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

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

Identification of an Enterococcus faecium strain isolated from raw bovine milk co-harbouring the oxazolidinone resistance genes optrA and poxtA in China.

Oxazolidinones are potent antimicrobial agents used to treat human infections caused by multidrug-resistant Gram-positive bacteria. The growing resistance to oxazolidinones poses a significant threat to public health. In August 2021, a linezolid-resistant Enterococcus faecium BN83 was isolated from a raw milk sample of cow in Inner Mongolia, China. This isolate exhibited a multidrug resistance phenotype and was resistant to most of drugs tested including linezolid and tedizolid. PCR detection showed that two mobile oxazolidinones resistance genes, optrA and poxtA, were present in this isolate. Whole genome sequencing analysis revealed that the genes optrA and poxtA were located on two different plasmids, designated as pBN83-1 and pBN83-2, belonging to RepA_N and Inc18 families respectively. Genetic context analysis suggested that optrA gene on plasmid pBN83-1 was located in transposon Tn6261 initially found in E. faecalis. Comprehensive analysis revealed that Tn6261 act as an important horizontal transmission vector for the spread of optrA in E. faecium. Additionally, poxtA-bearing pBN83-2 displayed high similarity to numerous plasmids from Enterococcus of different origin and pBN83-2-like plasmid represented a key mobile genetic element involved in movement of poxtA in enterococcal species. The presence of optrA- and poxtA-carrying E. faecium in raw bovine milk represents a public health concern and active surveillance is urgently warranted to investigate the prevalence of oxazolidinone resistance genes in animal-derived food products.

Investigation of some changes and clonal relationship in enterococci isolates due to relocation of a hospital.

Objectives: To investigate the isolation rates, antimicrobial resistance rates, minimum inhibitory concentration values of antimicrobial agents, and clonal relationships of Enterococcus faecalis and Enterococcus faeciumdue to the relocation of a hospital to a newly constructed building. METHODS: The comparative, prospective study was conducted at adult general intensive care units of the Mus State Hospital, Mus, Turkey, in two phases; before the relocation from January 25 to December 1, 2014, and after the relocation from February 10 to May 24, 2015. Rectal swab samples were collected 72 hours post-hospitalisation. Identification of Enterococcus faecalis and Enterococcus faeciumisolates was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and antimicrobial resistance with minimum inhibitory concentration values was detected with Vitek 2 system. The clonal relatedness among the strains was investigated by pulsed-field gel electrophoresis. Data was analysed using SPSS 23. RESULTS: Of the 69 patients, 37(53.62%) were related to pre-relocation phase; 20(54.1%) females and 17(45.9%) males with mean age 62.81±21.71 years. There were 32(46.37%) patients in the post-relocation phase; 13(40.6%) females and 19(59.4%) males with mean age 62.69±21.35 years (p>0.05). Of the 84 enterococci strains isolated, 51(60.7%) were Enterococcus faecium; 28(55%) before relocation and 23(45%) after relocation (p=0.77). The remaining 33(39.3%) isolates were Enterococcus faecalis; 16(48.5%) before relocation and 17(51.5%) after relocation (p=0.73). Multiple strains were located in 7(18.9%) patients before relocation and in 7(21.9%) after relocation. In 1(3.1%) patient after relocation, 2(8.7%) Enterococcus faecium isolates with different resistance and pulsed-field gel electrophoresis patterns were detected. There were no significant differences between the isolation and antibiotic resistance rates before and after relocation (p>0.05), and a clonal relation between the isolates was not detected (p>0.05). Decreased minimum inhibitory concentration values were noted for some antibiotics. CONCLUSIONS: Clonal relationship between the isolates and change in the rates of isolation and antimicrobial resistance of Enterococcus faecalis and Enterococcus faecium was not detected due to relocation. Minimum inhibitory concentration values could be used to reveal relocation-related changes in isolates obtained from patients hospitalised in intensive care units.

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.

Characterization of Weissella Species during a 3-Year Observational Study - An Emerging Threat.

