Genes efaA Research Articles

Antimicrobial resistance patterns, virulence genes, and biofilm formation in enterococci strains collected from different sources

BackgroundCurrently, antibiotic-resistant strains of Enterococcus are considered to be one of the critical health challenges globally. This study aimed to investigate the antibiotic susceptibility pattern, biofilm formation capacity, and virulence genes of enterococci isolated from different sources.MethodsIn this cross-sectional study, environmental and fecal samples were collected from the hospital environment, volunteers, and hospital staff from October 2018 to August 2019. The isolates were identified by morphological and biochemical tests (gram staining, catalase, bile resistance, esculin hydrolysis, carbohydrate fermentation, growth in 6.5% NaCl, Pyrrolidonyl arylamidase, arginine dehydrolase), and PCR for ddl gene. An antimicrobial susceptibility test was performed by the standard disk agar diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Quantitative microplate assays were used to assess biofilm production. The bacterial DNAs were extracted by alkaline lysis method and polymerase chain reaction technique was used detect the esp, ace, and efaA virulence genes.ResultsOut of 145 isolates, 84 (57.9%) were identified as E. faecalis and 61 (42.1%) as E. faecium. Resistance to kanamycin and quinupristin-dalfopristin was 82.1% (69/84) and 85.7% (72/84), respectively, in E. faecalis isolates. Out of 61 E. faecalis isolates, 38 (62.4%) were resistant to kanamycin. Among the E. faecalis isolates, esp was the most dominant virulence gene (73.80%), followed by efaA, and ace, which were detected in 60.71%, and 30.95% isolates, respectively. In total, 68.27% of the strains were biofilm producers. Further, esp and efaA genes were more frequently found among E. faecalis strains with moderate and strong biofilm biomass.ConclusionsAccording to the findings of our study, enterococci strains isolated from different samples possess distinctive patterns of virulence genes. The esp, ace, and efaA genes were more prevalent among E. faecalis than E. faecium. Besides, the high level antibiotic resistance of normal flora and environmental enterococci strains is alarming the researchers.

Clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants

In this study, we evaluated the antimicrobial susceptibility, the presence of gene-encoding virulence factors and CRISPR systems, as well as the ability to produce lytic enzymes among clinical E. faecalis and E. faecium isolates (n = 44). All enterococci isolates showed phenotypes of multidrug resistance. E. faecalis and E. faecium isolates exhibited high-level aminoglycoside resistance phenotype, several of them harboring the aac(6′)Ie-aph(2″)Ia and aph(3′)-IIIa genes. The gene vanA was the most frequent among vancomycin-resistant E. faecium. High prevalence of the virulence genes esp and efaA were observed; hyl gene was more associated with E. faecium, while ace and efaA genes were more frequently detected in E. faecalis. Caseinase activity was frequently detected among the isolates. Gelatinase and DNAse activities predominated among E. faecalis, while hemolytic capability was frequent among E. faecium isolates. Twenty-nine isolates showed at least one CRISPR system investigated. Several enterococci isolates harbored the aac(6′)-Ie-aph(2″)-Ia or aph(3’)-IIIa genes and a CRISPR loci. CRISPR loci were positively correlated to efaA and gelE genes, and gelatinase and DNAse activities, while CRISPR loci absence was related to hyl gene presence. These results show that clinical isolates of E. faecalis and E. faecium harboring virulence genes show the concomitant presence of CRISPR loci and antibiotic resistance determinants.

Enterococcus faecalis and Enterococcus faecium in pasteurized milk: Prevalence, genotyping, and characterization of virulence traits

To assess the prevalence of enterococci in pasteurized milk and characterize their virulence traits, 416 pasteurized milk samples were purchased from retail stores in the Northwest of Iran. Enterococci were isolated by conventional methods and confirmed by genus-specific PCR. Amongst the samples, 269 were identified as either Enterococcus faecalis or E. faecium, which were then subjected to genotypic and phenotypic analyses. Using enterobacterial repetitive intergenic consensus (ERIC)-PCR, E. faecalis and E. faecium strains were effectively grouped into 15 and 16 clusters, respectively. Nonetheless, ERIC-PCR was unable to accurately cluster E. faecalis and E. faecium strains according to their virulence or phenotypic characteristics. Based on the results, most of the isolates, especially E. faecalis strains, carried gelE, hyl, asa1, esp, cylA, ace, and efaA genes. Results also revealed that E. faecalis and E. faecium isolates were highly resistant to most of the tested antibiotics. The resistance to vancomycin for E. faecalis and E. faecium was estimated at 71.9% and 77.6%, respectively. Moreover, 81% and 69% of E. faecalis and E. faecium strains were able to produce biofilm. It was concluded that pasteurized milk could be an intermediate vehicle for the transmission of multidrug-resistant virulent enterococci strains to humans.

