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.

Benchmarking of virome metagenomic analysis approaches using a large, 60+ members, viral synthetic community.

IMPORTANCE: We report here efforts to benchmark performance of two widespread approaches for virome analysis, which target either virion-associated nucleic acids (VANA) or highly purified double-stranded RNAs (dsRNAs). This was achieved using synthetic communities of varying complexity levels, up to a highly complex community of 72 viral agents (115 viral molecules) comprising isolates from 21 families and 61 genera of plant viruses. The results obtained confirm that the dsRNA-based approach provides a more complete representation of the RNA virome, in particular, for high complexity ones. However, for viromes of low to medium complexity, VANA appears a reasonable alternative and would be the preferred choice if analysis of DNA viruses is of importance. Several parameters impacting performance were identified as well as a direct relationship between the completeness of virome description and sample sequencing depth. The strategy, results, and tools used here should prove useful in a range of virome analysis efforts.

Genetic analysis of vancomycin-variable Enterococcus faecium clinical isolates in Italy.

PURPOSE: To investigate the occurrence of vancomycin-variable enterococci (VVE) in a hospital in central Italy. METHODS: vanA positive but vancomycin-susceptible Enterococcus faecium isolates (VVE-S) were characterized by antibiotic susceptibility tests, molecular typing (PFGE and MLST), and WGS approach. The reversion of VVE-S to a resistant phenotype was assessed by exposure to increasing vancomycin concentrations, and the revertant isolates were used in filter mating experiments. qPCR was used to analyze the plasmid copy number. RESULTS: Eleven putative VVE-S were selected. WGS revealed two categories of vanA cluster plasmid located: the first type showed the lack of vanR, the deletion of vanS, and an intact vanH/vanA/vanX cluster; the second type was devoid of both vanR and vanS and showed a deletion of 544-bp at the 5'-end of the vanH. Strains (n = 7) carrying the first type of vanA cluster were considered VVE-S and were able to regain a resistance phenotype (VVE-R) in the presence of vancomycin, due to a 44-bp deletion in the promoter region of vanH/vanA/vanX, causing its constitutive expression. VVE-R strains were not able to transfer resistance by conjugation, and the resistance phenotype was unstable: after 11 days of growth without selective pressure, the revertants were still resistant but showed a lower vancomycin MIC. A higher plasmid copy number in the revertant strains was probably related to the resistance phenotype. CONCLUSION: We highlight the importance of VVE transition to VRE under vancomycin therapy resulting in a potential failure treatment. We also report the first-time identification of VVE-S isolates pstS-null belonging to ST1478.

Genomic analysis of enterococci carrying optrA, poxtA, and vanA resistance genes from wild boars, Italy.

AIMS: To investigate enterococci carrying linezolid and vancomycin resistance genes from fecal samples recovered from wild boars. METHODS AND RESULTS: Florfenicol- and vancomycin-resistant enterococci, isolated on selective agar plates, were screened by PCR for the presence of linezolid and vancomycin resistance genes. Five isolates carried optrA or poxtA linezolid resistance genes; one strain was resistant to vancomycin for the presence of vanA gene. All isolates were tested for their antibiotic susceptibility and subjected to Whole Genome Sequencing (WGS) analysis. In Enterococcus faecalis (E. faecalis) V1344 and V1676, the optrA was located on the new pV1344-optrA and pV1676-optrA plasmids, respectively, whereas in Enterococcus faecium (E. faecium) V1339 this gene was on a 22 354-bp chromosomal genetic context identical to the one detected in a human E. faecium isolate. In both E. faecium V1682 and E. durans V1343, poxtA was on the p1818-c plasmid previously found in a human E. faecium isolate. In E. faecium V1328, the vanA gene was on the Tn1546 transposon in turn located on a new pV1328-vanA plasmid. Only E. faecium V1682 successfully transferred the poxtA gene to an enterococcal recipient in filter mating assays. CONCLUSIONS: The occurrence of genetic elements carrying linezolid and vancomycin resistance genes in enterococci from wild boars is a matter of concern, moreover, the sharing of plasmids and transposons between isolates from wild animals, human, and environment indicates an exchange of genetic material between these settings.

Detection of linezolid and vancomycin resistant Enterococcus isolates collected from healthy chicken caecum.