Weissella species are mistaken for Lactobacillus or Leuconostoc because of their Gram-staining property and resistance to vancomycin. In this study, we aimed to evaluate the demographic pattern, presenting symptoms, risk factors, associated pathologies, and clinical outcomes in patients with Weissella infection. We also analyzed the antibiotic susceptibility pattern of the Weissella species isolated. This retrospective observational study was done from January 2021 to August 2023 in a tertiary care referral center in Uttar Pradesh. All Weissella species isolated from blood cultures or cerebrospinal fluid (CSF) samples during this period were included in the study. Twenty-four-hour growth from a positive blood culture or CSF sample was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Demographic and clinical details of the patients were extracted from the hospital information system. Kirby-Bauer disk diffusion was used for antibiotic susceptibility testing. During the 3-year study, 13 isolates of Weissella species were identified in our laboratory. Among the patients, male predominance was seen. The age range of the patients was 5-53 years. The samples were blood samples (n = 11) and CSF samples (n = 2). Of these isolates, 12 (92.3%) were identified as Weissella confusa and 1 (7.69%) was identified as Weissella viridescens. Common risk factors were anemia, prolonged hospital stay, indwelling catheter, chemotherapy, and chronic kidney disease. All the isolates in this study were sensitive to amikacin, daptomycin, amoxicillin-clavulanate, minocycline, and linezolid. Death was attributed to Weissella infection in three patients. Weissella species are opportunistic organisms that need to be identified quickly and precisely to guarantee the right course of treatment.

Epidemiology and outcomes associated with enterococcal blood stream infection among liver and kidney transplant recipients.

Bloodstream infections (BSIs) account for 18% of bacterial infections in the first year after solid organ transplantation (SOT). Enterococcus accounts for up to 20% of BSIs in this population, with vancomycin-resistant enterococcus (VRE) posing a particular risk. This is a retrospective, case-control study of adult liver and kidney transplant recipients between 01/01/2016 and 06/30/2021 that characterizes the epidemiology and outcomes of enterococcal BSIs in liver and kidney transplantations at a single institution. Subjects with an enterococcal BSI within the first 6 months post-transplant were compared to those with non-enterococcal BSIs in the same period. We identified 26 subjects with enterococcal BSIs and 28 controls with non-enterococcal BSIs (n = 54; 10.3%). Cases were mostly liver transplant recipients (n = 20; 77%) with a median MELD at transplant of 33 (range 14-43); controls included 14 KT recipients (50%). Groups differed significantly (all p < .05) by factors including perioperative transfusion requirements, need for reoperation, and number of interventions post-transplant. Cases had a median time of 25.5 days to infection and controls 100.5 days (p < .0001). There were no differences in 1-year mortality between the groups. Enterococcus faecium was the predominant species of Enterococcus (n = 23; 88.5%), with a majority (91.3%) of the isolates being VRE. In our liver and kidney transplants, enterococcal BSIs occurred early among liver transplant recipients. The high incidence of VRE among E. faecium isolates in this population warrants further investigation into the optimal approach to empiric antimicrobials for bacteremia in the early post-transplant period.

Comparative analysis of IR-Biotyper, MLST, cgMLST, and WGS for clustering of vancomycin-resistant Enterococcus faecium in a neonatal intensive care unit.

