocin A53 and Microbisporicin (NAI-107), against vanB-resistant Enterococcus faecium using a Galleria mellonella model. Our in vitro experiments demonstrated the efficacy of all five bacteriocins against vanB-resistant E. faecium with only NAI-107 demonstrating in vivo efficacy. Notably, NAI-107 exhibited efficacy across a range of tested doses, with the highest efficacy observed at a concentration of 16µg/mL. Mortality rates in the group treated with 16µg/mL NAI-107 were lower than those observed in the linezolid-treated group. These findings strongly suggest that NAI-107 holds promise as a potential alternative therapeutic agent for treating infections caused by resistant E. faecium and warrants further investigation.

Factors affecting vancomycin-resistant Enterococcus faecium colonization of in-hospital patients in different wards.

The prevalence of vancomycin-resistant Enterococcus faecium (VRE) infection at a medical center in Eastern Taiwan rose to 80.6%, exceeding the average prevalence of 55.6% among all medical centers nationwide during the same period. In recent years, the number of cases of VRE infection detected among hospitalized patients has increased annually. However, most of these patients in different wards are asymptomatic carriers. Therefore, restricting active screening to high-risk units will not improve the current situation, and it is necessary to review the risk factors for VRE colonization to provide a reference for future infection control policies. Between 2014 and 2019, there were 3188 VRE-positive cultures reported at our institution, as per the electronic medical records system. In the medical and surgical wards, patients who received penicillin (odds ratios [ORs]: 2.84 and 4.16, respectively) and third-generation cephalosporins (ORs: 3.17 and 6.19, respectively) were at higher risk of VRE colonization. In intensive care units, the use of carbapenems (OR: 2.08) was the most significant variable. This study demonstrated that the risk factors for VRE colonization differed between wards. Thus, policies should be established according to the attributes of patients in each ward, and active screening tests should be performed according to individual risks, instead of a policy for comprehensive mass screening.

An Outbreak of Vancomycin-Resistant Enterococci in a City Hospital Intensive Care Unit: Molecular Characterization of Resistance.

Background and Objectives: Vancomisin-resistant Enterococci (VRE), is a resistant microorganism that colonizes and causes infections in hospitalized patients. The aim of this study was to show the spread of vancomycin-resistant Enterococcus faecium (VREfm) step-by-step in all intensive care units, which started with the growth of VREfm on 2 December 2021 in the blood culture of a patient hospitalized in the anesthesia intensive care unit of our hospital and was found to have reached epidemic size in the surveys. Materials and Methods: Rectal swab samples were taken from all patients hospitalized in intensive care units, VRE colonization was determined, the VanA and VanB resistance genes associated with the vancomycin resistance of VREfm isolates were determined by PCR method, and clonal association analysis was performed by Arbitrarily Primed-PCR (AP-PCR) and PFGE (pulsed-field gel electrophoresis). Results: In our study, VRE were detected in 61 of 2601 rectal swab samples. In total, fifty-four (85.52%) of the VRE isolates were Enterococcus faecium, three (4.91%) was Enterococcus faecalis, three (4.91%) was Enterococcus gallinorum, and one (1.63%) was Enterococcus casseliflavus. It was determined that all of the 54 VREfm isolates, which were the most detected among all VRE isolates, carried the vanA gene. In the clonal association analysis of the isolates by AP-PCR and PFGE methods, it was found that they had 12 different genotypes, 48 of them were included in any cluster, the clustering rate was 88.8%, and the largest cluster was the genotype 1 cluster, with 36 isolates. Of the 54 patients with VREfm isolated recently, 18.51 percent of the clinical samples were isolated before the survey, and 9.25% were isolated after the survey. It was determined that 100% of VREfm isolates were resistant to ampicillin, levofloxacin, ciprofloxacin, high-level gentamicin, trimethoprimsulfamethoxazole, and teicoplanin, 7.4% to tigecycline, and 1.85% to linezolid. Conclusions: In our study, in the clonal association analysis performed by isolating VREfm in rectal swab samples, it was found that 88.8% of the samples were indistinguishably similar, and that the increase in the number of VREfm infections after the index case in our hospital was associated with the epidemic. VREfm infections cause long-term hospitalization, costs and also deaths, which shows the seriousness of the event, and the importance of the combination of epidemiological and molecular analysis in epidemic research.