AIM: The poultry industry represents an important economic sector in Tunisia. This study aims to determine the antimicrobial resistance phenotypes and genotypes and virulence factors of enterococci collected from chicken caecum in Tunisia. METHODS AND RESULTS: Forty-nine composite chicken caecum samples were recovered in 49 different Tunisian farms (December 2019-March 2020). Each composite sample corresponds to six individual caecum from each farm. Composite samples were plated on Slanetz-Bartley agar both supplemented (SB-Van) and not supplemented (SB) with vancomycin and isolates were identified by matrix-assisted laser desorption/ionization time-of-flight. Antibiotic resistance and virulence genes were tested by Polymerase Chain Reaction (PCR) and sequencing and multilocus-sequence-typing of selected enterococci was performed. One hundred sixty seven enterococci of six different species were recovered. Acquired linezolid resistance was detected in 6 enterococci of 4/49 samples (8.1%): (A) four optrA-carrying Enterococcus faecalis isolates assigned to ST792, ST478, and ST968 lineages; (B) two poxtA-carrying Enterococcus faecium assigned to ST2315 and new ST2330. Plasmid typing highlighted the presence of the rep10, rep14, rep7, rep8, and pLG1 in these strains. One vancomycin-resistant E. faecium isolate (typed as ST1091) with vanA gene (included in Tn1546) was detected in SB-Van plates. The gelE, agg, esp, and hyl virulence genes were found in linezolid- and vancomycin-resistant enterococci. High resistance rates were identified in the enterococci recovered in SB plates: tetracycline [74.8%, tet(M) and tet(L) genes], erythromycin [65.9%, erm(B)], and gentamicin [37.1%, aac(6')-Ie-aph(2″)-Ia]. CONCLUSION: The detection of emerging mechanisms of resistance related to linezolid and vancomycin in the fecal enterococci of poultry farms has public health implications, and further surveillance should be carried out to control their dissemination by the food chain.

New viruses of Cladosporium sp. expand considerably the taxonomic structure of Gammapartitivirus genus.

Despite the fact that Cladosporium sp. are ubiquitous fungi, their viromes have been little studied. By analysing a collection of Cladosporium fungi, two new partitiviruses named Cladosporium cladosporioides partitivirus 1 (CcPV1) and Cladosporium cladosporioides partitivirus 2 (CcPV2) co-infecting a strain of Cladosporium cladosporioides were identified. Their complete genome consists of two monocistronic dsRNA segments (RNA1 and RNA2) with a high percentage of pairwise identity on 5' and 3' end. The RNA directed RNA polymerase (RdRp) of both viruses and the capsid protein (CP) of CcPV1 display the classic characteristics required for their assignment to the Gammapartitivirus genus. In contrast, CcPV2 RNA2 encodes for a 41 KDa CP that is unusually smaller when aligned to CPs of other viruses classified in this genus. The structural role of this protein is confirmed by electrophoresis on acrylamide gel of purified viral particles. Despite the low percentage of identity between the capsid proteins of CcPV1 and CcPV2, their three-dimensional structures predicted by AlphaFold2 show strong similarities and confirm functional proximity. Fifteen similar viral sequences of unknown function were annotated using the CcPV2 CP sequence. The phylogeny of the CP was highly consistent with the phylogeny of their corresponding RdRp, supporting the organization of Gammapartitiviruses into three distinct clades despite stretching the current demarcation criteria. It is proposed that a new subgenus be created within the genus Gammapartitivirus for this new group.

Targeted elimination of Vancomycin resistance gene vanA by CRISPR-Cas9 system.

OBJECTIVE: The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system. METHODS: Two specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance. RESULTS: In the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA. CONCLUSION: In conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.

Prevalence of Vancomycin-resistant enterococci (VRE) in Egypt (2010-2022): a systematic review and meta-analysis.

