Identification of enterococcal isolates by temperature gradient gel electrophoresis and partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions.

Based on partial sequence analysis of PCR-amplified 16S rDNA variable V6 regions of 14 enterococcal type strains, Enterococcus faecalis, Enterococcus mundtii, Enterococcus gallinarum, Enterococcus avium, Enterococcus raffinosus and Enterococcus saccharolyticus showed characteristic sequence motifs which made it possible to separate them into six individual species lines. Furthermore, two species cluster groups could be identified, including (i) Enterococcus faecium, Enterococcus durans, Enterococcus hirae, Enterococcus malodoratus, and (ii) Enterococcus casseliflavus/Enterococcus flavescens, Enterococcus pseudoavium, Enterococcus dispar and Enterococcus sulfureus. There were identical DNA sequences in the V6 region within each group. Temporal temperature gradient gel electrophoresis (TTGE) of the PCR products from 16 type strains, 12 enterococcal reference strains and 8 clinical isolates revealed that a single nucleotide divergence in DNA sequences was sufficient for separation, identification and division of the studied enterococcal strains into corresponding TTGE profiles. It was concluded that partial DNA sequence analysis and TTGE profiling of PCR-amplified 16S rDNA variable V6 regions provide useful tools for the identification of clinically important Enterococcus spp.     

Species identification of enterococci via intergenic ribosomal PCR.

Accurate species identification of enterococci has become important with the wide prevalence of acquired vancomycin resistance and the presence of less epidemiologically important, inherently vancomycin-resistant enterococci. Using a collection of enterococcal strains, we found that PCR amplification of the intergenic spacer (ITS-PCR) between the 16S and 23S rRNA genes can produce amplicon profiles characteristic of the enterococcus examined. The species examined were group I enterococci (Enterococcus avium, Enterococcus raffinosus, Enterococcus malodoratus, and Enterococcus pseudoavium), group II enterococci (Enterococcus faecalis, Enterococcus faecium, Enterococcus casseliflavus, Enterococcus mundtii, and Enterococcus gallinarum), and group III enterococci (Enterococcus durans and Enterococcus hirae). The enterococcal species in group I, as well as E. faecalis and two strains of E. hirae, were similar and therefore had to be differentiated from each other by Sau3A restriction digests. This produced patterns characteristic of each of these species. The remaining group II and group III enterococcal species produced amplicons characteristic of a particular species except E. gallinarum. The PCR products from E. gallinarum displayed strain-to-strain heterogeneity in the number and size of amplicons. To further test the utility of this technique, 11 phenotypically aberrant strains which had been assigned species identification based on Facklam and Collins-type strain reactions (R.R. Facklam and M.D. Collins, J. Clin. Microbiol. 27:731-734, 1989) were subjected to ITS-PCR. ITS-PCR of the phenotypically aberrant strains identified six strains with reactions consistent with those of type strains. However, five strains were characterized as follows: two strains originally identified as E. mundtii were identified by ITS-PCR as E. casseliflavus, one strain originally identified as E. raffinosus was identified by ITS-PCR as E. durans, one strain originally identified as E. hirae was identified by ITS-PCR as E. faecium [corrected]. We conclude that amplification of the intergenic 23S and 16S rRNA gene regions of enterococci provides a reliable technique for species identification of enterococci.     

Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci

Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 438-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 19 enterococcal type strains (Enterococcus avium, Enterococcus casseliflavus, Enterococcus cecorum, Enterococcus columbae, Enterococcus dispar, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus hirae, Enterococcus malodoratus, Enterococcus mundtii, Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus solitarius, and Enterococcus sulfureus). Sequence analysis of the sodA(int) fragments enabled reliable identification of 18 enterococcal species, including E. casseliflavus-E. flavescens and E. gallinarum. The sodA(int) fragments of E. casseliflavus and E. flavescens were almost identical (99.5% sequence identity), which suggests that they should be associated in a single species. Our results confirm that the sodA gene constitutes a more discriminative target sequence than 16S rRNA gene in differentiating closely related bacterial species.     

