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Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice.

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.

Animals↗

A six amino acid deletion, partially overlapping the VanSB G2 ATP-binding motif, leads to constitutive glycopeptide resistance in VanB-type Enterococcus faecium.

Enterococcus faecium clinical isolate BM4524, resistant to vancomycin and susceptible to teicoplanin, harboured a chromosomal vanB cluster, including the vanSB/vanRB two-component system regulatory genes. Enterococcus faecium strain BM4525, isolated two weeks later from the same patient, was resistant to high levels of both glycopeptides. The ddl gene of BM4525 had a 2 bp insertion leading to an impaired d-alanine:d-alanine ligase. Sequencing of the vanB operon in BM4525 also revealed an 18 bp deletion in the vanSB gene designated vanSBDelta. The resulting six amino acid deletion partially overlapped the G2 ATP-binding domain of the VanSBDelta histidine kinase leading to constitutive expression of the resistance genes. Sequence analysis indicated that the deletion occurred between two tandemly arranged heptanucleotide direct repeats, separated by 11 base-pairs. The VanSB, VanSBDelta and VanRB proteins were overproduced in Escherichia coli and purified. In vitro autophosphorylation of the VanSB and VanSBDelta histidine kinases and phosphotransfer to the VanRB response regulator did not differ significantly. However, VanSBDelta was deficient in VanRB phosphatase activity leading to accumulation of phosphorylated VanRB. Increased glycopeptide resistance in E. faecium BM4525 was therefore a result of the lack of production of d-alanyl-d-alanine ending pentapeptide and to constitutive synthesis of d-alanyl-d-lactate terminating peptidoglycan precursors, following loss of d-alanine:d-alanine ligase and of VanSB phosphatase activity respectively. We suggest that the heptanucleotide direct repeat in vanSB may favour the appearance of high level constitutively expressed vancomycin resistance through a 'slippage' type of genetic rearrangement in VanB-type strains.

Bacterial Proteins↗

In-vitro selection of resistance to vancomycin and teicoplanin in Enterococcus faecium and Enterococcus faecalis compared with Staphylococcus epidermidis.

Clinical isolates of Enterococcus faecium, Enterococcus faecalis and Staphylococcus epidermidis were studied for their ability to develop resistance by two selection methods, one in broth and one on agar. The MICs for enterococci after exposure to vancomycin ranged from 0.5 mg/L to 4 mg/L, and those after exposure to teicoplanin ranged from 0.25 mg/L to 1 mg/L. No significant increases occurred for E. faecalis, whilst two isolates of E. faecium showed increases of up to eight-fold (from 0.125 mg/L to 1 mg/L). Vancomycin MICs ranged from 1 mg/L to 4 mg/L and teicoplanin MICs reached 32 mg/L for S. epidermidis. Pulsed-field gel electrophoresis analysis of SmaI-digested whole chromosomal DNA was performed to compare the genomic DNA of glycopeptide-exposed and wild-type strains. In-vitro exposure to vancomycin did not alter MICs significantly except in one S. epidermidis isolate for which the MIC reached 4 mg/L, whereas the teicoplanin-exposed cultures of S. epidermidis showed increases of up to 64 times the original MIC. Comparable results were achieved with the two selection methods.

Anti-Bacterial Agents↗

Comparative genomic characterization and antimicrobial resistance of bacteremia-causing Enterococcus faecium and Enterococcus faecalis in a Chinese hospital.