Development of VRE meningitis due to haematogenous spread while on high-dose daptomycin therapy.

Vancomycin-resistant Enterococcus faecium (VRE) meningitis accounts for only 0.3%-4.0% of bacterial meningitis cases and typically occurs following neurosurgical intervention. We describe a rare case of a patient without neurological devices in situ or a recent neurological procedure who developed VRE meningitis via haematogenous spread. Optimal treatment for VRE meningitis is unknown. A 67-year-old male with end-stage liver failure underwent liver transplantation complicated by VRE bacteraemia and subsequently developed VRE meningitis while on high-dose daptomycin therapy (12 mg/kg/day). Due to clinical and microbiological failure with daptomycin, he was switched to linezolid and symptoms resolved rapidly. He completed 2 weeks of linezolid, fully recovered, and continued to do well without complications at the 5 month follow-up. This case highlights the severity of VRE infections in solid organ transplant recipients and raises concerns about daptomycin penetration into the CNS. Linezolid could be considered the preferred treatment for VRE CNS infections rather than high-dose daptomycin.

Increasing trend of healthcare-associated infections due to vancomycin-resistant Enterococcus faecium (VRE-fm) paralleling escalating community-acquired VRE-fm infections in a medical center implementing strict contact precautions: An epidemiologic and pathogenic genotype analysis and its implications.

To clarify whether there were clandestine intra-hospital spreads of vancomycin-resistant Enterococcus faecium (VRE-fm) isolates that led to specific strain of VRE lingering in the hospital and/or developing outbreaks that rendered a progressively increasing trend of healthcare-associated infections due to VRE-fm (VRE-fm-HAIs). Despite implementing strict contact precautions for hospitalized patients with VRE-fm-infection/colonization, number of VRE-fm-HAIs in a medical centre in southern Taiwan were escalating in 2009-2019, paralleling an increasing trend of community-acquired VRE-fm- infections. We analyzed epidemiologic data and genotypes of non-duplicate VRE-fm isolates each grown from a normally sterile site of 89 patients between December 2016 and October 2018; multilocus sequence typing (MLST) and pulse-field gel electrophoresis (PFGE) typing were performed. Totally 13 sequence types (STs) were found, and the 3 leading STs were ST17 (44％), ST78 (37％), and ST18 (6％); 66 pulsotypes were generated by PFGE. Four VRE-fm isolates grouped as ST17/pulsotype S, 2 as ST17/pulsotype AS, 2 as ST17/pulsotype AU, and 3 as ST78/pulsotype V grew from clinical specimens sampled less than one week apart from patients staying at different wards/departments and/or on different floors of the hospital. Despite possible small transitory clusters of intra-hospital VRE-fm spreads, therewas no specific VRE-fm strain lingering in the hospital leading to increasing trend of VRE-fm-HAIs during the study period. Strict contact precautions were able to curb intra-hospital VRE-fm spreads, but unable to curb the increasing trend of VRE-fm-HAIs with the backdrop of progressively increasing VRE-fm-infections/colorizations in the community.

Impact of a new hospital with close to 100% single-occupancy rooms on environmental contamination and incidence of vancomycin-resistant Enterococcus faecium colonization or infection: a genomic surveillance study