Comparison of susceptibility to antimicrobials of the enterococcal species isolated from pigeons (Columba livia)

Antimicrobial resistance profiles were assessed in Enterococcus faecium, Enterococcus faecalis, and Enterococcus columbae strains isolated from racing pigeons (Columba livia). E. columbae is the most prevalent enterococcal species in the intestines of pigeons. Acquired resistance against the antimicrobials bambermycin, vancomycin, monensin, narasin, virginiamycin (cross-resistant with quinupristin/dalfopristin), avilamycin (cross-resistant with evernimicin), and ampicillin was not seen. Only for the antimicrobials tetracycline and tylosin was a frequent occurrence of resistance found in all three enterococcal species tested. Resistance to the fluoroquinolone antimicrobial enrofloxacin was present in E. columbae. Bacitracin resistance was seen in the species E. faecalis and E. faecium but not in E. columbae. High-level streptomycin resistance was found in E. columbae and in one E. faecium strain, whereas high-level gentamicin resistance was mainly associated with E. faecalis. The differences noted between the different species illustrate the difficulties related to the choice of indicator bacteria for antimicrobial resistance in the intestinal flora.     

Genome-based insights into the evolution of enterococci

It is now 15 years since the first genome of a free-living organism was sequenced. Subsequent to this milestone, a veritable avalanche of genome sequence data has revolutionized many aspects of microbiology. In this review, we discuss recent progress on the genomics of Enterococcus faecalis and Enterococcus faecium, which are the two enterococcal species that cause the large majority of enterococcal infections. We focus on the genome-based analysis of enterococcal diversity and phylogeny. Studies based on comparative genome hybridization have shown that both species exhibit considerable inter-strain genomic diversity, which is mainly linked to the variable presence of phages, plasmids, pathogenicity islands and conjugative elements. We also discuss how the advent of next-generation sequencing technologies allows for a comprehensive characterization of the gene repertoire of multiple isolates, which can be used for extremely robust analyses of diversity and population structure.     

Diversity of domain V of 23S rRNA gene sequence in different Enterococcus species

The highly conserved central loop of domain V of 23S RNA (nucleotides 2042 to 2628; Escherichia coli numbering) is implicated in peptidyltransferase activity and represents one of the target sites for macrolide, lincosamide, and streptogramin B antibiotics. DNA encoding domain V (590 bp) of several species of Enterococcus was amplified by PCR. Twenty enterococcal isolates were tested, including Enterococcus faecium (six isolates), Enterococcus faecalis, Enterococcus avium, Enterococcus durans, Enterococcus gallinarum, Enterococcus casseliflavus (two isolates of each), and Enterococcus raffinosus, Enterococcus mundtii, Enterococcus malodoratus, and Enterococcus hirae (one isolate of each). For all isolates, species identification by biochemical testing was corroborated by 16S rRNA gene sequencing. The sequence of domain V of the 23S rRNA gene from E. faecium and E. faecalis differed from those of all other enterococci. The domain V sequences of E. durans and E. hirae were identical. This was also true for E. gallinarum and E. casseliflavus. E. avium differed from E. casseliflavus by 23 bases, from E. durans by 16 bases, and from E. malodoratus by 2 bases. E. avium differed from E. raffinosus by one base. Despite the fact that domain V is considered to be highly conserved, substantial differences were identified between several enterococcal species.     

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.     

Native microbial colonization of Drosophila melanogaster and its use as a model of Enterococcus faecalis pathogenesis

Enterococci are commensal organisms of the gastrointestinal (GI) tracts of a broad range of mammalian and insect hosts, but they are also leading causes of nosocomial infection. Little is known about the ecological role of enterococci in the GI tract consortia. To develop a tractable model for studying the roles of these organisms as commensals and pathogens, we characterized the Drosophila melanogaster microflora and examined the occurrence of enterococci in the gastrointestinal consortium of Drosophila. In a survey of laboratory-reared Drosophila and wild-captured flies, we found that Drosophila was naturally colonized by representatives of five bacterial phyla. Among these organisms were several species of enterococci, including Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinaraum, and Enterococcus durans, as well as a previously detected but uncultured Enterococcus species. Drosophila could be cured of enterococcal carriage by antibiotic treatment and could be reassociated with laboratory strains. High-level colonization by a well-characterized strain expressing the enterococcal cytolysin was found to be detrimental to Drosophila compared to the effect of an isogenic, noncytolytic control. The anatomical distribution of enterococci in the Drosophila GI tract was determined by immunohistochemical staining of thin sections of naturally colonized and reassociated flies.     