Enterococci are common commensals of the human gut and important opportunistic pathogens, with Enterococcus faecium and Enterococcus faecalis being the most clinically prevalent species. A significant epidemiological shift has emerged with an increasing clinical burden of E. faecium. To compare genomic evolution of E. faecium and E. faecalis, we performed whole-genome sequencing on 93 E. faecium and 32 E. faecalis isolates causing bloodstream infections at a single hospital (2022-2024). Analysis of patient demographics revealed that E. faecium infections originated from fewer sources than E. faecalis, with a higher proportion deriving from intra-abdominal infections. Multilocus sequence typing identified ST78 and ST789 as the predominant sequence types for E. faecium, whereas ST16 and ST179 were most common for E. faecalis. E. faecium carried more antimicrobial resistance genes and putative virulence marker (PVM)-type virulence genes than E. faecalis, with vancomycin resistance predominantly mediated by vanHAX (33/93, 35.5%) and a single E. faecalis isolate also carrying vanHAX (1/32, 3.1%); the structurally incomplete vanHMX gene cluster was detected in 11 E. faecium isolates. Pan-genome analysis indicated a larger core genome in E. faecalis compared to E. faecium, consistent with greater plasmid replicon diversity in the latter. Intra-host comparisons showed that two E. faecalis pairs from the same patient were clonally related, with one isolate acquiring a vanHAX plasmid conferring vancomycin resistance. In contrast, E. faecium isolates exhibited marked genomic diversity even among clonally related pairs. These findings suggest that E. faecium possesses greater genomic plasticity and adaptive potential to the clinical environment.IMPORTANCEThis study provides a detailed comparison of clinical and genomic features between Enterococcus faecium and Enterococcus faecalis from the same hospital setting. We show that E. faecium isolates, mainly ST78/ST789, carry more antimicrobial resistance genes and a higher number of putative virulence marker (PVM) genes than E. faecalis, reflecting their hospital-adapted nature. E. faecium also exhibits a smaller core genome and greater diversity of plasmid replicon types, indicating higher genomic plasticity and capacity for horizontal gene transfer. By contrast, E. faecalis retains a larger core genome and a set of classical virulence factors, and its within-host isolates are clonally related. These distinct genomic profiles help to understand how the two species adapt to clinical environments and may inform more targeted infection control strategies and resistance surveillance.

Enterococcus faecium↗

A PCR assay for identification of Enterococcus faecium.

Enterococcus faecium has recently emerged as a serious nosocomial pathogen. The prevalence and severity of enterococcal infections, the mortality rate from such infections, and the antibiotic resistance of enterococci are often species dependent. Since conventional biochemical methods fail to differentiate E. faecium from certain newly described enterococcal species, a PCR-based assay was developed for the rapid identification of E. faecium.

Enterococcus faecium↗

Mechanisms of resistance to imipenem in imipenem-resistant, ampicillin-sensitive Enterococcus faecium.

Enterococcus faecium has six penicillin-binding proteins (PBP), where PBP5 seems to be the main target for beta-lactam antibiotics. The PBP profiles of three imipenem-resistant, ampicillin-sensitive E. faecium strains, isolated from the same patient, were studied using biotinylated ampicillin and chemiluminescence detection. Imipenem resistance in these strains was found to be associated with hyperproduction of PBP5 compared to the ampicillin- and imipenem-susceptible strain ATCC 19434. PBP5 in the imipenem-resistant strains (S1, B2) exhibited a selectively decreased affinity for imipenem. An 854 bp DNA fragment, corresponding to the penicillin-binding domain of pbp5fm, was studied in the resistant strains and the reference strain. Four amino acid substitutions were observed in the resistant strains compared to the susceptible one. The contribution of these substitutions to the increased production of PBP5 in these strains is unclear since the substitution was observed also in a strain without increased production of PBP5. Our results suggest that the moderate imipenem resistance observed in these strains is associated with increased production of PBP5 with relatively decreased affinity for imipenem, and that evolution of imipenem resistance in E. faecium is dinstinct from that of the other beta-lactams such as ampicillin.

Ampicillin↗

Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium.

Enterococcus faecalis and Enterococcus faecium infections are increasingly difficult to treat due to high levels of resistance to antibiotics. PlyV12, a bacteriophage lytic enzyme, was isolated and shown to effectively kill both E. faecalis and E. faecium (including vancomycin-resistant strains), as well as other human pathogens. We propose its development and use as an alternative therapeutic tool.

Amino Acid Sequence↗

Organization of heat shock dnaK and groE operons of the nosocomial pathogen Enterococcus faecium.

Enterococcus faecium is a frequently antibiotic-resistant opportunistic pathogen that is commonly recovered from hospitalized patients. The genetic organization of the dnaK operon was analyzed and was shown to consist of at least four heat shock genes, hrcA-grpE-dnaK-dnaJ. The dnaK/J intergenic region was 140 bp shorter than in E. faecalis. The dnaK operon was expressed from a putative sigma(A)-type promoter (PhrcA) upstream of the hrcA start codon and was preceded by two conserved CIRCE sequences. Northern hybridization revealed the presence of multiple mRNAs in the dnaK operon. Conversely, the groE operon was transcribed as a single mRNA. Induction of dnaK and groEL genes occurred in response to either heat shock or exposure to other stress agents.

Bacterial Proteins↗

Molecular detection of linezolid resistance in Enterococcus faecium and Enterococcus faecalis by use of 5' nuclease real-time PCR compared to a modified classical approach.

