vanA-mediated high-level glycopeptide resistance in Enterococcus faecium from animal husbandry

Abstract Glycopeptide-resistant Enterococcus faecium strains were isolated from a pig farm and a poultry farm both using avoparcin as a food additive. Such organisms were not isolated in a hen's eggs-producing farm not using avoparcin. Glycopeptide-resistant enterococci were also detected in broiler chicken carcasses that were delivered to a hospital's kitchen. The resistance was determined by the vanA gene as indicated by the detection of the inducible 39-kDa cytoplasmic membrane protein and of a vanA -specific DNA sequence amplified by polymerase chain reaction. Genomic DNA fragment patterns of strains from animal sources were different from each other and also from those of strains isolated in hospitals and from sewage treatment plants. This findings suggest the dissemination of the vanA determinant among different enterococcal strains of distinct ecological origin.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Conjugative transfer of glycopeptide and macrolide resistant genes among Enterococci and from Enterococcus faecalis to Staphylococcus aureus

The resistance determinants were transferred from clinical strains of enterococci to Staphylococcus aureus strains. As recipients methicillin-resistant and methicillin-susceptible strains were used and the filter-mating procedure was performed. The transconjugants resistant to erythromycin were obtained in the case of all recipients, in one case the vanA determinant conferring the resistance to vancomycin was transferred together with erythromycin resistance. However the resistance was very unstable and the level was not as high as in the case of Enterococcus faecalis donor strain. The vanA determinant was easily transferred between enterococcal strains by the conjugation and a transfer occurred of vanA alone or together with erythromycin resistance.     

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.     

粪肠、屎肠球菌及相近种部分持家基因的系统发育分析

【目的】利用16S rRNA、clpX和recA基因分子标记研究Enterococcus faecalis、Enterococcus faecium及相近种间的种系发育关系,并比较这些基因序列对E.faecalis、E.faecium及相近种的区分能力。【方法】以分离自传统乳制品中的9株E.faecium和1株E.durans分离株为研究对象,以clpX和recA基因片段为标记,通过PCR扩增、测序,结合已公布的近缘种相应序列构建系统发育树并与16S rRNA基因进行比较。【结果】在基于clpX和recA基因的进化树中,10株试验菌株与E.faecalis始终处于同一分支。与该物种这两个基因的平均相似性为99.6%和98.6%,与另一分支的Faecium-group(E.durans和E.faecium)的平均相似性仅为61.5%和33.5%。相近种E.durans和E.hirae间这两个基因的差异性为20.3%和39.0%;在基于16S rRNA基因的进化树中,试验菌株与Faecium-group(E.lactis、E.faecium、E.durans、E.hirae)处于同一分支。与这些成员间该基因的相似性大于99.6%,与E.faecalis基因的平均相似性可达98.4%。相近种间该基因相似性无明显差异。【结论】按照10株试验菌株clpX和recA基因的分析结果可将由传统生理生化和16S rRNA基因序列鉴定的9株E.faecium和1株E.durans归类为E.faecalis,clpX和recA基因可用于部分相近种的分类鉴定。     

The tyrosine decarboxylation test does not differentiate Enterococcus faecalis from Enterococcus faecium

According to the current edition of the Bergey's Manual of Systematic Bacteriology [11] the tyrosine decarboxylation test allows the differentiation of enterococci. Tyrosine is decarboxylated to the biogenic amine tyramine by E. faecalis and not by E. faecium strains. In the present study we sequenced the16S rDNA of two tyramine-producing strains, BIFI-56 and BIFI-58, presumptively classified as E. faecalis. Their 16S rDNA were identical to the same fragment from the E. faecium type strain. Several E. faecium strains were then checked for their ability to decarboxylate tyrosine and also a putative tyrosine decarboxylase-coding gene was PCR amplified from these strains. All the strains confirmed as E. faecium produced tyramine and possessed a DNA fragment coding for a putative tyrosine decarboxylase. The concordance of the two methods allows us to conclude that the tyrosine decarboxylase test cannot be used in the differentiation of E. faecalis from E. faecium since at least some E. faecium strains are tyramine producers.     