Identification and characterization of resistance and pathogenicity of Enterococcus spp. in samples of donor breast milk.

BACKGROUNDBreast milk is the primary source of nutrition for newborns. Hospitalized babies frequently need nutritional support from Human Milk Banks. As bacterial species of the genus Enterococcus are part of the microbiota of healthy donors, they may contaminate samples of pumped breast milk. AIMTo identify and characterize the bacterial virulence and resistance in samples isolated from the nipple-areolar region, hands, and breast milk aliquots from donors at the Human Milk Bank of Municipal Hospital Esaú Matos in the city of Vitória da Conquista, Bahia State, Brazil.METHODSThe personal hygiene and sanitation of donors were analyzed with the aim of identifying possible reasons for contamination of pumped milk. Cutaneous samples as well as aliquots of unpasteurized and pasteurized milk from 30 participants were obtained. Each Enterococcus spp. isolate underwent a disk diffusion susceptibility test and molecular biology techniques to determine resistance and virulence genes.RESULTS Enterococcus spp. were identified in 30% of donors (n = 9), and 11 specimens were isolated. Resistance to tetracycline was highly prevalent, being detectable in 63% of the isolates (n = 7) and followed by intermediate sensitivity to ciprofloxacin, observed in 27% of the specimens (n = 3). The efaA gene was found in 63% (n = 7) of the isolates, while the ace gene was detected in 27% (n = 3). CONCLUSIONThis study illustrates the importance of microbiological monitoring by Human Milk Banks and the need for alternatives to prevent the presence of Enterococcus spp. in hospital settings.

Antisense peptide nucleic acids againstftsZ andefaA genes inhibit growth and biofilm formation of Enterococcusfaecalis

Enterococcus faecalis is one of the important causes of nosocomial infections. Nowadays, increasing prevalence of antibiotic-resistant bacteria and slow progress in recognizing new antimicrobial agents has limited the efficiency of conventional antibiotics, which cause to find novel strategies to overcome bacteria. Therefore, in this study, we aimed to assess the role of efaA gene in the biofilm formation and the role of ftsZ gene in the controlling of bacterial growth by the anti-sense PNAs(Peptide Nucleic Acid).E. faecalis ATCC® 29212™was used for the study of PNAs designed to targeting the start codon section of the ftsZ andefaA genes. PNA attachment to RNA was confirmed by blotting. Electroporation technique was used for the intracellular transfer of anti-ftsZ PNAs. The spot-plating method was used to the assessment of alteration in bacterial growth. Biofilm formation assay and real-time PCR were used for detection of biofilm inhibitory effect of cell penetrating peptide (CPP) conjugated to anti-efaA PNAs.ByftsZ PNAs treatment, no growth was seen from the strain in agar by a spot plating method and the inhibition zone of anti-ftsZ PNAs was not seen. PNAs against the efaA gene decreased by 95% the expression of the efaA gene and biofilm formation. In addition, the(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) MTT assay showed no toxicity on MCF7 cells for both of anti-ftsZand anti-efaA PNAs.This study used new genetic and molecular tools to inhibit pathogenicity and infection by E. faecalis. In this study, we suggested that efaA gene plays a critical role in the biofilm formation and anti-efaA PNAs could decrease the formation of biofilm, as well as, anti-ftsZ PNAs could eliminate bacterial growth.

Does Enterococcus faecalis from Traditional Raw Milk Cheeses Serve as a Reservoir of Antibiotic Resistance and Pathogenic Traits?