A nucleotide transversion from guanine to uracil in the 23S rRNA confers linezolid resistance. We describe a real-time PCR using two Taqman probes that detects a single mutated allele among the genomes of Enterococcus faecium and Enterococcus faecalis. Results were confirmed by a classical approach involving LabChip technology assayed with an Agilent Bioanalyzer 2100.

Acetamides↗

Transferable vancomycin and teicoplanin resistance in Enterococcus faecium.

Enterococcus faecium BM4165 and BM4178, isolated from immunocompromised patients, one treated with vancomycin, were inducibly resistant to high levels of the glycopeptide antibiotics vancomycin and teicoplanin but susceptible to the new lipopeptide daptomycin (LY146032). Strain BM4165 was also resistant to macrolidelincosamide-streptogramin B-type (MLS) antibiotics. The genes conferring resistance to glycopeptides and to MLS antibiotics in strain BM4165 were carried on plasmids pIP819 and pIP821, respectively; pIP819 also carried genes that encoded resistance to MLS antibiotics. The two plasmids, which were distinct although related, were self-transferable to other E. faecium strains. Plasmid pIP819 could also conjugate to E. faecalis, Streptococcus sanguis, S. pyogenes, S. lactis, and Listeria monocytogenes, in which it conferred inducible glycopeptide resistance, but not to S. aureus. Glycopeptide-inactivating activity was not detected, and the biochemical mechanism of resistance remains unknown. Based on this first report of transferable resistance to glycopeptides, we anticipate dissemination of resistance to these antibiotics in gram-positive cocci and bacilli in which it can be phenotypically expressed.

Adult↗

Conjugative transfer of the virulence gene, esp, among isolates of Enterococcus faecium and Enterococcus faecalis.

OBJECTIVES: The enterococcal surface protein gene, esp, is a major putative pathogenicity marker in clinical isolates of Enterococcus faecium and Enterococcus faecalis. This study demonstrates in vitro conjugative transfer of the esp gene among E. faecium and E. faecalis. MATERIALS AND METHODS: Enterococcal isolates from clinical samples, positive for esp, were mated on filters with enterococcal recipients. Transconjugants were checked for transfer of antibiotic resistance determinants and co-mobilization of the esp gene. They were also characterized by PCR and plasmid profiling/PFGE typing including Southern hybridizations with labelled esp probes. Transfer as triggered by excision was tested using Taqman PCR. RESULTS: Two of five E. faecalis and five of nine E. faecium transferred antibiotic resistance determinants into a recipient. Of the transconjugants analysed by PCR for acquisition of esp, only isolates from two E. faecalis and a single E. faecium mating were positive. In the donor strains, the esp gene was located on the chromosome. Molecular analysis revealed a plasmid localization of esp in the E. faecium transconjugant and chromosome-to-chromosome transfer in E. faecalis. CONCLUSION: The esp gene is transferable by conjugation among enterococcal isolates.

Conjugation, Genetic↗

Phenotypic distinction in Enterococcus faecium and Enterococcus faecalis strains between susceptibility and resistance to growth-enhancing antibiotics.

Susceptibility of Enterococcus faecium and Enterococcus faecalis strains from animals and foods to growth-promoting antibiotics used in animal feed was tested by the agar dilution technique. Acquired resistance to bacitracin, narasin, tylosin, and virginiamycin was seen for both species, and for E. faecium, resistance to avilamycin and avoparcin was also seen. Drawing the distinction between susceptibility and resistance based on frequency distributions of MICs was easy with avoparcin, avilamycin, and tylosin but difficult with virginiamycin and to some extent also with bacitracin and narasin.

Animals↗

[The lactic acid enterococci Enterococcus faecium and Enterococcus durans: nucleotide sequence diversity in 16S rRNA genes].

Among the strains used as starters for making sour milk products on the territory of the CIS, the bacteria Enterococcus faecium and Enterococcus durans are frequently found. In this work, we studied a new collection of lactic acid enterococci and also obtained more complete data on the nucleotide sequences of 16S rRNA genes in some strains studied earlier and found that most strains had certain distinctions in their 16S rRNA genes as compared with the E. durans and E. faecium genes available in the NCBI database. Based on these data, it is suggested that the strains of lactic acid enterococci represent new, earlier unknown taxa of enterococci that use milk as an ecological niche.