Vancomycin-resistant Enterococcus faecium (VRE) is a leading cause of nosocomial infection, driven by its ability to spread between patients and persist in the hospital environment. To investigate the impact of a long-established cardiothoracic hospital moving to new premises with close to 100% single-occupancy rooms on the rates of environmentalcontamination and infection or colonization by VRE. Prospective environmental surveillance for VRE was conducted at five time-points between April and November 2019, once in the original building, and four times in the new building. Incidence rate ratios (IRRs) of VRE infection/colonization were determined for the one-year period before and after the hospital move, and compared to a nearby hospital. In the original location, the first environmental screen found 29% VRE positivity. The following four screens in the new location showed a significant reduction in positivity (1-6%; P<0.0001). The VRE infection/colonization rates were halved in the new location (IRR: 0.56; 95% confidence interval: 0.38-0.84), compared to the original location,contrasting with an increase in a nearby hospital (1.62; 1.17-2.27) over the same time-period. Genomic analysis of the environmental isolates was consistent with reduced transmission in the new hospital. The use of single-occupancy rooms was associated with reducedenvironmental contamination with VRE, and lower transmission and isolation of VRE from clinical samples. The cost-effectiveness of single-occupancy room hospitals in reducing healthcare-associated infections should be reassessed in the context of operational costs of emerging pandemic and increasing antimicrobial resistance threats.

An Economic Evaluation Estimating the Clinical and Economic Burden of Increased Vancomycin-Resistant Enterococcus faecium Infection Incidence in Japan.

While incidence rates of vancomycin-resistant Enterococcus faecium have remained comparatively low in Japan, there have been increasing reports of more vancomycin-resistant Enterococcus (VRE) outbreaks, requiring costly measures to contain. Increased incidence of VRE in Japan may lead to more frequent and harder to contain outbreaks with current control measures, causing a significant burden to the healthcare system in Japan. This study aimed to demonstrate the clinical and economic burden of vancomycin-resistant E. faecium infections to the Japanese healthcare system and the impact of increasing rates of vancomycin resistance. A de novo deterministic analytic model was developed to assess the health economic outcomes of treating hospital-acquired VRE infections; patients are treated according to a two-line treatment strategy, dependent on their resistance status. The model considers hospitalisation costs and the additional cost of infection control. Scenarios investigated the current burden of VRE infections and the additional burden of increased incidence of VRE. Outcomes were assessed over a 1-year and 10-year time horizon from a healthcare payer's perspective in a Japanese setting. Quality-adjusted life years (QALYs) were valued with a willingness-to-pay threshold of ¥5,000,000 ($38,023), and costs and benefits were discounted at a rate of 2%. Current VRE incidence levels in enterococcal infections in Japan equates to ¥130,209,933,636 ($996,204,669) in associated costs and a loss of 185,361 life years (LYs) and 165,934 QALYs over 10 years. A three-fold increase (1.83%) is associated with an additional ¥4,745,059,504 ($36,084,651) in total costs on top of the current cost burden as well as an additional loss of 683 LYs over a lifetime, corresponding to 616 QALYs lost. Despite low incidence rates, VRE infections already represent a substantial economic burden to the Japanese healthcare system. The substantial increase in costs associated with a higher incidence of VRE infections could result in a significant economic challenge for Japan.

Molecular epidemiology of bacteraemic vancomycin-resistant Enterococcus faecium isolates and in vitro activities of SC5005 and other comparators against these isolates collected from a medical centre in northern Taiwan, 2019-2020.

The global prevalence of vancomycin-resistant Enterococcus faecium (VREfm) highlights the need for new anti-enterococcal agents. Here, we assessed the molecular epidemiology of clinical VREfm bacteraemic isolates from a medical centre in northern Taiwan in 2019-2020 and to evaluate their susceptibility to last-line antibiotics and a new antimicrobial agent, SC5005. The molecular epidemiology of VREfm was investigated using van genotyping, MLST and PFGE. The susceptibilities of VREfm strains to antibiotics and SC5005 were determined using the agar dilution and broth microdilution methods. The capability of E. faecium to develop resistance to antibiotics and SC5005 was evaluated using frequency of resistance and multipassage resistance assays. The mode of action of SC5005 was assessed by time-kill, bacterial membrane integrity and membrane potential assays. All 262 VREfm isolates harboured vanA gene, and the most prevalent sequence type was ST17 (51%, n = 134, 84 pulsotypes), followed by ST78 (25%, n = 65, 54 pulsotypes). Additionally, we identified four new STs (ST2101, ST2102, ST2135 and ST2136) and observed the arrival of multidrug-resistant ST1885 in Taiwan. Moreover, SC5005 was effective against all VREfm isolates, including those non-susceptible to last-line antibiotics. SC5005 can disrupt and depolarize the bacterial membrane to kill E. faecium without detectable resistance. The findings provide insights into the latest epidemiology and resistance profiles of bacteraemic-causing VREfm in northern Taiwan. Additionally, SC5005 has the potential for development as a new therapeutic to treat VREfm infections.