Use of the Streptotest for identification of enterococci and viridans-type streptococci]

The new identification system STREPTOtest (fy. Lachema, Brno) was evaluated. A total 118 well-known strains of genus Enterococcus, Streptococcus, Aerococcus, Stomatococcus and Gemella were tested. The STREPTOtest system rapidly and reliably distinguished genus Enterococcus from genus Streptococcus. This method identified correctly to the species level 65.9% of enterococci strains and 52.5% of streptococci strains. The STREPTOtest was used for identification of A. viridans and S. mucilaginosus and it was possible to separate these strains from similar ones. A new differentiation chart for identification of all 13 recently described enterococcal species was proposed.     

Fatal meningitis caused by vancomycin-resistant enterococci: report of two cases from south India

Vancomycin-resistant enterococci rarely cause meningitis and present a therapeutic challenge. Antimicrobial susceptibility testing was done for strains of Enterococcus species isolated from CSF samples of patients with meningitis by phenotypic methods. Multiplex polymerase chain reaction was performed to determine the genetic basis of vancomycin resistance of such isolates. We report here two cases of enterococcal meningitis caused by vancomycin-resistant Enterococcus species. One of the isolates was identified as Enterococcus faecalis and the other as Enterococcus gallinarum. We also report the simultaneous presence of vanC1 and vanA resistance genes in the strain of E. gallinarum. To the best of our knowledge, this is the first report of vanA resistance gene in an isolate of E. gallinarum from the Indian subcontinent. This is also the first Indian report of vancomycin-resistant Enterococcus causing meningitis.     

Isolation of Enterococcus mundtii from normally sterile body sites in two patients.

Enterococcus mundtii, a recently described nonmotile, yellow-pigmented enterococcal species, was isolated from a chronic thigh abscess and from operatively obtained sinus mucosa. It is emphasized that this species may be encountered in clinical specimens and that the correct species identification may be missed when commercially available identification systems are relied on.     

Antibiotic resistance patterns of enterococci isolated from coastal bathing waters.

Recreational water should be considered a risk for enterococcal infections in regions with high utilisation and long exposure periods. A total of 1113 enterococcal isolates was obtained from 1670 bathing water samples from 120 bathing areas of seven prefectures in northern Greece. Enterococcus avium, E. raffinosus and E. faecium were the most prevalent species. Single, double and multiple antibiotic resistance patterns were observed in 33.5% 31.0% and 22.8% of the isolates, respectively. Resistance to erythromycin occurred most frequently, in 57.3% isolates, many of which also exhibited resistance to ciprofloxacin and rifampicin as well as high-level resistance to kanamycin and streptomycin. The results suggest that bathing water may contribute to the dissemination of uncommon enterococcal species that exhibit resistance to several antibiotics which are used to treat community-acquired infections.     

Spontaneous peritonitis caused by Enterococcus faecium.

Three cases of spontaneous peritonitis caused by Enterococcus faecium are presented. The underlying condition was alcoholic cirrhosis in each case. This enterococcal species has never before been reported as a cause of spontaneous bacterial peritonitis. Two patients responded to therapy. The development of enterococcal peritonitis and the cases documented in the literature are briefly reviewed. Taxonomic problems with pathogenic, clinical, and therapeutic implications are discussed.     

Distribution of endo-beta-N-acetylglucosaminidase amongst enterococci.