Enterococcus faecalis is not only a prevalent species among dairy microbial community but also a well-documented opportunistic pathogen. Food safety should exclude the possibility of consumer exposure to its virulence traits through consumption of dairy products. In this study, an integrated approach based on both phenotypic and genotypic methods was applied to investigate the incidence of antibiotic resistance and pathogenicity potential in 40 E. faecalis isolated from 10 Italian raw milk cheeses over a 13-year period (1997-2009). Among the 14 tested antibiotics, resistance to tetracycline, rifampicin, chloramphenicol, and erythromycin was observed, whereas vancomycin-resistant enterococci were not found. A high incidence (90% of strains) of the tet(M) gene emerged, whereas tet(K), tet(S), tet(L), int, and ermB genes were occasionally amplified (12.5%, 10%, 7.5%, 2.5% and 30%, respectively). No strain was positive for vancomycin-resistant determinants. Among the seven virulence determinants considered, the asa1, gelE, esp, and efaA genes were harbored. No other gene encoding for either different virulence factors (cylA, hyl, and ace) or amino acid decarboxylase activity (hdc, tdc, and odc) was detected. Consequently, E. faecalis isolated from raw milk cheeses does not represent a substantial reservoir of antimicrobial resistance and virulence factors if compared with clinical strains. However, this species occasionally harbors detrimental traits; thus, the possibility that it could be a route for transmission of pathogenic genes through dairy products should never be disregarded.

Effect of acidic and alkali shocks on expression of efaA gene in Enterococcus faecalis, isolated from root canal infection

Enterococcus faecalis is an important opportunistic infectious agent involving the oral cavity and endodontics. The aim of this study was to evaluate the expression ratio of efaA gene in biofilm producer E. faecalis before and after receiving acidic and alkali shocks. One hundred E. faecalis isolates were gathered from 170 infectious root canals. After analysis of biofilm formation by the Microtiterplate method, the presence of efaA gene was examined by PCR and its expression was evaluated by Real-time PCR, one before applying any stressed to isolates and another by applying acidic and alkali shock. Chi-square method was used for statistical analysis. Eighty-two percent of samples had efaA gene. Evaluation of biofilm formation, 49% of the isolates were strong biofilm producer, 42% moderate and 10 % of them had no biofilm. 59% overexpression of efaA gene was observed in biofilm producer isolates, while there were no significant changes in samples with acidic stress and decreased expression after alkali shock. Findings of the present study, indicates importance of efaA gene in biofilm formation and pathogenesis of E. faecalis. Acid had no effect of expression of this gene but alkali reduced expression of this gene in a significant level. These results indicate the importance of efaA and acidic conditions in biofilm production by E. faecalis.

Genotypic characterization of Enterococcus species isolated from the oral cavity and their pattern of antibiotic susceptibility

Background: Enterococci comprise a small share of oral flora and occur as contaminants in food such as meat and cheese. They are commonly encountered in patients with infections of oral tissues such as necrotic pulp, root canals and periodontitis. Objectives: To characterize the genotypes of Enterococcus isolated from patients with oral infection and to determine their antibiotic susceptibility. Methods: We genotypically characterized a number of isolates of Enterococcus species derived from patients with various oral infections. All the isolates were tested for the presence of putative virulence genes; efaA (gene for endocarditis), gelE (gene for gelatinase), ace (gene for collagen binding antigen), asa (gene for aggregation substance), cylA (gene for cytolysin activator), and esp (gene for surface adhesin) of E. faecalis and E. faecium as described in previous studies. Results: E. faecalis dominated in oral infections compared with E. faecium. Isolates were susceptible to most antibiotics (only four were resistant to vancomycin). To our knowledge, the first report from this region. Conclusions: Treatment of enterococcal infections of the oral mucosal and deep oral areas necessitate microbiological diagnosis and identification of resistance strains. Keywords: Antibiotic susceptibility pattern, Enterococcus species, genotypic characterization, oral infections

Virulence factors, antimicrobial resistance and biofilm formation in Enterococcus spp. isolated from retail shrimps

A total of 60 samples from retail raw, cooked and ready-to-eat shrimps were collected and the prevalence of Enterococcus spp, their virulence factors, antibiotic resistance and biofilm-forming ability was determined. Most of the strains were isolated from ready-to-eat shrimps (16/20; 80%) followed by raw shrimps (11/20; 55%) and cooked shrimps (8/20; 40%). Among the 35 isolates, Enterococcus faecalis (62.9%) and Enterococcus faecium (28.6%) were the dominant species. More than half of all investigated isolates (65.7%) were resistant to at least one class of antibiotic, and 16 (45.7%) of the strains were classified as multidrug resistant. A high percentage of isolates were resistant to tetracycline (48.6%), followed by tigecycline and fosfomycin (45.7%). All of the enterococci isolates, carried between 5 and 9 tested virulence genes. The presence of esp (100%), gelE (88.6%), efaA (77.1%) genes and sex pheromones cpd (100%) cob (94.3%), ccf (94.3%) were found most frequently in all of the tested enterococci species. Shrimp strains showed a moderate or weak ability to produce biofilm. Our data indicate that shrimps can be considered a reservoir of antibiotic resistant, virulence strains from the genus Enterococcus.