Healthcare-associated infections caused by vancomycin-resistant Enterococcus faecium (VREFM) pose a significant threat to healthcare. Confirming the relatedness of the bacterial isolates from different patients is challenging. We aimed to assess the efficacy of IR-Biotyper, multilocus sequencing typing (MLST), and core-genome MLST (cgMLST) in comparison with whole-genome sequencing (WGS) for outbreak confirmation in the neonatal intensive care unit (NICU). Twenty VREFM isolates from four neonates and ten control isolates from unrelated patients were analyzed. Genomic DNA extraction, MLST, cgMLST, and WGS were performed. An IR-Biotyper was used with colonies obtained after 24 h of incubation on tryptic soy agar supplemented with 5% sheep blood. The optimal clustering cutoff for the IR-Biotyper was determined by comparing the results with WGS. Clustering concordance was assessed using the adjusted Rand and Wallace indices. MLST and cgMLST identified sequence types (ST) and complex types (CT), revealing suspected outbreak isolates with a predominance of ST17 and CT6553, were confirmed by WGS. For the IR-Biotyper, the proposed optimal clustering cut-off range was 0.106-0.111. Despite lower within-run precision, of the IR-Biotyper, the clustering concordance with WGS was favorable, meeting the criteria for real-time screening. This study confirmed a nosocomial outbreak of VREFM in the NICU using an IR-Biotyper, showing promising results compared to MLST. Although within-run precision requires improvement, the IR-Biotyper demonstrated high discriminatory power and clustering concordance with WGS. These findings suggest its potential as a real-time screening tool for the detection of VREFM-related nosocomial outbreaks. IMPORTANCE: In this study, we evaluated the performance of the IR-Biotyper in detecting nosocomial outbreaks caused by vancomycin-resistant Enterococcus faecium, comparing it with MLST, cgMLST, and WGS. We proposed a cutoff that showed the highest concordance compared to WGS and assessed the within-run precision of the IR-Biotyper by evaluating the consistency in genetically identical strain when repeated in the same run.

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

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

Risk factors and outcome associated with the acquisition of MDR linezolid-resistant Enterococcus faecium: a report from tertiary care centre.

OBJECTIVE: The aim of the study was to determine the resistance profile of linezolid-resistant Enterococcus faecium (LREfm) and to investigate risk factors and outcomes associated with LREfm infections. MATERIAL AND METHODS: A prospective case-control study was undertaken (2019 to 2022) and included 202 patients with LREfm infections (cases) and 200 controls with LSEfm infections. Clinical data was prospectively collected and analysed for risk factors and outcomes. Antimicrobial susceptibility was performed, and resistance profile was studied using WHOnet. RESULTS: Risk factors associated with LREfm infection were site of infection UTI (OR 5.87, 95% CI 2.59-13.29, p ≤ 0.001), prior use of carbapenem (OR 2.85 95% CI 1.62-5.02, p ≤ 0.001) and linezolid (OR 10.13, 95% CI 4.13-24.82, p ≤ 0.001), use of central line (OR 5.54, 95% CI 2.35-13.09, p ≤ 0.001), urinary catheter (OR 0.29, 95% CI 0.12-0.70, p ≤ 0.001) and ventilation (OR 14.87, 95% CI 7.86-28.11, p ≤ 0.007). The hospital stay 8-14 days (< 0.001) prior to infection and the mortality rate (p = 0.003) were also significantly high among patients with LREfm infections. Linezolid and vancomycin resistance coexisted; further, MDR, XDR and PDR phenotypes were significantly higher among LREfm. CONCLUSION: This study provided insight into epidemiology of MDR LREfm in a setting where linezolid use is high. The main drivers of infections with LREfm are multiple, including use of carbapenems and linezolid. Invasive procedures and increased hospital stay facilitate spread through breach in infection control practises. As therapeutic options are limited, ongoing surveillance of LREfm and VRE is critical to guide appropriate use of linezolid and infection control policies.

Vancomycin-resistant Enterococcus faecium: impact of ending screening and isolation in a Danish University hospital.