BACKGROUND: Vancomycin-resistant Enterococci (VRE) represent a critical medical and public health concerns due to their association with serious nosocomial infections and a high risk of mortality. We aimed to reveal the pooled prevalence of VRE and antimicrobial resistance profiles among enterococci clinical isolates in Egypt. METHODS: A PubMed, Scopus, Google Scholar, and Web of Science literature search was carried out in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. Only published studies documenting the prevalence of VRE between 2010 and 2022 were included. Using the random effects model and the 95% confidence intervals, the pooled estimate of VRE was calculated by MedCalc Version 20.113. Cochran's Q and I2 tests were used to evaluate the degree of heterogeneity, and publication bias was examined by visually examining the funnel plot and its associated tests (Begg's and Egger's tests). RESULTS: The pooled prevalence of VRE among enterococci clinical isolates in Egypt was estimated to be 26% (95% CI 16.9 to 36.3). E. faecalis had a greater pooled prevalence than E. faecium, with 61.22% (95% CI 53.65 to 68.53) and 32.47% (95% CI 27 to 38.2), respectively. The VanA gene is more frequent than the VanB gene among VRE, with a pooled prevalence of 63.3% (95% CI 52.1 to 73.7) and 17.95% (95% CI 7.8 to 31), respectively. The pooled resistance rate of linezolid was substantially lower than that of ampicillin and high-level gentamicin (HLG) 5.54% (95% CI 2.33 to 10%), 65.7% (95% CI 50.8 to 79.2%), and 61.1% (95% CI 47.4 to 73.9), respectively. CONCLUSION: The prevalence of VRE is alarmingly high in Egypt. It is imperative that antimicrobial stewardship activities and infection control programs are strictly adhered to and implemented to prevent further escalation of the problem.

Activity of Oritavancin against Gram-Positive Pathogens Causing Bloodstream Infections in the United States over 10 Years: Focus on Drug-Resistant Enterococcal Subsets (2010-2019).

Oritavancin displayed potent and stable activity (MIC90 range of 0.06 to 0.5 mg/L) over a 10-year period (2010 to 2019) against Gram-positive pathogens that cause bloodstream infections (BSI), including methicillin-resistant Staphylococcus aureus (MRSA) and resistant subsets of Enterococcus spp. Daptomycin and linezolid were also active against methicillin-resistant S. aureus and vancomycin-resistant Enterococcus (VRE). Only oritavancin and linezolid remained active against Enterococcus faecium isolates displaying an elevated daptomycin MIC (i.e., 2 to 4 mg/L). Proportions of methicillin-resistant S. aureus and vancomycin-resistant Enterococcus within the respective S. aureus and enterococcal populations decreased over this period.

Genotypic characterization of vancomycin-resistant Enterococcus causing urinary tract infection in northern India.

Background & objectives: Infections caused by vancomycin-resistant Enterococci are difficult to treat given the limited therapeutic alternatives. Different gene clusters are known to confer vancomycin resistance. vanA and vanB genes are transferable and are clinically relevant. This cross-sectional study aimed to identify the vancomycin-resistant genotypes in the strains causing urinary tract infection and also to test the in vitro efficacy of linezolid and pristinamycin against the vancomycin-resistant isolates. Methods: Antimicrobial resistance profile of 118 enterococcal isolates was evaluated. Minimum inhibitory concentration of vancomycin, teicoplanin and high-level gentamicin (HLG) was determined by micro broth dilution. The vancomycin-resistant isolates were tested against linezolid and pristinamycin by micro-broth dilution and E strip method. The presence of vancomycin-resistant genes was detected by multiplex polymerase chain reaction and was sequenced and analyzed. Results: Most commonly isolated species were Enterococcus faecalis (76.9%) and Enterococcus faecium (16.9%). It was found that 43 per cent of the isolates were resistant to HLG and 16.9 per cent to vancomycin. Higher resistance was seen against fluoroquinolones, erythromycin, tetracycline and ß-lactam drugs. However, 5.08 per cent strains were resistant to tigecycline. All vancomycin-resistant strains were sensitive to pristinamycin and one was resistant to linezolid. vanA and vanB gene were found in 15 and five isolates, respectively. The gene sequences were submitted to NCBI gene bank and accession numbers were obtained. Interpretation & conclusions: The present study showed prevalence of vanA and vanB genes carrying Enterococcus in a tertiary care centre in north India. The emergence of resistance against drugs such as tigecycline and linezolid is a topic of concern as it will be a therapeutic challenge for physicians.

Effect of Vancomycin on Cytoplasmic Peptidoglycan Intermediates and van Operon mRNA Levels in VanA-Type Vancomycin-Resistant Enterococcus faecium.