A High Daptomycin Dose Is Associated with Better Bacterial Clearance in Infections Caused by Vancomycin-Resistant Enterococcus faecium Regardless of Daptomycin Minimum Inhibitory Concentration in a Rat Infective Endocarditis Model.

ABSTRACTA high daptomycin dose has been suggested for treating vancomycin-resistant Enterococcus faecium (VREf) infections. However, even a 12 mg/kg daptomycin dose might be insufficient for treating VREf with high daptomycin minimum inhibitory concentrations (MICs). Additionally, animal pharmacodynamic and infection models to confirm the efficacy of 12 mg/kg daptomycin are lacking. Male Wistar rats were used for pharmacokinetic profiling and for the development of an infective endocarditis (IE) model. Daptomycin-susceptible dose-dependent VREf (DSE) (MIC of 0.5 mg/L) and daptomycin nonsusceptible VREf (DNSE) (MIC of 8 mg/L) were used for the IE models. The bacterial load of vegetation was the primary outcome and was evaluated after 3 days of daptomycin treatment. Daptomycin administered subcutaneously (s.c.) at 45 and 90 mg/kg, which corresponded to maximum serum concentrations (Cmax) of 122.6 mg/L and 178.5 mg/L, respectively, was equivalent to doses of 8 mg/kg and 12 mg/kg, respectively, in humans. The Cmax/MIC value was correlated with the bacterial load of vegetation after treatment (r = −0.88, P < 0.001). The 90 mg/kg s.c. group showed a significantly lower bacterial load of vegetation (log10 CFU/g) than the 45 mg/kg s.c. group against DSE (0 versus 4.75, P < 0.001) and DNSE (5.12 versus 6.98, P = 0.002). The 90 mg/kg s.c. group did not sterilize the vegetation against DNSE. Although the human equivalent dose of 12 mg/kg daptomycin was more effective than the smaller dose in reducing the bacterial load in DSE and DNSE IE, the dose could not sterilize the vegetation during a DNSE treatment. Further treatment strategies by which to manage severe VREf infections, especially at high daptomycin MICs, are urgently needed.IMPORTANCE Using a rat IE model with pharmacokinetic analysis, the treatment response of VREf IE was found to be daptomycin dose-dependent, presented as Cmax/MIC or as the 24 h area under the concentration-time curve (AUC0–24)/MIC. Daptomycin 90 mg/kg s.c. significantly reduced the bacterial load against DSE and DNSE. It also showed significant activity against DSE and DNSE, compared to 45 mg/kg s.c. Although daptomycin 90 mg/kg can eradicate the bacterial load after 3 days of treatment against DSE, eradication cannot be achieved with 90 mg/kg daptomycin against DNSE.

Rapid in vivo development of resistance to daptomycin in vancomycin-resistant Enterococcus faecium due to genomic alterations.

Daptomycin is a cyclic lipopeptide used in the treatment of vancomycin-resistant Enterococcus faecium (VREfm). However, the development of daptomycin-resistant VREfm challenges the treatment of nosocomial VREfm infections. Resistance mechanisms of daptomycin are not fully understood. Here, we analyzed the genomic changes leading to a daptomycin-susceptible VREfm isolate becoming resistant after 50 days of daptomycin and linezolid combination therapy. A total of seven isogenic VREfm isolates from the same patient (daptomycin-susceptible and daptomycin-resistant) were analyzed using Illumina whole genome sequencing, and two isolates were further characterized with Nanopore sequencing. One nonsynonymous SNP in the rpoC gene previously shown to harbor mutations in daptomycin-resistant VREfm was identified in the daptomycin-resistant isolates. Whole genome comparative analysis identified the loss of a 46.5 kb fragment, duplication of a 29.7 kb fragment, and integration of two plasmids upon acquisition of daptomycin resistance. Transmission electron microscopy showed similar alterations in cell morphology and cell wall structure as have previously been described in daptomycin-resistant E. faecalis.