Detection of penicillin-binding proteins immunologically related to penicillin-binding protein 5 of Enterococcus hirae ATCC 9790 in Enterococcus faecium and Enterococcus faecalis

Penicillin-binding protein (PBP) 5 of Enterococcus hirae ATCC 9790 belongs to the class of the high-molecular mass, low-affinity PBPs which have been correlated with penicillin resistance in most Enterococcus species. Polyclonal antibodies were raised against PBP 5 and used to detect immunologically related membrane proteins in E. faecium and E. faecalis strains. Several strains of both species were found to have a membrane protein of similar molecular mass to E. hirae PBP 5 which reacted with the antibodies. Some E. faecium strains did not react with antibodies but their derivatives with increased penicillin minimal inhibitory concentrations did. In some E. faecalis strains the lack of a PBP 5-related protein was associated with failure to select stable penicillin-resistant derivatives.     

High level of aminoglycoside resistance among Enterococcus faecalis and Enterococcus faecium strains

Enterococcus sp. strains are believed as important reason of serious nosocomial infections currently. These infections are cured by using combination of beta-lactams and aminoglycosides for their treatment. Enterococcus sp. resistant to high-level doses of aminoglycosides, beta-lactams and vancomycin are responsible for therapeutic failure. The aim of our study was to evaluate the incidence of isolation and susceptibility to antibiotics of HLAR Enterococcus sp. strains isolated between 2007 and 2010 from the patients of University Hospital No. 1 of dr A. Jurasz Collegium Medicum of L. Rydygier in Bydgoszcz Nicolaus Copernicus University in Toruń. Amongst 6137 Enterococcus sp. strains 1124 (18,3%) presented HLAR phenotype; 53,1% of them was identified as E. faecalis and 46,9% as E. faecium. The highest percentage of all examined strains was isolated from the patients of different surgery clinics, Intensive Care Units, and Pediatrics, Hematology and Oncology Clinic. HLAR and HLSR phenotypes were noted in E. faecalis, for 45,7% and 27,5% strains, in E. faecium - 29,8% and 9,5%, respectively. HLGR phenotype was presented twice more often in E. faecium than E. faecalis. Highest percentages of E. faecium resistant to glycopeptides and rifampicin were observed when compared with E. faecalis. The highest percentages of strains intermediate, resistant to vancomycin and resistant to glycopeptides were noted for E. faecium strains with phenotypes HLAR, HLGR and HLSR.     

Species composition and antimicrobial resistance genes of Enterococcus spp,isolated from integrated and traditional fish farms in Thailand

Integrated fish farming uses the manure from animal husbandry as fertilisers in the fish ponds. A total of 410 enterococcal isolates, from integrated and traditional fish farms in Thailand, were collected to assess whether the input of manure from chickens receiving feed containing growth promoters and antimicrobial treatments influenced the species composition and the bacterial antimicrobial resistance in the fish pond environment. Enterococcus faecium and E. faecalis were the predominate species isolated from the integrated farms, whereas E. casseliflavus and E. mundtii isolates were most prevalent in traditional farms. Enterococcus faecalis and E. faecium demonstrated the highest prevalence of resistance, whereas E. mundtii isolates were susceptible to all antimicrobials tested. All the enterococci species isolated from the integrated farms, generally demonstrated higher resistance phenotypes to the tested antimicrobials compared with the same species from traditional farms. The erm(B) and tet(M) genes, associated with resistance to erythromycin and tetracycline, respectively, were found in 87% of the erythromycin-resistant and 95% of the oxytetracycline-resistant enterococci isolates respectively. These results suggest that the species composition and antimicrobial resistance of enterococci in tropical aquatic environments are influenced by faecal and antimicrobial pollution.     

Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium isolates from hospitals in Tehran

The prevalence of glycopeptides, aminoglycosides and erythromycin resistance among Enterococcus faecalis and Enterococcus faecium was investigated. The susceptibility of 326 enterococcal hospital isolates to amikacin, kanamycin, netilmicin and tobramycin were determined using disk diffusion method. The minimum inhibitory concentration (MIC) of vancomycin, teicoplanin, gentamicin, streptomycin, and erythromycin were determined by microbroth dilution method. The genes encoding aminoglycoside modifying enzymes described as AMEs genes, erythromycin-resistant methylase (erm) and vancomycin-resistant were targeted by multiplex-PCR reaction. High level resistance (HLR) to gentamicin and streptomycin among enterococci isolates were 52% and 72% respectively. The most prevalent of AMEs genes were aac (6')-Ie aph (2") (63%) followed by aph (3')-IIIa (37%). The erythromycin resistance was 45% and 41% of isolates were positive for ermB gene. The ermA gene was found in 5% of isolates whereas the ermC gene was not detected in any isolates. The prevalence of vancomycin resistant enterococci (VRE) was 12% consisting of E. faecalis (6%) and E. faecium (22%) and all of them were VanA Phenotype. The results demonstrated that AMEs, erm and van genes are common in enterococci isolated in Tehran. Furthermore our results show an increase in the rate of vancomycin resistance among enterococci isolates in Iran.     

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.     

Identification and characterization of enterococci from bryndza cheese

AIMS: To identify enterococci isolated from sheep milk cheese--bryndza, and to compare differences in the composition of enterococcal microflora affected by the season, and to evaluate the potential presence of vancomycin resistance and virulence determinants. METHODS AND RESULTS: Bacterial strains were isolated during analysis of bryndza cheese and identified on the genus and species level by phenotypic methods and with commercial biochemical sets. The identification of the species, Enterococcus faecium, Ent. durans and Ent. faecalis, was confirmed by PCR using species-specific primers for ddl genes. PCR was also used for assessment of presence of vanA and vanB genes and virulence determinants gelE, agg and cytolysin genes namely: cylL(L), cylL(S), cylM, cylB and cylA. Among 308 Enterococcus sp. strains, 177 isolates were proved to be Ent. faecium, 59 to be Ent. durans and 41 to be Ent. faecalis. Vancomycin resistance genes vanA and vanB were not detected. Agar plate testing confirmed their absence. Gene gelE, however, was found in 20 Ent. faecalis isolates, but only 13 of them showed gelatinase-positive phenotype. Seven isolates had five cytolysin genes, but none of the isolates exhibited a positive haemolytic phenotype. Four isolates possessed the agg gene. The prevalence of Ent. faecium species was highest in samples from the winter season harvest. CONCLUSIONS: Ent. faecium is the dominant enterococcal species in bryndza cheese and the most prevalent in the winter season product. None of the Enterococcus sp. strains was proved to have vanA or vanB genes and the vancomycin resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of enterococcal microflora in bryndza cheese and its evaluation for the presence of vanA and vanB genes as well as virulence determinants.     

血流感染粪肠球菌和屎肠球菌的临床分布及耐药研究

回顾性分析上海市某三甲医院血培养阳性标本中粪肠球菌和屎肠球菌的临床分布及对抗菌药物的耐药特征,为临床治疗其所致感染奠定基础。收集上海市某三甲医院2012年2月—2016年9月血流感染患者血液标本中的粪肠球菌和屎肠球菌,采用法国生物梅里埃公司的VITEK 2Compact全自动细菌鉴定和药敏分析系统进行细菌鉴定及药敏测定,研究细菌临床分布特点及对常用抗菌药物的耐药特征。共分离获得30株粪肠球菌和17株屎肠球菌。粪肠球菌样本主要来自泌尿科、消化科和血液科,所占比例分别为13.33%、16.67%和10.00%。粪肠球菌对青霉素、氨苄西林、环丙沙星、左氧氟沙星、四环素和红霉素的耐药率分别为13.33%、10.00%、36.67%、33.33%、66.67%和60.00%。屎肠球菌样本主要来自消化科(29.41%),其对以上抗菌药物的耐药率分别为88.24%、82.35%、88.24%、76.47%、23.53%和70.59%。屎肠球菌对青霉素、氨苄西林、环丙沙星和左氧氟沙星的耐药率显著高于粪肠球菌,而对四环素的耐药率显著低于粪肠球菌。两者均对替加环素、利奈唑胺和万古霉素敏感,但万古霉素对屎肠球菌的最低抑菌浓度显著低于粪肠球菌。结果提示,屎肠球菌对青霉素、氨苄西林、环丙沙星、左氧氟沙星的耐药率高于屎肠球菌,对万古霉素敏感,且其万古霉素最低抑菌浓度低于粪肠球菌。本研究为治疗这两种细菌所致感染的经验性用药提供了数据支持。     