Resistance in VanA-type vancomycin-resistant Enterococcus faecium (VREfm) is due to an inducible gene cassette encoding seven proteins (vanRSHAXYZ). This provides for an alternative peptidoglycan (PG) biosynthesis pathway whereby D-Ala-D-Ala is replaced by D-Ala-d-lactate (Lac), to which vancomycin cannot bind effectively. This study aimed to quantify cytoplasmic levels of normal and alternative pathway PG intermediates in VanA-type VREfm by liquid chromatography-tandem mass spectrometry before and after vancomycin exposure and to correlate these changes with changes in vanA operon mRNA levels measured by real-time quantitative PCR (RT-qPCR). Normal pathway intermediates predominated in the absence of vancomycin, with low levels of alternative pathway intermediates. Extended (18-h) vancomycin exposure resulted in a mixture of the terminal normal (UDP-N-acetylmuramic acid [NAM]-l-Ala-D-Glu-l-Lys-D-Ala-D-Ala [UDP-Penta]) and alternative (UDP-NAM-l-Ala-γ-D-Glu-l-Lys-D-Ala-D-Lac [UDP-Pentadepsi]) pathway intermediates (2:3 ratio). Time course analyses revealed normal pathway intermediates responding rapidly (peaking in 3 to 10 min) and alternative pathway intermediates responding more slowly (peaking in 15 to 45 min). RT-qPCR demonstrated that vanA operon mRNA transcript levels increased rapidly after exposure, reaching maximal levels in 15 min. To resolve the effect of increased van operon protein expression on PG metabolite levels, linezolid was used to block protein biosynthesis. Surprisingly, linezolid dramatically reduced PG intermediate levels when used alone. When used in combination with vancomycin, linezolid only modestly reduced alternative UDP-linked PG intermediate levels, indicating substantial alternative pathway presence before vancomycin exposure. Comparison of PG intermediate levels between VREfm, vancomycin-sensitive Enterococcus faecium, and methicillin-resistant Staphylococcus aureus after vancomycin exposure demonstrated substantial differences between S. aureus and E. faecium PG biosynthesis pathways. IMPORTANCE VREfm is highly resistant to vancomycin due to the presence of a vancomycin resistance gene cassette. Exposure to vancomycin induces the expression of genes in this cassette, which encode enzymes that provide for an alternative PG biosynthesis pathway. In VanA-type resistance, these alternative pathway enzymes replace the D-Ala-D-Ala terminus of normal PG intermediates with D-Ala-D-Lac terminated intermediates, to which vancomycin cannot bind. While the general features of this resistance mechanism are well known, the details of the choreography between vancomycin exposure, vanA gene induction, and changes in the normal and alternative pathway intermediate levels have not been described previously. This study comprehensively explores how VREfm responds to vancomycin exposure at the mRNA and PG intermediate levels.

Thirty years of VRE in Germany - "expect the unexpected": The view from the National Reference Centre for Staphylococci and Enterococci.

Enterococci are commensals of the intestinal tract of many animals and humans. Of the various known and still unnamed new enterococcal species, only isolates of Enterococcus faecium and Enterococcus faecalis have received increased medical and public health attention. According to textbook knowledge, the majority of infections are caused by E. faecalis. In recent decades, the number of enterococcal infections has increased, with the increase being exclusively associated with a rising number of nosocomial E. faecium infections. This increase has been accompanied by the dissemination of certain hospital-acquired strain variants and an alarming progress in the development of antibiotic resistance namely vancomycin resistance. With this review we focus on a description of the specific situation of vancomycin resistance among clinical E. faecium isolates in Germany over the past 30 years. The present review describes three VRE episodes in Germany, each of which is framed by the beginning and end of the respective decade. The first episode is specified by the first appearance of VRE in 1990 and a country-wide spread of specific vanA-type VRE strains (ST117/CT24) until the late 1990s. The second decade was initially marked by regional clusters and VRE outbreaks in hospitals in South-Western Germany in 2004 and 2005, mainly caused by vanA-type VRE of ST203. Against the background of a certain "basic level" of VRE prevalence throughout Germany, an early shift from the vanA genotype to the vanB genotype in clinical isolates already occurred at the end of the 2000s without much notice. With the beginning of the third decade in 2010, VRE rates in Germany have permanently increased, first in some federal states and soon after country-wide. Besides an increase in VRE prevalence, this decade was marked by a sharp increase in vanB-type resistance and a dominance of a few, novel strain variants like ST192 and later on ST117 (CT71, CT469) and ST80 (CT1065). The largest VRE outbreak, which involved about 2,900 patients and lasted over three years, was caused by a novel and until that time, unknown strain type of ST80/CT1013 (vanB). Across all periods, VRE outbreaks were mainly oligoclonal and strain types varied over space (hospital wards) and time. The spread of VRE strains obviously respects political borders; for instance, both vancomycin-variable enterococci which were highly prevalent in Denmark and ST796 VRE which successfully disseminated in Australia and Switzerland, were still completely absent among German hospital patients, until to date.