Enterococci are becoming increasingly important nosocomial pathogens, a fact mainly attributed to their antimicrobial resistance profiles. However, the enzymic activities required for these organisms to proliferate in vivo have received little attention. Enterococcus faecalis has been shown previously to produce an endo-beta-N-acetylglucosaminidase activity which cleaves high mannose-type glycans in glycoproteins between the N-acetylglucosamine residues of the pentasaccharide core. This study investigated the distribution of this endoglycosidase activity amongst the other enterococcal species. Ribonuclease B, a high mannose-type glycoprotein, was used as a substrate and endoglycosidase activity was demonstrated by a combination of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and high pH anion-exchange chromatography. Endo-beta-N-acetylglucosaminidase activity was present in 10 of the 18 enterococcal species isolated from both human and animal sources, including all E. faecalis strains. The most notable exception was the lack of this activity in all E. faecium isolates tested. All enterococcal species possessing endoglycosidase activity utilised the liberated glycans to support bacterial growth.     

Species identities of enterococci isolated from clinical specimens.

Conventional tests and commercially available systems were used to determine the species identities of clinical isolates of enterococci. Strict adherence to the conventional test scheme of Facklam and Collins (R. R. Facklam and M. D. Collins, J. Clin. Microbiol. 27:731-734, 1989) resulted in the misidentification of lactose-negative Enterococcus faecalis isolates as Enterococcus solitarius, but this problem was overcome by the application of additional tests. The commercially available systems tested were unable to recognize some of the more recently described enterococcal species. E. faecalis accounted for 87.1% of 302 consecutive isolates. Enterococcus faecium (8.6%), Enterococcus avium (0.7%), Enterococcus durans (0.3%), Enterococcus gallinarum (1.0%), Enterococcus casseliflavus (1.0%), Enterococcus hirae (0.3%), and Enterococcus raffinosus (0.3%) isolates were also identified. None of the isolates produced beta-lactamase, but 15.4% of 235 isolates tested, including 1 strain of E. gallinarum, displayed high-level resistance to gentamicin.     

Evaluation of MicroScan for identification of Enterococcus species.

Emerging drug resistance of the enterococci necessitates differentiation from group D streptococci and accurate species identification. MicroScan (Baxter Healthcare Corp., West Sacramento, Calif.) has recently developed a microdilution system for identification and antibiotic susceptibility testing of gram-positive cocci. To evaluate the ability of this system to identify Enterococcus species, 100 isolate identified as enterococci by MicroScan were tested by conventional media and 60 isolates of streptococci were tested by MicroScan. Incubation times for conventional and MicroScan methods were 96 and 18 to 24 h, respectively. For 94 strains of enterococci (77 Enterococcus faecalis, 14 Enterococcus faecium, and 3 Enterococcus durans), identification by conventional media and MicroScan agreed. Of the remaining six isolates, four were identified as E. faecalis and two were identified as E. durans by MicroScan, whereas by conventional media the four E. faecalis isolates were identified as Enterococcus solitarius and the two E. durans isolates were identified as Enterococcus hirae. None of the 60 streptococci were identified as enterococci. MicroScan is a reliable method for identification of the commonly encountered enterococcal species E. faecalis and E. faecium; however, modifications of the system are necessary for identification of other Enterococcus species.     

Characterization of three new enterococcal species, Enterococcus sp. nov. CDC PNS-E1, Enterococcus sp. nov. CDC PNS-E2, and Enterococcus sp. nov. CDC PNS-E3, isolated from human clinical specimens