BACKGROUND: Substantial resources are used in hospitals worldwide to counteract the ever-increasing incidence of vancomycin-resistant and vancomycin-variable Enterococcus faecium (VREfm and VVEfm), but it is important to balance patient safety, infection prevention, and hospital costs. AIM: To investigate the impact of ending VREfm/VVEfm screening and isolation at Odense University Hospital (OUH), Denmark, on patient and clinical characteristics, risk of bacteraemia, and mortality of VREfm/VVEfm disease at OUH. The burden of VREfm/VVEfm bacteraemia at OUH and the three collaborative hospitals in the Region of Southern Denmark (RSD) was also investigated. METHODS: A retrospective cohort study was conducted including first-time VREfm/VVEfm clinical isolates (index isolates) detected at OUH and collaborative hospitals in the period 2015-2022. The intervention period with screening and isolation was from 2015 to 2021, and the post-intervention period was 2022. Information about clinical isolates was retrieved from microbiological databases. Patient data were obtained from hospital records. FINDINGS: At OUH, 436 patients were included in the study, with 285 in the intervention period and 151 in the post-intervention period. Ending screening and isolation was followed by an increased number of index isolates. Besides a change in van genes, only minor non-significant changes were detected in all the other investigated parameters. Mortality within 30 days did not reflect the VREfm/VVEfm-attributable deaths, and in only four cases was VREfm/VVEfm infection the likely cause of death. CONCLUSION: Despite an increasing number of index isolates, nothing in the short follow-up period supported a reintroduction of screening and isolation.

Global spread of the linezolid-resistant Enterococcus faecalis ST476 clonal lineage carrying optrA.

OBJECTIVES: To investigate the global distribution of an optrA-harbouring linezolid-resistant Enterococcus faecalis ST476 clonal lineage. METHODS: Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed. RESULTS: Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage. CONCLUSIONS: Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage.

Epidemiology and infection control of vancomycin-resistant enterococci at a German university hospital: A three-year retrospective cohort study.

Vancomycin-resistant enterococci (VRE) occur in hospitalized patients, causing both infection and colonization. In recent years, there has been an increase in VRE in German and other hospitals, raising the question of how to control this epidemic best. To better understand the specific epidemiology and to guide infection control, we conducted a retrospective cohort study analyzing all patients with VRE at Hannover Medical School, a tertiary university clinic in Germany that specializes in solid organ transplantation. Epidemiologic and clinical characteristics of patients with VRE from 2015-2017 were collected. Basic epidemiologic parameters, including VRE incidence and incidence density, were calculated. Independent risk factors for nosocomial VRE infection compared to colonization were assessed using a logistic regression model. There were 1,492 VRE cases corresponding to 822 individual patients. The incidence was 0.8 VRE cases per 100 cases. A total of 536 (35.9%) of the 1,492 VRE cases were acquired nosocomially. Of the 1,492 cases, 912 cases had VRE-positive samples (894 Enterococcus (E.) faecium and 18 E. faecalis) in our hospital laboratory and the remaining cases were known VRE carriers. The vanB-phenotype was observed in 369 of the 894 (41.3%) E. faecium isolates and in 6 of the 18 (33.3%) E. faecalis isolates. There was an increase over time in the vanB-phenotype proportion in E. faecium (2015: 63 of 171, 36.8%, 2016: 115 of 322, 35.7% and 2017: 191 of 401, 47.6%). A total of 107 cases had a VRE infection (7.2% of all VRE cases) according to the criteria of the German National Reference Center for Surveillance of Nosocomial Infections. The remaining cases were only colonized. Among other factors, leukocytopenia (<1,000/µL), the use of a central venous catheter and the visceral surgery medical specialty were independently associated with nosocomial VRE infection. VRE imposed a relevant and increasing infection control burden at our hospital. Nosocomial VRE infection was predominantly found in certain medical specialties, such as hematology and oncology and visceral surgery. Infection control efforts should focus on these highly affected patient groups/specialties.

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

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

Comparison of morbidity and mortality after bloodstream infection with vancomycin-resistant versus -susceptible Enterococcus faecium: a nationwide cohort study in Denmark, 2010-2019.