Phytovirome Analysis of Wild Plant Populations: Comparison of Double-Stranded RNA and Virion-Associated Nucleic Acid Metagenomic Approaches.

Metagenomic studies have indicated that the diversity of plant viruses was until recently far underestimated. As important components of ecosystems, there is a need to explore the diversity and richness of the viruses associated with plant populations and to understand the drivers shaping their diversity in space and time. Two viral sequence enrichment approaches, double-stranded RNA (dsRNA) and virion-associated nucleic acids (VANA), have been used and compared here for the description of the virome of complex plant pools representative of the most prevalent plant species in unmanaged and cultivated ecosystems. A novel bioinformatics strategy was used to assess viral richness not only at the family level but also by determining operational taxonomic units (OTU) following the clustering of conserved viral domains. A large viral diversity dominated by novel dsRNA viruses was detected in all sites, while a large between-site variability limited the ability to draw a clear conclusion on the impact of cultivation. A trend for a higher diversity of dsRNA viruses was nevertheless detected in unmanaged sites (118 versus 77 unique OTUs). The dsRNA-based approach consistently revealed a broader and more comprehensive diversity for RNA viruses than the VANA approach, whatever the assessment criterion. In addition, dissimilarity analyses indicated both approaches to be largely reproducible but not necessarily convergent. These findings illustrate features of phytoviromes in various ecosystems and a novel strategy for precise virus richness estimation. These results allow us to reason methodological choices in phytovirome studies and likely in other virome studies where RNA viruses are the focal taxa.IMPORTANCE There are today significant knowledge gaps on phytovirus populations and on the drivers impacting them but also on the comparative performance-methodological approaches for their study. We used and compared two viral sequence enrichment approaches, double-stranded RNAs (dsRNA) and virion-associated nucleic acids (VANA), for phytovirome description in complex pools representative of the most prevalent plant species in unmanaged and cultivated ecosystems. Viral richness was assessed by determining operational taxonomic units (OTU) following the clustering of conserved viral domains. There is some limited evidence of an impact of cultivation on viral populations. These results provide data allowing us to reason the methodological choices in virome studies. For researchers primarily interested in RNA viruses, the dsRNA approach is recommended because it consistently provided a more comprehensive description of the analyzed phytoviromes, but it understandably underrepresented DNA viruses and bacteriophages.

Characterization of clinical enterococci isolates, focusing on the vancomycin-resistant enterococci in a tertiary hospital in China: based on the data from 2013 to 2018.

BACKGROUND: Vancomycin-resistant Enterococcus spp. (VRE) have spread all over the world. The present study aims to investigate the species distribution, specimen type and susceptibilities of Enterococcal species collected from Nanjing Drum Tower Hospital from 2013 to 2018. Additionally, distribution of VRE and prevalence of van gene among VRE isolates were also analyzed. METHODS: The susceptibilities of 3913 Enterococcus isolates were retrospectively investigated. Among these strains, 60 VRE strains were further anazlyed in this study. The minimum inhibitory concentrations (MICs) of the VRE strains towards vancomycin, teicoplanin and linezolid were determined by E-test. Polymerase chain reaction (PCR) and DNA sequencing were used to investigate the prevalence of van genes among VRE. Furthermore, the sequence types (STs) of VRE strains were explored by multi-locus sequence typing (MLST). RESULTS: Among the 3913 enterococci isolates, Enterococcus faecalis (n = 1870, 47.8%) and Enterococcus faecium (1738, 44.4%) were the main isolates. These Enterococcus strains were mainly isolated from urine (n = 1673, 42.8%), followed by secretions (n = 583, 14.9%) and ascites (n = 554, 14.2%). VRE displayed a decreasing trend year by year. Molecular analysis revealed that 49 out of 60 VRE isolates carried vanA gene, 10 carried vanM, and 1 carried both vanA and vanM genes. Sixteen distinct STs were identified among the 58 VREM, with ST78 (n = 16), ST192 (n = 8) and ST570 (n = 7) being the most dominant ones. CONCLUSIONS: E. faecalis and E. faecium were the major enterococci strains which are the main pathogens of urinary traction infections; vanA and vanM were the main determinants conferring resistance to vancomycin; ST78, ST192 and ST570 were the leading STs of VREM which displayed a decreasing trend of prevalence year by year.