Preliminary characterization of bacteriocins produced by Enterococcus faecium and Enterococcus faecalis isolated from pig faeces

A total of 92 enterococci, isolated from the faeces of minipigs subjected to an in vivo feeding trial, were screened for the production of antimicrobial substances. Bacteriocin production was confirmed for seven strains, of which four were identified as Enterococcus faecalis and three as Enterococcus faecium, on the basis of physiological and biochemical characteristics. The bacteriocins produced by the Ent. faecalis strains showed a narrow spectrum of activity, mainly against other Enterococcus spp., compared with those from the Ent. faecium strains showing a broader spectrum of activity, against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp. and Propionibacterium spp. The bacteriocins of all seven Enterococcus strains were inactivated by alpha-chymotrypsin, proteinase K, trypsin, pronase, pepsin and papain, but not by lipase, lysozyme and catalase. The bacteriocins were heat stable and displayed highest activity at neutral pH. The molecular weight of the bacteriocins, as determined by tricine SDS-PAGE, was approximately 3.4 kDa. Only the strains of Ent. faecalis were found to contain plasmids. PCR detection revealed that the bacteriocins produced by Ent. faecium BFE 1170 and BFE 1228 were similar to enterocin A, whereas those produced by Ent. faecium BFE 1072 displayed homology with enterocin L50A and B.     

Drug resistance of Enterococcus species isolated from the urogenital system

Despite low virulence of enterococci, they have become important nosocomial pathogens. This has been correlated with the increased use of broad-spectrum antibiotics, particularly cephalosporins. Many strains of enterococci exhibit multiple drug resistance; the most important being high-level resistance (HLR) to penicillin (MIC > 100 mg/l) and gentamicin (MIC > 500 mg/l and 2000 mg/l) and/or streptomycin (MIC > 2000 mg/l). The investigation was performed on 92 strains, isolated from genito-urinary tract and recognised as Enterococcus sp. All strains were obtained from several microbiological laboratories of Gdańsk, Gdynia and Tczew. On biochemical reaction profiles species of enterococci were identified as: E. faecalis (72.8%), E. faecalis varians (9.8%), E. durans (7.6%) and E. faecium (9.8%). The minimal inhibitory concentration (MICs) of penicillin, ampicillin, azlocillin, imipenem, gentamicin, amicacin, ciprofioxacin and vancomycin were determined by the agar dilution method. None of these 92 enterococcal strains was vancomycin resistant. 22.2% of E. faecium and 7.5% of E. faecalis showed high-level resistance to penicillin. None of these strains were produced beta-lactamase. High-level resistance to streptomycin and gentamicin was detected. Both--high-level resistance to streptomycin and gentamicin--were found in 6% E. faecalis; 11.1% E. faecalis varians and 22.2% E. faecium.     

Effect of the probiotic Enterococcus faecium NCIMB10415 on cell numbers of total Enterococcus spp., E. faecium and E. faecalis in the intestine of piglets

Sows and their piglets were fed a diet supplemented with or without the probiotic E. faecium NCIMB10415 (also known as SF68). Piglets were sacrificed 14, 28, 35 and 56 days after birth and DNA from intestinal segments was extracted and purified. A real time PCR assay was used to distinguish Enterococcus spp. (16s rDNA based), E. faecium (Efaafm gene), E. faecalis (Efaafs gene) as well as the probiotic strain (unique plasmid sequence). Extracts of autoclaved sow feces inoculated with E. faecium and E. faecalis cultures were used to calibrate real time PCR results. The probiotic strain was detected in 14 day old suckling piglets before the piglets had access to the starter diet. In piglets of the probiotic group, probiotic E. faecium cell counts were always a significant proportion of total E. faecium cells in stomach digesta (4-20%), however only a small fraction of the total Enterococcus spp. cell number on day 14 and 28 in all intestinal segments (0.1-0.7%). Compared to control samples, the probiotic E. faecium strain significantly (p < or = 0.05) decreased the amount of total Enterococcus spp. and E. faecalis cells in the colon of 14 day old suckling piglets as well as in jejunum and colon samples one week after weaning. E. faecium cell counts were not modified on any sampling day or intestinal segment. This study showed that the presence of probiotic E. faecium NCIMB10415 coincided with reduced total E. faecalis, but not total E. faecium cell numbers in the intestine of piglets. In view of unchanged cell numbers and ratios in sow feces, modifications must have taken place within the intestine of suckling piglets.     