Gentamicin induces efaA expression and biofilm formation in Enterococcus faecalis

Enterococci have been ranked among the leading causes of nosocomial bacteremia and urinary tract infection. This study aimed to investigate the effect of ampicillin, vancomycin, gentamicin and ceftizoxime on biofilm formation and gene expression of colonization factors on Enterococcus faecalis. Twelve clinical isolates of E. faecalis were used to investigate the effect of antibiotics on biofilm formation and gene expression of efaA, asa1, ebpA, esp and ace. Flow system assay and Microtiter plates were used for biofilm assay. Two hundred clinical isolates were used for confirming the effect of antibiotics on biofilm formation. Ampicillin, vancomycin and ceftizoxime did not have any significant effect on biofilm formation, but gentamicin induced biofilm formation in 89% of isolates. In twelve selected isolate gentamicin increased expression of esp (+50.9%) and efaA (+33.9%) genes and reduced or maintained expression of others (asa1:−47.4%, ebpA: 0, ace:−19.2%). Vancomycin increased expression of esp (+89.1%) but reduced the others (asa1: −34.9%, ebpA:−11%, ace:−30%, efaA:−60%). Ceftizoxime increased slightly ebpA (+19.7%) and reduced others (asa1:−66.2%, esp:−35%, ace:−28.1%, efaA:−38.4%). and ampicillin strongly increased expression of ace (+231%), esp (+131%) and ebpA (+83%) but reduced others (asa1:−85.5%, efaA:−47.4%). The findings of the present study showed that antibiotics may have a role in biofilm formation and sustainability of enterococci, especially in case of gentamicin. efaA gene may have an important role, especially in antibiotic induced biofilm formation by gentamicin. Experiments with efaA mutants are needed to investigate the exact effect of efaA on biofilm formation with antibiotic induced cells.

<b>Investigation <em>in vivo</em> of <em>Enterococcus faecalis</em> in endodontic retreatment by phenotypic and genotypic methods

This study aimed to investigate the prevalence of E. faecalis in root-filled canals using culture and molecular approaches. It was evaluated the antimicrobial susceptibility to different antibiotics and the virulence factors of E. faecalis isolates. Microbial samples were taken from thirty root-filled canals. Culture methods and 16S rDNA assay were used to identify E. faecalis . The antimicrobial susceptibility of E. faecalis was determined by MIC values using the E test. Cultivable strains of E. faecalis were investigated for virulence factors by PCR technique. E. faecalis were detected by culture (7/30), traditional PCR assay (13/30) and nested PCR (23/30). Both PCR were significantly more effective than culture in detecting E. faecalis (p < 0.05). All tested E. faecalis were highly sensitive to amoxicillin. Some strains of E. faecalis were resistant to antibiotics such as rifampicin (4/12), erythromycin (3/12) and azythromycin (8/12). The genes efaA and ace were detected in all isolates. The other virulence genes were found in 91.6 ( gelE ), 83.3 ( asa ), 25 ( esp ) and 16.6% ( cylA ). Strains of E. faecalis isolated from root-filled canals showed virulence factors related to adherence. They also showed resistance to some antibiotics commonly used in dentistry.

Assessment of biofilm formation by enterococci isolates from urinary tract infections with different virulence profiles

This study aimed to investigate possible associations between virulence profiles and biofilm formation in Clinical UTI isolates. Isolates were collected from five university hospitals and identified and characterized for the presence of virulence factors by PCR. Biofilm assays were conducted in 96 well microtiter plates by reading the OD570 after crystal violet staining. 75% of isolates had esp gene, 38.77% had asa1, 84.18% had ace, 81.63% had efaA, 93.36% had ebpR, 34.18% had cylA, 81.63% had gelE and 17.35% had hyl. Biofilm experiences were done and isolates having asa1 or efaA genes produced more biofilms than negative ones (P=0.011, P=0.008), but the presence of esp, ace, cylA or gelE genes in isolates had no effect on biofilm formation. Isolates possessing hyl had much lower biofilm formation (P=0.000). Present study showed that the esp, ace, gelE and cylA genes do not seem to be necessary nor sufficient for the production of biofilm in enterococci but the presence of efaA and asa1 correlates with increased biofilm formation of urinary tract isolates. Also the low prevalence of hyl among enterococci isolated from UTIS and its association with poor biofilm production indicate that the absence of this gene can be an advantage in the pathogenesis of UTIs.