Detection of Genetic Elements Carrying vanA in Vancomycin-Resistant Enterococcus saigonensis VE80T Isolated from Retail Chicken Meat.

In this study, we aimed to detect genetic elements carrying vanA in Enterococcus saigonensis VE80T isolated from retail chicken in Vietnam. The structures of vancomycin-resistance determinants and the location of vancomycin-resistance genes were detected by sequencing the vanA gene cluster, Southern hybridization analyses, and whole-genome sequence analyses. The Tn1546-related elements harboring vanA clusters, which exhibited a characteristic structure with five point mutations compared with the prototype Tn1546, were located on the 76-kb plasmid pVE80-1 of VE80T. The vanS sequence of VE80T harboring three point mutations was 100% identical to those of vancomycin-resistant enterococci isolated from poultry in Taiwan and Japan, indicating that the element may be prevalent in poultry production farms in Asia.

Detection of vancomycin nonsusceptible strains in clinical isolates of Staphylococcus aureus in northern Iran.

Glycopeptides, particularly the cell wall-acting antibiotic vancomycin, are the safest cure for methicillin-resistant Staphylococcus aureus. The aim of this study was to evaluate nonsusceptibility of clinical isolates of S. aureus to vancomycin and investigate mutations in vraSR, a cell wall synthesis regulator gene, in vancomycin-resistant strains. Susceptibility of 110 clinical strains of S. aureus to methicillin and vancomycin were determined using disc diffusion method and determination of minimum inhibitory concentration, respectively. Presence of mecA and vanA genes was determined by PCR. Determination of spa types and mutations of the vraSR gene in vancomycin nonsusceptible isolates were assessed by PCR-sequencing analyses. In total, 47 isolates (42.73%) were recognized as MRSA, three (2.73%) strains were resistant to vancomycin, and eight (7.27%) strains were vancomycin intermediates. The MIC of vancomycin was 4-64 µg/ml in these isolates. All vancomycin nonsusceptible S. aureus strains were mecA positive and one isolate was positive for the vanA gene. Spa type t030 was found as the most common type. In vraSR sequence analysis, all 11 vancomycin nonsusceptible isolates had the D59E mutation in the vraR and E45G in vraS genes. R117H, R121S, and R121I are the other identified missense mutations in the vraR gene. The identification of a high percentage of MRSA and presence of VRSA and VISA isolates is a serious warning about the treatment of future MRSA infections and reveals the need for new and effective therapeutic agents.

Species, antibiotic susceptibility profiles and van gene frequencies among enterococci isolated from patients at Mulago National Referral Hospital in Kampala, Uganda.

BACKGROUND: The increase in drug resistance to affordable antibiotics used to treat Gram positive bacterial infections has complicated the management of enterococcal infections. Resistance to vancomycin, one of the most powerful antibiotics, is of utmost concern as both intrinsic and acquired forms of resistance do occur in enterococci. This cross-sectional study aimed to determine the species, antibiotic susceptibility profiles and vanA/vanB gene frequencies among enterococci isolated from patients at Mulago Hospital in Kampala, Uganda. METHODS: Between November 2011 and October 2012, stool, urine, sputum and blood samples, as well as vaginal, endocervical, pus, ear and urethra swabs from 3229 patients were processed for isolation of bacteria, yielding 162 enterococci of which 115 were available for analysis (one isolate per specimen/patient). Species-level confirmation and susceptibility testing were determined with the Phoenix™ AST/ID Automated System, while vanA/vanB gene carriage was determined by PCR. RESULTS: Species-level identification revealed 72 isolates of E. faecalis, 20 E. gallinarum/casseliflavus, 5 E. faecium, 4 E. raffinosus and 2 isolates each for E. hirae and E. durans. Ten isolates could not be identified to species level. Antibiotic resistance was generally low especially to ampicillin, quinolones, nitrofurantoin, glycopeptides and linezolid, but high for erythromycin and tetracycline. Equally, vanA and vanB gene frequencies were low (i.e. 15.8 and 7.9%, respectively) and detected only in E. casseliflavus/gallinarum species that are intrinsically resistant to vancomycin. Vancomycin resistant isolates of E. faecalis and E. faecium were not detected. CONCLUSIONS: Enterococcus species are frequent in clinical specimens at Mulago Hospital but they are highly susceptible to common antibiotics especially ampicillin. While vancomycin resistant enterococcal (VRE) isolates of E. faecium and E. faecalis are rare in the hospital and frequency of multidrug resistance is low, non-faecium and non-faecalis VRE isolates (i.e. E. gallinarum/casseliflavus) are frequent, some with VanA/VanB (high-level) vancomycin resistance. Therefore, species-level identification of enterococci is necessary in resource limited settings to guide infection control and treatment of enterococcal infections.