Armenia↗

Molecular characterization of clinical and environmental isolates of vancomycin-resistant Enterococcus faecium and Enterococcus faecalis from a teaching hospital in Wales.

The present study describes the first molecular characterization of environmental and clinical isolates of vancomycin-resistant enterococci (VRE) in Wales. Over a 3-month period (May-July 2000), 134 isolates of VRE (89 Enterococcus faecium and 45 Enterococcus faecalis) were isolated from the patient environment of the University Hospital of Wales (UHW) in Cardiff, Wales, UK. In addition, over the same time-period, 24 clinical isolates of VRE (20 isolates of E. faecium and four isolates of E. faecalis) were obtained from 14 patients. All study isolates were subjected to PFGE typing and their van genotypes were determined by using multiplex PCR. The vanA PCR product (231 bp) was evident in 146 (92 %) of 158 VRE isolates; the remaining 12 isolates (8 %) were positive for the vanB gene. All isolates of E. faecalis were found to be vanA-positive. In total, 16 PFGE banding profiles (pulsotypes) were observed for environmental isolates of E. faecium, whilst eight pulsotypes were found for isolates of E. faecalis. Some of these pulsotypes were isolated from multiple sites, whereas others were more restricted in their distribution. Eleven pulsotypes were evident for clinical isolates and eight of these (representing 11 isolates) were also encountered in environmental isolates. Eleven clinical isolates of E. faecium (55 %) shared an identical pulsotype that was not detected in environmental isolates. These results demonstrate a heterogeneous environmental population of VRE and an association of certain strains with clinical isolates. Predominance of a single pulsotype (not detected in the environment) amongst clinical isolates suggests non-environmental transmission between patients.

Bacterial Proteins↗

Incomplete cross resistance against ionophores in Enterococcus faecium and Enterococcus faecalis strains from pigs and poultry.

Thirty-two Enterococcus faecium strains and 33 Enterococcus faecalis strains were tested for their susceptibility to the ionophore antibiotics salinomycin, narasin, monensin, and lasalocid. Enterococcal strains originated from poultry in which these products are in use as coccidiostats, and from pigs in which these products are allowed as growth promoters. Resistance against salinomycin and narasin in enterococci was frequent among poultry strains, whereas in pig strains, resistance was less common. No resistance was found against monensin and lasalocid. Full cross resistance between salinomycin and narasin was evident. There was no cross resistance between these two ionophores and monensin and lasalocid.

Animals↗

Glycopeptide susceptibility among Danish Enterococcus faecium and Enterococcus faecalis isolates of animal and human origin and PCR identification of genes within the VanA cluster.

The MICs of vancomycin and avoparcin were determined for isolates of Enterococcus faecium and isolates of Enterococcus faecalis recovered from the feces of humans and animals in Denmark. Two hundred twenty-one of 376 (59%) isolates of E. faecium and 2 of 133 (1.5%) isolates of E. faecalis were resistant to vancomycin (MICs, 128 to > or = 256 micrograms/ml), and all vancomycin-resistant isolates were resistant to avoparcin (MICs, 64 to > or = 256 micrograms/ml). All vancomycin-resistant isolates examined carried the vanA, vanX, and vanR genes, suggesting that a gene cluster similar to that of the transposon Tn1546 was responsible for the resistance.

Animals↗

Enterococcus faecium and Enterococcus faecalis bacteremia: acquisition and outcome.

The incidence of enterococcal bacteremia due to Enterococcus faecium is increasing. To understand the clinical significance of E. faecium bacteremia, we compared 16 patients who were bacteremic due to E. faecium to 56 patients who were bacteremic due to Enterococcus faecalis. E. faecium bacteremia developed most frequently in severely ill patients with fever or hypothermia accompanied by CNS, cardiovascular, and/or pulmonary dysfunction, while E. faecalis bacteremia occurred most often in less seriously ill patients. Nosocomial acquisition, cancer, neutropenia, renal insufficiency, current corticosteroid therapy, and previous treatment with broad-spectrum antibiotics were significantly more frequently associated with E. faecium bacteremia. Mortality was significantly higher among patients infected with E. faecium than among those infected with E. faecalis (50% vs. 11%; P = .001); this was true particularly among patients with monomicrobial or nosocomial bacteremia, those who had previously received antibiotic treatment, and those with cancer. Death due to enterococcal bacteremia was observed only among severely ill patients. These findings suggest that E. faecium often infects debilitated patients and that such infection appears to be a significant factor contributing to mortality.

Bacteremia↗