Molecular detection and occurrence of vancomycin resistance genes (van A, B, C1, C2/C3) among Enterococcus species isolated from farm ostriches.

BACKGROUND: Evaluating the prevalence of vancomycin resistance genes (van genes) in enterococcal isolates from food-producing animals is an important public health issue because of the possibility of resistance genes spread to human. OBJECTIVES: The present study aimed to determine the occurrence of vancomycin resistance genes among Enterococcus species obtained from ostrich faecal samples. METHODS: One hundred and twenty-five faecal samples of apparently healthy ostriches from five different farms were investigated. Genes encoding vancomycin resistance were studied by multiplex-PCR, and susceptibility to six antibiotics was evaluated by disk-diffusion method. RESULTS: In total, 107 Enterococcus spp. isolates were obtained and confirmed by biochemical and molecular tests. Enterococcus faecium was the prevailing species (56 isolates of 107; 52.3%), followed by E. hirae (24 isolates; 22.4%) and E. gallinarum (12 isolates; 11.2%). Of the 107 recovered isolates, 44% harboured at least a type of van genes. vanA, vanC2/3 and vanC1 were identified in 34 (31.7%), 13 isolates (12.1%) and 4 (3.7%) isolates respectively. Additionally, four isolates (E. gallinarum, E. rafinosus) co-harboured the the vanA and vanC1 or vanA and vanC2/3. Enterococcus faecium and Enterococcus hirae strains with the vanA genotype were the most frequent van-carrying enterococci from ostrich faecal samples. Among van-carrying enterococcal isolates, 23.4% were phenotypically resistant to vancomycin. This study revealed a relatively high prevalence (44%) of van-carrying enterococci in ostrich faecal samples. CONCLUSIONS: Results of the present study suggest that ostrich faeces could be considered as a reservoir of vancomycin resistance genes, especially vanA containing enterococci that could be potentially transferred to human through the food chain.

Vancomycin-Nonsusceptible Enterococci Mediated by vanC at a Large Children's Hospital: Prevalence, Susceptibility, and Impact on Care of Enterococcal Bacteremia.

Enterococcus gallinarum and casseliflavus have inherent vancomycin resistance and, though known as pathogens, have not been well characterized in pediatric patients. We identified a significant prevalence of these enterococcal species among immunocompromised patients at a large pediatric institution and describe the impact on patient care, antibiotic stewardship, and infection control.

Dispersion of the Vancomycin Resistance Genes vanA and vanC of Enterococcus Isolated from Nile Tilapia on Retail Sale: A Public Health Hazard.

Although normally regarded harmless commensals, enterococci may cause a range of different infections in humans, including urinary tract infections, sepsis, and endocarditis. The acquisition of vancomycin resistance by enterococci (VRE) has seriously affected the treatment and infection control of these organisms. VRE are frequently resistant to all antibiotics that are effective treatment for vancomycin-susceptible enterococci, which leaves clinicians treating VRE infections with limited therapeutic options. With VRE emerging as a global threat to public health, we aimed to isolate, identify enterococci species from tilapia and their resistance to van-mediated glycopeptide (vanA and vanC) as well as the presence of enterococcal surface protein (esp) using conventional and molecular methods. The cultural, biochemical (Vitek 2 system) and polymerase chain reaction results revealed eight Enterococcus isolates from the 80 fish samples (10%) to be further identified as E. faecalis (6/8, 75%) and E gallinarum (2/8, 25%). Intraperitoneal injection of healthy Nile tilapia with the eight Enterococcus isolates caused significant morbidity (70%) within 3 days and 100% mortality at 6 days post-injection with general signs of septicemia. All of the eight Enterococcus isolates were found to be resistant to tetracycline. The 6/6 E. faecalis isolates were susceptible for penicillin, nitrofurantoin, gentamicin, and streptomycin. On the other hand 5/6 were susceptible for ampicillin, vancomycin, chloramphenicol, and ciprofloxacin. The two isolates of E. gallinarum were sensitive to rifampicin and ciprofloxacin and resistant to vancomycin, chloramphenicol, and erythromycin. Molecular characterization proved that they all presented the prototypic vanC element. On the whole, one of the two vancomycin resistance gene was present in 3/8 of the enterococci isolates, while the esp virulence gene was present in 1/8 of the enterococci isolates. The results in this study emphasize the potential role that aquatic environments are correlated to proximity to anthropogenic activities in determining the antimicrobial resistance patterns of Enterococcus spp. recovered from fish in the river Nile in Giza, Elmounib, Egypt as a continuation of our larger study on the reservoirs of antibiotic resistance in the environment.