Methicillin-resistant Staphylococcus aureus and glycopeptide-resistant enterococci in fecal samples of birds from South-Eastern Poland.

BACKGROUND: The incidence of human infection and colonization with methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) has increased in the recent years. Environmental sources, including bird droppings, might play an important role as resistance reservoirs. RESULTS: Fresh fecal samples were collected from rooks and wild-living birds during the autumn-winter period of 2016/2017, and tested for the presence of bacteria associated with human diseases. Besides bacteria representing the genera Enterococcus, Campylobacter, Escherichia, and Staphylococcus, Enterobacter, Citrobacter, Proteus, Hafnia, and Pseudomonas were also identified. The susceptibility of S. aureus and Enterococcus spp. isolates to methicillin, and vancomycin and teicoplanin, respectively, was analyzed to assess the avian wildlife as a reservoir of MRSA and VRE strains. Twenty-two percent of all S. aureus isolates were methicillin-resistant. These strains were screened by polymerase chain reaction (PCR), using the most widely used primer sets specific for the mecA gene. Twenty percent of all Enterococcus strains were phenotypically vancomycin-resistant. The presence of van resistance genes in these strains was investigated by PCR using vanA and vanB gene-specific primers. A good correlation between mecA gene detection and disc diffusion data was observed, while some discrepancy was noted between the PCR data and the vancomycin/teicoplanin phenotypic resistance pattern. The incidence of strains resistant to methicillin and glycopeptide antibiotics in wild-living birds was twice that in rooks. CONCLUSIONS: The study suggests that rooks from urban areas and passerine birds from the natural habitat carry antibiotic-resistant Enterococcus spp. and S. aureus strains, probably reflecting the presence of such isolates in the environmental food sources.

Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. Invasive VRE infections are difficult to treat since common therapeutic options including ampicillin and glycopeptides often fail. In vitro, most VRE remain susceptible to last-resort antibiotics such as linezolid, tigecycline and daptomycin. However, neither tigecycline nor linezolid act in a bactericidal manner, and daptomycin has proven activity only at high dosages licensed for treating enterococcal endocarditis. Despite these pharmacological and therapeutic limitations, reports on resistance to these last-resort drugs in VRE, and enterococci in general, have increased in recent years. In this review, we briefly recapitulate the current knowledge on the mode of action as well as the known and novel mechanisms of resistance and describe surveillance data on resistance to linezolid, tigecycline and daptomycin in enterococci. In addition, we also suggest a common nomenclature for designating enterococci and VRE with resistances to these important last-resort antibiotics.

Surveillance of vancomycin-resistant enterococci reveals shift in dominating clones and national spread of a vancomycin-variable vanA Enterococcus faecium ST1421-CT1134 clone, Denmark, 2015 to March 2019.

We describe clonal shifts in vanA Enterococcus faecium isolates from clinical samples obtained from patients in Denmark from 2015 to the first quarter (Q1) of 2019. During Q1 2019, the vancomycin-variable enterococci (VVE) ST1421-CT1134 vanA E. faecium became the most dominant vanA E. faecium clone and has spread to all five regions in Denmark. Among 174 E. faecium isolates with vanA, vanB or vanA/vanB genes in Q1 2019, 44% belonged to this type.