The emergence of bloodstream infections (BSI) caused by vancomycin-resistant Enterococci (VRE) has caused concern. Nonetheless, it remains unclear whether these types are associated with an excess risk of severe outcomes when compared with infections caused by vancomycin-susceptible Enterococci (VSE). This cohort study included hospitalized patients in Denmark with Enterococcus faecium-positive blood cultures collected between 2010 and 2019 identified in the Danish Microbiology Database. We estimated 30-day hazard ratio (HR) of death or discharge among VRE compared to VSE patients adjusted for age, sex, and comorbidity. The cohort included 6071 patients with E. faecium BSI (335 VRE, 5736 VSE) among whom VRE increased (2010-13, 2.6%; 2014-16, 6.3%; 2017-19; 9.4%). Mortality (HR 1.08, 95%CI 0.90-1.29; 126 VRE, 37.6%; 2223 VSE, 37.0%) or discharge (HR 0.89, 95%CI 0.75-1.06; 126 VRE, 37.6%; 2386 VSE, 41.6%) was not different between VRE and VSE except in 2014 (HR 1.87, 95% CI 1.18-2.96). There was no interaction between time from admission to BSI (1-2, 3-14, and >14 days) and HR of death (P = 0.14) or discharge (P = 0.45) after VRE compared to VSE, despite longer time for VRE patients (17 vs. 10 days for VSE, P < 0.0001). In conclusion, VRE BSI was not associated with excess morbidity and mortality. The excess mortality in 2014 only may be attributed to improved diagnostic- and patient-management practices after 2014, reducing time to appropriate antibiotic therapy. The high level of mortality after E. faecium BSI warrants further study.

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

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

Detection of Cutibacterium (Propionibacterium) acnes in orthopaedic surgery: serious problem or contamination?

PURPOSE: Bone and joint infections are an important and increasing problem. Whether intraoperatively detected bacteria should be considered relevant or not is often difficult to assess. This retrospective cohort study analyzes the relevance of C. acnes cultured from deep intraoperative specimens. METHODS: All deep tissue samples collected intraoperatively between 2015 and 2020 from a quartiary care provider were evaluated for detection of C. acnes and its therapeutical consequences. Infection rates were determined according to a standardized definition and protocol and analyzed in dependence of patient's demographic data (age and gender), operative parameters (type of surgery, body region/location of surgery, and impression of the surgeon), and initiated therapy. RESULTS: In 270 cases of more than 8500 samples, C. acnes was detected. In 30%, the detection was considered an infection. The number of samples taken and tested positive for C. acnes correlated significantly with its classification as a cause of infection. If more than one sample of the patient was positive, the detection was significantly more likely to be treated as infection (p < 0.001). In 76% of cases, a consultation to the infectious diseases (ID) department took place regarding the classification of the pathogen detection and the therapy to be carried out. Almost all of the tested isolates demonstrated the wild-type susceptibility for penicillin and clindamycin. CONCLUSION: Intraoperative detection of skin-colonizing bacteria such as C. acnes is not always synonymous with infection. In particular, if other examination results contradict an infection (pathological sample without evidence of an infectious event, detection of malignant cells, etc.), the situation must be considered in a very differentiated manner. Interdisciplinary boards, for example, are suitable for this purpose. Care should be taken to obtain a sufficiently large number of tissue samples for microbiological examination to be able to better classify the result.

Concerning emergence of a new vancomycin-resistant Enterococcus faecium strain ST1299/CT1903/vanA at a tertiary university centre in South Germany.