Vancomycin resistant Enterococcus spp. from crows and their environment in metropolitan Washington State, USA: Is there a correlation between VRE positive crows and the environment?

Vancomycin-resistant enterococci [VRE] have been isolated from municipal, hospital and agricultural wastewater, recreational beaches, wild animals, birds and food animals around the world. In this study, American crows (Corvus brachyrhynchos) from sewage treatment plants (WWTP), dairy farms, and a large roost in a restored wetland with corresponding environmental samples were cultured for VRE. A total of 245 samples [156 crows, 89 environmental] were collected and screened for acquired vanA, vanB and/or intrinsic vanC1 genes. Samples were enriched overnight in BHI supplemented with 20µg/mL aztreonam, 4µg/mL vancomycin and plated on m-Enterococcus agar media supplemented with 6µg/mL vancomycin. Selected colonies were grown on BHI media supplemented with 18µg/mL vancomycin. Of these, 24.5% of the crow and 55% the environmental/cow samples were VRE positive as defined by Enterococcus spp. able to grow on media supplemented with 18µg/mL vancomycin. A total of 122 VRE isolates, 43 crow and 79 environmental isolates were screened, identified to species level using 16S sequencing and further characterized. Four vanA E. faecium and multiple vanC1 E. gallinarum were identified from crows isolated from three sites. E. faecium vanA and E. gallinarum vanC1 along with other Enterococcus spp. carrying vanA, vanB, vanC1 were isolated from three environments. All enterococci were multidrug resistant. Crows were more likely to carry vanA E. faecium than either the cow feces or wetland waters/soils. Comparing E. gallinarum vanC1 from crows and their environment would be useful in determining whether crows share VRE strains with their environment.

Characterization of fecal vancomycin-resistant enterococci with acquired and intrinsic resistance mechanisms in wild animals, Spain.

The objectives were to evaluate the presence of vancomycin-resistant enterococci with acquired (VRE-a) and intrinsic (VRE-i) resistance mechanisms in fecal samples from different wild animals, and analyze their phenotypes and genotypes of antimicrobial resistance. A total of 348 cloacal/rectal samples from red-legged partridges (127), white storks (81), red kites (59), and wild boars (81) (June 2014/February 2015) were inoculated in Slanetz-Bartley agar supplemented with vancomycin (4 µg/mL). We investigated the susceptibility to 12 antimicrobials and the presence of 19 antimicrobial resistance and five virulence genes. In addition, we performed multilocus sequence typing, detection of IS16 and studied Tn1546 structure. One VRE-a isolate was identified in one wild boar. This isolate was identified as Enterococcus faecium, harbored vanA gene included into Tn1546 (truncated with IS1542/IS1216), and belonged to the new ST993. This isolate contained the erm(A), erm(B), tet(M), dfrG, and dfrK genes. Neither element IS16 nor the studied virulence genes were detected. Ninety-six VRE-i isolates were identified (89 Enterococcus gallinarum and seven Enterococcus casseliflavus), with the following prevalence: red kites (71.2 %), white storks (46.9 %), red-legged partridges (7.9 %), and wild boars (4.9 %). Most E. gallinarum isolates showed resistance to tetracycline (66.3 %) and/or erythromycin (46.1 %). High-level resistance to aminoglycosides was present among our VRE-i isolates: kanamycin (22.9 %), streptomycin (11.5 %), and gentamicin (9.4 %). In general, VRE-i isolates of red kites showed higher rates of resistance for non-glycopeptide agents than those of other animal species. The dissemination of acquired resistance mechanisms in natural environments could have implications in the global spread of resistance with public health implications.