Epidemiology, Risk Factors and Outcome Due to Multidrug Resistant Organisms in Paediatric Liver Transplant Patients in the Era of Antimicrobial Stewardship and Screening.

(1) Background: Multidrug-resistant organisms (MDRO) are a growing problem in liver transplant recipients (LTR), associated with high morbidity and mortality. We reviewed the impact of antimicrobial stewardship (AMS) and active screening of MDRO on the epidemiology and outcomes in paediatric LTR. (2) Methods: Single-centre retrospective review of paediatric LTR from January 2017 to December 2018. (3) Results: Ninety-six children were included; 32 (33%) patients were colonised with ≥1 MDRO and 22 (23%) patients had MDRO infections. Median (IQR) duration for start of infection was 9.5 (1.8–16.0) days. Colonisation rate with Gram-positive MDRO was 15.6%, with infection rate of 6.2%; majority due to Vancomycin-Resistant Enterococcus faecium (VRE). Colonisation with Gram-negative MDRO was 27.0%, with infection rate of 16.6%; majority due to extended-spectrum β-lactamase producing Enterobacteriaceae. Colonisation and infection rate due to Carbapenem-resistant Enterobacteriaceae was 6% and 3%, respectively, during screening and AMS, compared to historical control of 25% and 30%, respectively, without screening and AMS. There was significant reduction in VRE and CRE infection during AMS period in comparison to historical control. Pre-transplant risk factors including bacterial infections pre-transplant (p < 0.01), diagnosis of biliary atresia (p = 0.03), exposure to antibiotics (p < 0.01), EBV viraemia (p = 0.01), and auxiliary transplantation (p < 0.01) were associated with post-transplant MDRO infections. Patients with MDRO infections had longer length of hospital and paediatric intensive care unit stay days (p < 0.01) but associated with no mortality. (4) Conclusions: Our results demonstrate low incidence of colonisation and infections with MDRO, which were associated with high morbidity but no mortality in paediatric LTR. There was significant reduction in MRSA, VRE, and CRE during AMS period compared to pre-AMS era. Some risk factors are unavoidable but antibiotic overuse, early initiation of appropriate antibiotic therapy and effective infection prevention strategies can be monitored with multifaceted approach of AMS and screening of MDRO. With limited therapeutic options for MDRO and efficacy data of newer antibiotics in paediatric LTR, robust infection control practices are of paramount importance.

Daptomycin susceptibility testing and therapeutic use in enterococcal bloodstream infection (EBSI) in a setting with high rates of vancomycin-resistant Enterococcus faecium (VREfm).

There is in vitro and clinical evidence to suggest daptomycin has good activity against Enterococcus. In 2019, CLSI produced clinical breakpoints for Enterococcus spp. To describe the distribution of MICs of daptomycin for enterococcal bloodstream infection (EBSI) isolates in a large Scottish health board, the indications for local daptomycin susceptibility testing and daptomycin doses used in vancomycin-resistant Enterococcus faecium (VREfm) infection. We investigated all EBSIs over a 21 month period and identified isolates tested against daptomycin. We recorded the distribution of MICs, as well as indications for daptomycin susceptibility testing and information on daptomycin dosing, where it was used. There were 293 blood culture isolates of Enterococcus spp., of which 37 had daptomycin susceptibility testing performed, from 31 individual patients. Of the 293 isolates, 103 were E. faecium, of which 63 were VREfm. Daptomycin testing was indicated by vancomycin resistance in 24/37 isolates. All E. faecium isolates tested were in the CLSI 'susceptible dose-dependent (SDD)' range of MICs. All other Enterococcus spp. tested were in the 'susceptible' range. Twelve patients received daptomycin, and dosing information was recovered for 10. Nine of these patients received 8-12 mg/kg/day dosing. There were no recorded adverse drug reactions. Ten of 12 patients were alive at the time of data collection. Daptomycin MIC distribution for EBSI isolates suggests a high local rate of susceptibility, according to CLSI breakpoints, in a population with high rates of VREfm. CLSI-recommended doses of daptomycin were used, with encouraging survival outcomes.