BACKGROUND: vanB-carrying vancomycin-resistant Enterococcus faecium (VREfm) of the sequence types 80 (ST80) and ST117 have dominated Germany in the past. In 2020, our hospital witnessed a sharp increase in the proportion of vanA-positive VREfm. AIM: To attempt to understand these dynamics through whole-genome sequencing (WGS) and analysis of nosocomial transmissions. METHODS: At our hospital, the first VREfm isolate per patient, treated during 2020, was analysed retrospectively using specific vanA/vanB PCR, WGS, multi-locus sequence typing (MLST), and core-genome (cg) MLST. Epidemiologic links between VRE-positive patients were assessed using hospital occupancy data. FINDINGS: Isolates from 319 out of 356 VREfm patients were available for WGS, of which 181 (56.7%) fulfilled the ECDC definition for nosocomial transmission. The high load of nosocomial cases is reflected in the overall high clonality rate with only three dominating sequence (ST) and complex types (CT), respectively: the new emerging strain ST1299 (100% vanA, 77.4% CT1903), and the well-known ST80 (90.0% vanB, 81.0% CT1065) and ST117 (78.0% vanB, 65.0% CT71). The ST1299 isolates overall, and the subtype CT1903 in particular, showed high isolate clonality, which demonstrates impressively high spreading potential. Overall, 152 out of 319 isolates had an allelic cgMLST difference of ≤3 to another, including 91 (59.6%) ST1299. Occupancy data identified shared rooms (3.7%), shared departments (6.2%), and VRE-colonized prior room occupants (0.6%) within 30 days before diagnosis as solid epidemiological links. CONCLUSION: A new emerging VREfm clone, ST1299/CT1903/vanA, dominated our institution in 2020 and has been an important driver of the increasing VREfm rates.

Perioperative daptomycin for prophylaxis of vancomycin-resistant Enterococcus infection in colonized liver transplant recipients.

BACKGROUND: Infection with vancomycin-resistant Enterococcus (VRE) in liver transplant recipients (LTR) has been associated with extended hospital stays, increased readmission rates, graft failure, and death. A tailored perioperative surgical prophylaxis regimen targeting VRE may reduce postoperative infections in VRE-colonized patients. This study investigated the outcomes of perioperative daptomycin in this patient population. METHODS: This retrospective, single-center cohort study included LTR ≥ 18 years old who were VRE-colonized from June 2018 to November 2022. VRE colonization was identified by a VRE rectal swab screen or a positive VRE culture prior to transplant. Two groups were analyzed: daptomycin versus no daptomycin. All LTR received perioperative piperacillin-tazobactam for 24 h. If VRE-colonized, one dose of daptomycin (6 mg/kg) was given pre- and postoperatively. Demographics, clinical characteristics, risk factors for VRE infection, and daptomycin dose were collected. The primary outcome was VRE infection at 14 days and 90 days post-transplant. RESULTS: There were 36 VRE-colonized LTR; 19 received daptomycin and 17 did not. Baseline characteristics and risk factors for VRE infection were similar between groups. More VRE infections occurred in the no daptomycin group within 14 days post-transplant (24% vs. 0%, p = .04), but at 90 days posttransplant there was no significant difference (29% vs. 16%, p = .43). The average daptomycin dose was 7.1 mg/kg. CONCLUSION: Perioperative daptomycin reduced the rate of VRE infections in VRE-colonized LTR within 14 days posttransplant but not at 90 days. Future studies should evaluate if higher doses and/or longer duration of perioperative daptomycin can reduce VRE infections beyond 14 days post-transplant.

Successful expansion of hospital-associated clone of vanA-positive vancomycin-resistant Enterococcus faecalis ST9 to an anthropogenically polluted mangrove in Brazil.

Mangrove ecosystems are hotspots of biodiversity, but have been threatened by anthropogenic activities. Vancomycin-resistant enterococci (VRE) are nosocomial bacteria classified as high priority by the World Health Organization (WHO). Herein, we describe the identification and genomic characteristics of a vancomycin-resistant Enterococcus faecalis strain isolated from a highly impacted mangrove ecosystem of the northeastern Brazilian, in 2021. Genomic analysis confirmed the existence of the transposon Tn1546-vanA and clinically relevant antimicrobial resistance genes, such as streptogramins, tetracycline, phenicols, and fluoroquinolones. Virulome analysis identified several genes associated to adherence, immune modulation, biofilm, and exoenzymes production. The UFSEfl strain was assigned to sequence type (ST9), whereas phylogenomic analysis with publicly available genomes from a worldwide confirmed clonal relatedness with a hospital-associated Brazilian clone. Our findings highlight the successful expansion of hospital-associated VRE in a mangrove area and shed light on the need for strengthening genomic surveillance of WHO priority pathogens in these vital ecosystems.