The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

Detection of vanC 1 gene transcription in vancomycin-susceptible Enterococcus faecalis

Here we report the presence and expression levels of the vanC 1 and vanC 2/3 genes in vancomycin-susceptible strains of Enterococcus faecalis. The vanC 1 and vanC 2/3 genes were located in the plasmid DNA and on the chromosome, respectively. Specific mRNA of the vanC 1 gene was detected in one of these strains. Additionally, analysis of the vanC gene sequences showed that these genes are related to the vanC genes of Enterococcus gallinarum and Enterococcus casseliflavus. The presence of vanC genes is useful for the identification of E. gallinarum and E. casseliflavus. Moreover, this is the first report of vanC mRNA in E. faecalis.

Risk factors for vancomycin-resistant enterococci colonisation in critically ill patients

Vancomycin-resistant enterococci (VRE) are important hospital pathogens and have become increasingly common in patients admitted to the intensive care unit (ICU). To determine the incidence and the risk factors associated with VRE colonisation among ICU patients, active surveillance cultures for VRE faecal carriages were carried out in patients admitted to the ICU of the University Hospital of Uberlândia, Minas Gerais, Brazil. Risk factors were assessed using a case-control study. Seventy-seven patients (23.1 percent) were found to be colonised with vanC VRE and only one patient (0.3 percent) was colonised with vanA VRE. Independent risk factors for VRE colonisation included nephropathy [odds ratio (OR) = 13.6, p < 0.001], prior antibiotic use (OR = 5.5, p < 0.03) and carbapenem use (OR = 17.3, p < 0.001). Our results showed a higher frequency (23.1 percent) of Enterococcus gallinarum and Enterococcus casseliflavus, species that are intrinsically resistant to low levels of vancomycin (vanC), without an associated infection, associated with prior antibiotic use, carbapenem use and nephropathy as comorbidity. This study is the first to demonstrate the risk factors associated with vanC VRE colonisation in ICU hospitalised patients. Although vanA and vanB enterococci are of great importance, the epidemiology of vanC VRE needs to be better understood. Even though the clinical relevance of vanC VRE is uncertain, these species are opportunistic pathogens and vanC VRE-colonised patients are a potential epidemiologic reservoir of resistance genes.

Clinical Features and Rate of Infective Endocarditis in Non-Faecalis and Non-faecium Enterococcal Bacteremia.

Non-faecalis and non-faecium enterococci are an occasional cause of bacteremia, and some cases of infective endocarditis caused by these pathogens have been reported. However, the rate of infective endocarditis in non-faecalis and non-faecium enterococcal bacteremia is still undetermined. We compared the clinical features and the rate of infective endocarditis of 70 cases of non-faecalis and non-faecium enterococcal bacteremia with those of 65 cases of Enterococcus faecalis bacteremia. Non-faecalis and non-faecium enterococcal bacteremia was more frequently associated with biliary tract infection and polymicrobial bacteremia, and was less frequently associated with infective endocarditis, than was E. faecalis bacteremia (57% vs. 28%, p<0.01; 47% vs. 31%, p=0.05; 1% vs. 14%, p<0.01, respectively).

Clinical Features and Rate of Infective Endocarditis in Non-Faecalis and Non-faecium Enterococcal Bacteremia / 전남의대학술지

Non-faecalis and non-faecium enterococci are an occasional cause of bacteremia, and some cases of infective endocarditis caused by these pathogens have been reported. However, the rate of infective endocarditis in non-faecalis and non-faecium enterococcal bacteremia is still undetermined. We compared the clinical features and the rate of infective endocarditis of 70 cases of non-faecalis and non-faecium enterococcal bacteremia with those of 65 cases of Enterococcus faecalis bacteremia. Non-faecalis and non-faecium enterococcal bacteremia was more frequently associated with biliary tract infection and polymicrobial bacteremia, and was less frequently associated with infective endocarditis, than was E. faecalis bacteremia (57% vs. 28%, p<0.01; 47% vs. 31%, p=0.05; 1% vs. 14%, p<0.01, respectively).