Optimising genomic approaches for identifying vancomycin-resistant Enterococcus faecium transmission in healthcare settings

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen. Identifying VREfm transmission dynamics permits targeted interventions, and while genomics is increasingly being utilised, methods are not yet standardised or optimised for accuracy. We aimed to develop a standardized genomic method for identifying putative VREfm transmission links. Using comprehensive genomic and epidemiological data from a cohort of 308 VREfm infection or colonization cases, we compared multiple approaches for quantifying genetic relatedness. We showed that clustering by core genome multilocus sequence type (cgMLST) was more informative of population structure than traditional MLST. Pairwise genome comparisons using split k-mer analysis (SKA) provided the high-level resolution needed to infer patient-to-patient transmission. The more common mapping to a reference genome was not sufficiently discriminatory, defining more than three times more genomic transmission events than SKA (3729 compared to 1079 events). Here, we show a standardized genomic framework for inferring VREfm transmission that can be the basis for global deployment of VREfm genomics into routine outbreak detection and investigation.

First report of nosocomial outbreak of vancomycin-resistant Enterococcus faecium infection among COVID-19 patients hospitalized in a non-intensive care unit ward in Slovakia.

Enterococcus species account for most of the human enterococcal HAI and multidrug-resistant infections andhave become a major threat to modern public health. We examine the risein the number of vancomycin resistantE. faeciumblood stream and urinary tractinfections in a COVID-19 department during an epidemiologic outbreak investigation to detect and eliminate nosocomial clusters of the bacteria. Strain identification was performed by classical isolation andbiochemical andcultivation methods. Antibiotic testing results were interpreted according to European committee on antimicrobial susceptibility testing (EUCAST) guidelines. Six isolatedsamples underwent the whole genome sequencing (WGS) during the outbreak investigation. Isolate relatedness was determined using the core genome multi-locus sequence typing. WGS revealed two genotypically distinct VRE clusters, one of which had genetically closely related patients and environmental isolates. The cluster was terminated by enhanced infection control strategies. This study provides the first description of an outbreak caused by vanB-ST117 and vanA-ST17E. faeciumstrains among COVID-19 patients in Slovakia. This study can help to raise the awareness about the need for strict adherence to infection control measures and the implementation of rational antimicrobial stewardship as a routine part of COVID-19 management (Tab. 3, Fig. 3, Ref. 27). Text in PDF www.elis.sk Keywords: vancomycin-resistantEnterococcus faecium, antibiotic resistant, COVID-19, SARS-CoV-2, bacterial outbreak, healthcare-associated infection.

Daptomycin Resistance Occurs Predominantly in vanA-Type Vancomycin-Resistant Enterococcus faecium in Australasia and Is Associated With Heterogeneous and Novel Mutations.

Healthcare associated infections caused by vancomycin-resistant Enterococcus faecium (VREfm) have a major impact on health outcomes. VREfm is difficult to treat because of intrinsic and acquired resistance to many clinically used antimicrobials, with daptomycin being one of the few last line therapeutic options for treating multidrug-resistant VREfm. The emergence of daptomycin-resistant VREfm is therefore of serious clinical concern. Despite this, the impact that daptomycin-resistant VREfm have on patient health outcomes is not clearly defined and knowledge on the mechanisms and genetic signatures linked with daptomycin resistance in VREfm remains incomplete. To address these knowledge gaps, phenotypic daptomycin susceptibility testing was undertaken on 324 E. faecium isolates from Australia and New Zealand. Approximately 15% of study isolates were phenotypically resistant to daptomycin. Whole genome sequencing revealed a strong association between vanA-VREfm and daptomycin resistance, with 95% of daptomycin-resistant study isolates harbouring vanA. Genomic analyses showed that daptomycin-resistant VREfm isolates were polyclonal and carried several previously characterised mutations in the liaR and liaS genes as well as several novel mutations within the rpoB, rpoC, and dltC genes. Overall, 70% of daptomycin-resistant study isolates were found to carry mutations within the liaR, rpoB, rpoC, or dltC genes. Finally, in a mouse model of VREfm bacteraemia, infection with the locally dominant daptomycin-resistant clone led to reduced daptomycin treatment efficacy in comparison to daptomycin-susceptible E. faecium. These findings have important implications for ongoing VREfm surveillance activities and the treatment of VREfm infections.