Genetic diversity and safety aspects of enterococci from slightly fermented sausages

AIMS: To determine the biodiversity of enterococci from slightly fermented sausages (chorizo and fuet) at species and strain level by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR and partial sequencing of 16S rRNA and sodA genes were used to identify enterococcal population. Enterococcus faecium was the most frequently isolated species followed by E. faecalis, E. hirae and E. durans. Randomly amplified polymorphic DNA (RAPD)-PCR revealed species-specific clusters and allowed strain typing. Sixty strains of 106 isolates exhibited different RAPD profiles indicating a high genetic variability. All the E. faecalis strains carried virulence genes (efaAfs, esp, agg and gelE) and all E. faecium isolates carried efaAfm gene. Enterococcus faecalis showed higher antibiotic resistance than the other species. Only one E. faecium strain showed vanA genotype (high-level resistance to glycopeptides) and E. gallinarum and E. casseliflavus/flavescens isolates showed vanC1 and vanC2/C3 genotypes (low-level resistance only to vancomycin) respectively. CONCLUSIONS: E. faecalis has been mainly associated with virulence factors and antimicrobial multi-resistance and, although potential risk for human health is low, the presence of this species in slightly fermented sausages should be avoided to obtain high quality products. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal population of slightly fermented sausages has been thoroughly characterized. Several relevant safety aspects have been revealed.     

Galleria mellonella as a model for studying Enterococcus faecium host persistence

Enterococcus faecium is an opportunistic pathogen responsible for numerous outbreaks worldwide. The basis for the colonization capacities, host persistence and environmental stress response of the hospital-adapted clones emerging from E. faecium are poorly understood. In this study, we propose the use of Galleria mellonella as a simple nonmammalian model to assess E. faecium host persistence. Various strains (n = 10), including hospital-adapted, commensal or animal isolates and a SodA-deficient strain were used to assess the relevance of this model. Compared to Enterococcus faecalis, E. faecium strains do not appear very lethal in a Galleria killing assay. The ability of E. faecium strains to overcome host-immune responses and multiply within the host system was evaluated by monitoring bacterial loads following Galleria infection. Among the E. faecium strains, two hospital-adapted isolates displayed increased colonization ability. In contrast, inactivation of sodA, encoding a putative manganese-dependent superoxide dismutase, significantly reduced survival of E. faecium to Galleria defenses. Galleria appears to be a suitable and convenient surrogate model to study E. faecium survival to host defenses and the role of suspected virulence factors in the colonization process.     

Antimicrobial resistance of Enterococcus species isolated from produce

The purpose of this study was to characterize the antibiotic resistance profiles of Enterococcus species isolated from fresh produce harvested in the southwestern United States. Among the 185 Enterococcus isolates obtained, 97 (52%) were Enterococcus faecium, 38 (21%) were Enterococcus faecalis, and 50 (27%) were other Enterococcus species. Of human clinical importance, E. faecium strains had a much higher prevalence of resistance to ciprofloxacin, tetracycline, and nitrofurantoin than E. faecalis. E. faecalis strains had a low prevalence of resistance to antibiotics used to treat E. faecalis infections of both clinical and of agricultural relevance, excluding its intrinsic resistance patterns. Thirty-four percent of the isolates had multiple-drug-resistance patterns, excluding intrinsic resistance. Data on the prevalence and types of antibiotic resistance in Enterococcus species isolated from fresh produce may be used to describe baseline antibiotic susceptibility profiles associated with Enterococcus spp. isolated from the environment. The data collected may also help elucidate the role of foods in the transmission of antibiotic-resistant strains to human populations.