As a reference laboratory, the Streptococcus Laboratory at the Centers for Disease Control and Prevention (CDC) is frequently asked to confirm the identity of unusual or difficult-to-identify catalase-negative, gram-positive cocci. In order to accomplish the precise identification of these microorganisms, we have systematically applied analysis of whole-cell protein profiles (WCPP) and DNA-DNA reassociation experiments, in conjunction with conventional physiological tests. Using this approach, we recently focused on the characterization of three strains resembling the physiological groups I (strain SS-1730), II (strain SS-1729), and IV (strain SS-1728) of enterococcal species. Two strains were isolated from human blood, and one was isolated from human brain tissue. The results of physiological testing were not consistent enough to allow confident inclusion of the strains in any of the known enterococcal species. Resistance to vancomycin was detected in one of the strains (SS-1729). Analysis of WCPP showed unique profiles for each strain, which were not similar to the profiles of any previously described Enterococcus species. 16S ribosomal DNA (rDNA) sequencing results revealed three new taxa within the genus ENTEROCOCCUS: The results of DNA-DNA relatedness experiments were consistent with the results of WCPP analysis and 16S rDNA sequencing, since the percentages of homology with all 25 known species of Enterococcus were lower than 70%. Overall, the results indicate that these three strains constitute three new species of Enterococcus identified from human clinical sources, including one that harbors the vanA gene. The isolates were provisionally designated Enterococcus sp. nov. CDC Proposed New Species of Enterococcus 1 (CDC PNS-E1), type strain SS-1728(T) (= ATCC BAA-780(T) = CCUG 47860(T)); Enterococcus sp. nov. CDC PNS-E2, type strain SS-1729(T) (= ATCC BAA-781(T) = CCUG 47861(T)); and Enterococcus sp. nov. CDC PNS-E3, type strain SS-1730(T) (= ATCC BAA-782(T) = CCUG 47862(T)).     

Identification of clinically relevant enterococcus species by direct sequencing of groES and spacer region

We determined the groESL sequences (groES, groEL, and the intergenic spacer) of 10 clinically relevant Enterococcus species and evaluated the feasibility of identifying Enterococcus species on the basis of these sequences. Seven common clinical Enterococcus species, E. faecalis, E. faecium, E. casseliflavus, E. gallinarum, E. avium, E. raffinosus, and E. hirae, and three less common Enterococcus species, E. cecorum, E. durans, and E. mundtii, were examined in this study. We found that the groES genes of these enterococcal species are identical in length (285 nucleotides) and contain an unusual putative start codon, GTG. The lengths and sequences of the intergenic regions (spacers between the groES and groEL genes) are quite variable (17 to 57 bp in length) among Enterococcus species but are conserved in strains within each species, with only a few exceptions. Considerable variation of groES or groEL sequences was also observed. The evolutionary trees of groES or groEL sequences revealed similarities among Enterococcus species. However, the overall intraspecies variation of groES was less than that of groEL. The high interspecies variation and low intraspecies variation indicate that the groES and spacer sequences are more useful than groEL for identification of clinically relevant Enterococcus species. The sequences of these two genetic traits, groES and spacer, can be determined by a single PCR and direct sequencing and may provide important information for the differentiation of closely related species of Enterococcus.     

Virulence Factors of Enterococcus faecalis and Enterococcus faecium Blood Culture Isolates

 Known and potential virulence factors of enterococcal blood culture isolates were studied using 89 Enterococcus faecalis and 24 Enterococcus faecium isolates. The prevalence of the respective factors was (Enterococcus faecalis vs. Enterococcus faecium): hemolysin 16% vs. 0%, gelatinase 55% vs. 0%, aggregation substance 63% vs. 13%, lipase 35% vs. 4%, hemagglutinin 97% vs. 0%. Deoxyribonuclease was not detected in any isolate. The study showed that hemagglutinin and lipase may represent additional virulence factors of Enterococcus faecalis but not Enterococcus faecium. The significance of these factors in the pathogenesis of enterococcal infection needs to be elucidated in further studies.     

Status of high level aminoglycoside resistant Enterococcus faecium and Enterococcus faecalis in a rural hospital of central India

Considering the emergence of high level aminoglycoside resistance (HLAR) in enterococci this study was undertaken to determine their status in a rural setting. HLAR by disc diffusion and agar dilution, beta lactamase by nitrocefin disc and vancomycin resistance by agar dilution was determined in 150 enterococcal isolates, as per NCCLS guidelines. Only two species, Enterococcus faecalis (85.5%) and Enterococcus faecium (14.7%) were recovered, mostly from blood. Forty six percent showed HLAR. Multi drug resistance and concomitant resistance of HLAR strains to beta lactams were quite high. None showed beta lactamase activity or vancomycin resistance.