Sequence type 17 is a predictor of subsequent bacteremia in vancomycin-resistant Enterococcus faecium-colonized patients: a retrospective cohort study

BackgroundSequence type (ST) 17 vancomycin-resistant Enterococcus faecium (VREF) is frequently isolated in nosocomial settings. The aim of this study was to identify whether ST17 contributes to subsequent bacteremia more often than other STs among hospitalized patients carrying VREF.MethodsA retrospective cohort study was conducted in patients carrying ST17 VREF and those with non-ST17 VREF. Rectal screening according to hospital policy was used to identify patients with VREF. Subsequent VREF bacteremia events within a year of detection of colonization were recorded. Cox regression analysis was used to adjust the covariates involved in determining the association between ST17 and subsequent bacteremia events.ResultsThe cohorts comprised 52 patients with ST17 and 169 patients with non-ST17 VREF. One-year VREF bacteremia-free rates were 85.9% and 90.2%, respectively. In multivariate analysis, ST17 was associated with subsequent bacteremia at an adjusted hazard risk (aHR) of 4.02 (95% confidence interval [CI], 1.32–12.29). Liver transplantation (aHR, 40.08; 95% CI, 4.87–329.76) and hematologic malignancy (aHR, 20.97; 95% CI, 4.87–87.82) were also significant. All cases of subsequent bacteremia in ST17 VREF carriers were caused by ST17; however, subsequent bacteremia in non-ST17 carriers was often caused by ST17 or another ST variant.ConclusionsA specific genotype, ST17 is a predictor of subsequent bacteremia in hospitalized patients carrying VREF. Patients with a hematologic malignancy and those receiving a liver transplant are also at high risk. More targeted strategies may be needed to prevent VREF infection in hospitals.

869. Outbreak of Vancomycin Resistant Enterococcus faecium (VREfm) in a Hematology Unit Identified Through Whole Genome Sequencing

BackgroundVREfm is a major cause of Hospital Acquired Infection in the United States. We analyzed all the VREfm infections that occurred in our institution between 2018 and 2019 using Whole Genome Sequencing (WGS) to understand epidemiological relationship between previously unidentified clusters. In this study we describe a cluster in our hematology oncology unit.MethodsA total of 109 discrete VREfm isolates from 66 patients were analyzed. VREfm isolates used in this study were identified from positive blood and urine cultures. Genomic deoxyribonucleic acid (DNA) was extracted from pure cultures. The purity and integrity of extracted DNA were determined using appropriate assays. Library construction and sequencing were conducted and Multi Locust Sequence Typing (MLST) obtained (image 1). Phylogenomic tree was plotted using the Interactive Tree of Life (image 2).Image 1 - methods Image 2 - Tree of Life ResultsTotal of 7 clusters were identified. Here we describe one cluster (image 3) with the highest genetic similarity which showed maximum difference of 5 Single Nucleotide Polymorphisms (zero between patient 1 and 2, image 4). The cluster is composed of 24 clinical strains of VREfm from 6 patients, over a 9 month time period (Image 5). All patients had hematologic malignancies; 4/6 patients had received recent chemotherapy and 5/6 patients were neutropenic. 4 patients were admitted in a single unit (labelled E7), 1 patient was on a sister unit (labelled F7); and 1 patient was in the cancer infusion center. All patients had central venous access placed by radiology at the time of diagnosis of infection and had visited our outpatient infusion center multiple times during this time frame.Image 3 - Close look at cluster 1 Image 4 - Dendrogram of 106 isolates performed with coreSNP(Single Nucleotide Polymorphisms) pairwise distances. • Dendogram shows different patients (same color for isolates that belong to the same patient) and the patient numbers. • Besides the patient number, the number of largest number SNPS that separate those isolates is shown. • Branches represent the number of coreSNPs that differ strains from that branch. As you see isolates from cluster 1 differ in a maximum of 5 SNPs but isolates of patient 1 and patient 2 differ in 0 SNPs between them. Cluster 1 is represented by a green square. Image 5 - Time period of infections ConclusionThe prolonged period in our cluster argues in favor of an environmental niche in the hospital unit. We are unable to elucidate pattern of transmission in a cluster of infections without knowing patient colonization of VREfm; because we are likely looking at the tip of the iceberg when analyzing infected cases. It is difficult to ascribe causality to any one of these exposures without concomitant surveillance cultures of environment and personnel. Retrospective WGS is of limited value in infection control. We now have third generation sequencing with the MinION device to do real time sequencing with which we also validated some of our samples.DisclosuresAtul Kothari, MD, Ansun Biopharma (Consultant)