Technological and safety properties of lactic acid bacteria isolated from Spanish dry-cured sausages

Technological and safety-related properties were analyzed in lactic acid bacteria isolated from Spanish dry-cured sausages in order to select them as starter cultures. In relation to technological properties, all the strains showed significative nitrate reductase activity; Lactobacillus plantarum, Lactobacillus paracasei and 52% of the Enterococcus faecium strains showed lipolytic activity and only Lactobacillus sakei strains (43%) were able to form biofilms. Related to safety aspects, E. faecium strains were the most resistant to antibiotics, whereas, L. sakei strains were the most sensitive. In relation to virulence factors, in the E. faecium strains analyzed, only the presence of efaA gene was detected. The analysis of biogenic amine production showed that most E. faecium strains and L. sakei Al-142 produced tyramine. In conclusion, L. paracasei Al-128 and L. sakei Al-143 strains possess the best properties to be selected as adequate and safe meat starter cultures.

Adhesion and virulence factor properties of Enterococci isolated from clinical samples in Iran

Enterococci rank among leading causes of nosocomial bacteremia, urinary tract infections and community acquired endocarditis. The aim of the present study was to investigate the presence of virulence factors in Enterococci strains isolated from clinical samples in Iranian Educational hospitals. Presence of aggregation substance (asa), extracellular surface protein (esp), Enterococcus faecalis antigen A (efaA), adhesin of collagen from E. faecalis (ace), endocarditis and biofilm-associated pilli (ebp) as colonization factors and cytolysin (cyl), gelatinase (gel) and hyaloronidase (hyl) as secretary factors were investigated in isolates. A total of 201 clinical isolates of Enterococci were collected in 2009-2010 from eight educational hospitals. After deoxyribonucleic acid extraction, they were examined for presence of virulence factors by polymerase chain reaction. E. faecalis and Enterococcus faecium were isolated from 56.9% to 43.1%, respectively. Resistance to vancomycin and gentamicin were 33.8% and 83.9% in E. faecium isolates and 16.3% and 88.1% in E. faecalis isolates respectively. Colonization factors were found to be more prevalent in E. faecalis isolates and almost all isolates of E. faecalis had ace, ebp and efaA genes. Esp gene had a higher rate of distribution in Enterococci isolates (75.1%) in this study compared with previous studies. One of E. faecalis isolates contained hyl gene, but 38.8% of E. faecium isolates had it. Mutual exclusive were present between hyl and efaA in all E. faecium isolates and 69.7% of E. faecium hyl - positive isolates were esp positive. According to these results, virulence genes were more prevalent in E. faecalis isolates and E. faecalis had more potential pathogenesis for initiating an infection; however because of E. faeciums higher antibiotic resistance, we have been facing higher E. faecium infections in hospitalized patients.

Characterization of Enterococcus faecium isolates and first report of vanB phenotype–vanA genotype incongruence in the Middle East

We aimed to characterize the vancomycin genotype/phenotype, carriage of putative virulence genes, and genetic relatedness of Enterococcus faecium isolates in Saudi Arabia. E. faecium isolated from inpatients at our medical center were studied. Sensitivity to ampicillin, linezolid, teicoplanin, quinupristin/dalfopristin, tetracycline, and ciprofloxacin was determined. The presence of van genes and virulence genes for aggregation substance (Asa-1), enterococcal surface proteins (esp), cytolysin (cylA, cylL, cylM), gelatinase (gelE), E. faecium endocarditis antigen (EfaA( fm )), hyaluronidase (hyl), and collagen adhesion (Ace) was assessed. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE). Twenty-nine E. faecium isolates were obtained and the majority of isolates (n/N = 22/29) were from stool specimens. PFGE analysis identified eight pulsotypes (A-H) based on 80% similarities. Isolates were represented in five major pulsotypes: type A (n = 5), type B (n = 3), type D (n = 6), type E (n = 5), and type F (n = 7). All isolates were vanA gene-positive. Thirteen isolates had vanA(+)/vanB(+) genotype. Of these, ten exhibited a vanB phenotype and three had a vanA phenotype. Eight isolates with vanA(+)/vanB(-) genotype exhibited vanB phenotype. Six of these eight isolates belonged to the same pulsotype. All isolates were positive for gelE, esp, and EfaA( fm ) genes. Five were CylA-positive and 24 had the hyl genes. Of the eight isolates harboring a combination of gelE, esp, EfaA( fm ), and hyl genes, five showed vanB phenotype-vanA genotype incongruence. This is the first report of vanB phenotype-vanA genotype incongruent E. faecium in the Middle East region. Molecular typing indicates clonal spread and high occurrence of virulence genes, especially esp genes, associated with epidemic clones.