Synergistic activity of fosfomycin and chloramphenicol against vancomycin-resistant Enterococcus faecium (VREfm) isolates from bloodstream infections

Vancomycin-resistant Enterococcus faecium (VREfm) infections are increasing. Current anti-VREfm options (linezolid and daptomycin) are suboptimal. Fosfomycin maintains good efficacy against VREfm and chloramphenicol is active against ≥ 90% of VREfm. We tested chloramphenicol + fosfomycin (CAF+FOS) against 10 VREfm isolated from blood. MICs were 64 to 512 µg/mL for fosfomycin and 8 to 16 µg/mL for chloramphenicol. The combination decreased both MICs, with a synergic effect in 50% of the isolates and an additive effect in the remaining 50%. Time-kill assays performed on fractional inhibitory concentration index ≤ 0.5 strains confirmed the synergism. The antibiotic combination at ¼ of minimum inhibitory concentrations (MICs) caused a ≥ 2 log10 reduction compared to the two antibiotics alone. Finally, we provided a proof of concept of the in vitro efficacy of CAF+FOS in G. mellonella. The survival of G. mellonella larvae treated with the combination was significantly higher. The activity of fosfomycin and chloramphenicol against VREfm increases when they are used in combination.

Clinical and molecular epidemiology of vancomycin-resistant Enterococcus faecium bacteremia from an Indian tertiary hospital.

Wedetermined the clinical and molecular epidemiology of emerging nosocomial vancomycin-resistant Enterococcus faecium (VREfm)-causing serious bloodstream infections (BSIs) and the correlations between antibiotic resistance and virulence determinantsamong isolates. Allisolates were confirmed by molecular methods (16SrRNA and E. faecium ddl genes) and tested for disk diffusion. PCRwas used to detect aac(6')-aph(2″), vanA and vanB resistance genes, and asa1, cylA, ace, esp, gelE and hyl virulence genes. VREfm and high-level gentamicin-resistant (HLGR) representative isolates were selected to characterize by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Of 173 isolates, 73(42.2%), 146(84.4%), and 0(0.0%) were vanA-containing VREfm, aac(6')-aph(2″)-positive HLGR, and vanB-positive. Independent predictors of VREfm infection were hematological malignancies (P = 0.001) and previous hospitalizations (P = 0.007). Observed mortality rate was 34.7%. Independent predictors of BSI-related mortality were endotracheal intubations (P < 0.001), gastrointestinal diseases (P = 0.002), and pulmonary disease (P < 0.001). All VREfm were resistant to vancomycin, teicoplanin, ciprofloxacin, and erythromycin. The esp, hyl, ace, asa1, cylA, and gelE genes were detected at 55.9, 22.5, 2.9, 2.3, 1.7, and 1.2%, respectively. The esp gene was significantly associated with VREfm compared toVSEfm (P = 0.001). PFGE analysis revealed 23 clones, with 7 major clones. The MLST analysis revealed the following five sequence types: ST80, ST17, ST117, ST132, and ST280, all belonging to CC17. The emergence and expansion of VREfm CC17 with limited antibiotic options in our hospital present a serious public health menace and represent challenges to infection control.