Comparative analysis of virulence determinants and mass spectral profiles of Finnish and Lithuanian endodontic Enterococcus faecalis isolates

Putative virulence factors of Enterococcus faecalis have been proposed by several workers and, by analogy, these have been linked to strains of endodontic origin. However, their distribution within the cell population is unknown. In the present study, isolates were taken from the dental root canals of two defined human populations, Lithuanian and Finnish, and examined for a range of virulence properties. In addition, surface-associated molecules and intracellular proteins were compared using matrix-assisted laser desorption-ionization/mass spectrometry (MALDI-TOF-MS) and ProteinChip capture/MS (SELDI-TOF-MS), respectively. Twenty-three Lithuanian and 35 Finnish dental root canal isolates were included. The esp, gelE, ace and efaA genes were detected by polymerase chain reaction, and cytolysin and gelatinase phenotypes were determined by hydrolysis of horse blood agar and gelatine agar, respectively. Protein extracts and surface-associated molecules of whole cells were analysed by SELDI-TOF-MS and MALDI-TOF-MS, respectively. Presence of esp (n = 15), cytolysin (n = 9), ace (n = 55) and efaA (n = 58) was not statistically different in the two samples, whereas gelE and gelatinase production was detected more frequently in the Finnish material (chi-squared, P < 0.01). Analysis of protein profiles by SELDI-TOF-MS showed clustering of cytolysin-producing strains, whereas MALDI-TOF-MS generated profiles that clustered according to the samples' origin and, furthermore, to atypical quinupristin-dalfopristin susceptibility. A high prevalence of virulence factors was demonstrated in both population types. SELDI-TOF-MS and MALDI-TOF-MS proved useful in distinguishing between different E. faecalis phenotypes and they may be useful technologies for elucidating the eco-distribution of E. faecalis in humans.

In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification

Disruption of the previously described efaA (from Enterococcus faecalis antigen A) gene was generated in E. faecalis strain OG1RF and loss of an ∼37-kDa immunoreactive band from the mutant was demonstrated in Western blots. In a mouse peritonitis model, mice infected with the efaA fs (fs=from Enterococcus faecalis) mutant showed more prolonged survival than mice infected with the parent strain OG1RF. These results suggest that efaA fs encodes a function important for infection of mice by enterococci. An efaA-like gene was also identified in E. faecium DNA libraries and its deduced amino acid sequence showed 73% similarity to EfaA of E. faecalis and 42–63% similarities to a group of streptococcal virulence and adhesion associated proteins that are components of ATP-binding cassette transport systems. Intragenic probes representing efaA fs, recA fs, efaA fm (fm=from E. faecium) and gyrA fm were tested for their ability to identify E. faecalis and E. faecium using colony lysates of 133 enterococci and one Streptococcus sp. Probes of E. faecium and E. faecalis origin hybridized to all isolates of E. faecium and E. faecalis, respectively, regardless of their clinical source but not to any of 29 other enterococci. These results suggest that the use of gene probes may prove helpful in identification of isolates of E. faecium and E. faecalis.

In vivo testing of an Enterococcus faecalis efaA mutant and use of efaA homologs for species identification.

Disruption of the previously described efaA (from Enterococcus faecalis antigen A) gene was generated in E. faecalis strain OG1RF and loss of an 37-kDa immunoreactive band from the mutant was demonstrated in Western blots. In a mouse peritonitis model, mice infected with the efaAfs (fs=from Enterococcus faecalis) mutant showed more prolonged survival than mice infected with the parent strain OG1RF. These results suggest that efaAfs encodes a function important for infection of mice by enterococci. An efaA-like gene was also identified in E. faecium DNA libraries and its deduced amino acid sequence showed 73% similarity to EfaA of E. faecalis and 42-63% similarities to a group of streptococcal virulence and adhesion associated proteins that are components of ATP-binding cassette transport systems. Intragenic probes representing efaAfs, recAfs, efaAfm (fm=from E. faecium) and gyrAfm were tested for their ability to identify E. faecalis and E. faecium using colony lysates of 133 enterococci and one Streptococcus sp. Probes of E. faecium and E. faecalis origin hybridized to all isolates of E. faecium and E. faecalis, respectively, regardless of their clinical source but not to any of 29 other enterococci. These results suggest that the use of gene probes may prove helpful in identification of isolates of E. faecium and E. faecalis.