Clinical Features and Rate of Infective Endocarditis in Non-Faecalis and Non-faecium Enterococcal Bacteremia / 전남의대학술지

Non-faecalis and non-faecium enterococci are an occasional cause of bacteremia, and some cases of infective endocarditis caused by these pathogens have been reported. However, the rate of infective endocarditis in non-faecalis and non-faecium enterococcal bacteremia is still undetermined. We compared the clinical features and the rate of infective endocarditis of 70 cases of non-faecalis and non-faecium enterococcal bacteremia with those of 65 cases of Enterococcus faecalis bacteremia. Non-faecalis and non-faecium enterococcal bacteremia was more frequently associated with biliary tract infection and polymicrobial bacteremia, and was less frequently associated with infective endocarditis, than was E. faecalis bacteremia (57% vs. 28%, p<0.01; 47% vs. 31%, p=0.05; 1% vs. 14%, p<0.01, respectively).

An Enterococcus gallinarum Strain Carrying Both vanA and vanC1 Genes / 대한임상병리학회지

Enterococcus gallinarum carrying both vanA and vanC1 genes were detected from a surveillance culture from a patient staying at the surgical intensive care unit for a few years. E. gallinarum, SI04, was highly resistant to vancomycin (MIC of >or=256ng/mL) and teicoplanin (MIC of >or=256ng/mL). Multiplex PCR for vanA, vanB, vanC1 and vanC2/3 genes revealed SI04 to be positive for both vanA and vanC1 genes. This finding supports the fact that genotyping is needed to classify vancomycin-resistant enterococci (VRE). This is the first report on VanC VRE accompanying vanA gene in Korea.

Comparison of Antimicrobial Susceptibility Testing Methods to Detect Glycopeptide Resistance in Enterococci: E-test, Vitek, Disk Diffusion and Agar Dilution Method / 대한임상병리학회지

BACKGROUNDS: The emergence of resistant strains to glycopeptide in enterococci(GRE) is increasingly serious problem in the worldwide. Automated methods and disk diffusion test have difficulties in detecting vancomycin resistance of some strains of vancomycin-resistant enterococci(VRE), especially having vanC genotypes. And a few studies have been done assessing the ability of antimicrobial susceptibility testing methods to detect teicoplanin resistance in enterococci. METHODS: We evaluated the abilities of two commercial kits including Vitek GPS-IZ(BioMerieux, Vitek, Inc., USA) and E-test(AB Biodisk, USA), and disk diffusion test to detect glycopeptide resistance using 34 strains of vanA and 15 strains of vanC1/C2 VRE. We compared the results with those of standard agar dilution test. RESULTS: In detecting vancomycin resistance, no very major or major errors were seen, and minor error rates were observed with disk diffusion(25%), Vitek GPS-IZ(20%) and E-test(8%). Overall sensitivities of all three methods in detecting vancomycin resistance of vanA VRE were 97-100%, but sensitivities in detecting vancomycin resistance of vanC VRE were 20% in disk diffusion, 87% in E-test and 87% in Vitek GPS-IZ. In detecting teicoplanin resistance, very major error rate was high in Vitek GPS-IZ(47%), but no very major or major errors were seen in disk diffusion and E-test; minor error rates of 2% and 6% were seen in Vitek GPS-IZ and E-test, respectively. CONCLUSION: All three methods detect vancomycin resistance of vanA VRE, but they continue to demonstrate problems in detecting low-level vancomycin resistance and the Vitek GPS-IZ is difficult to detect teicoplanin resistance in enterococci.

Biochemical Tests for Differential Identification of Enterococci with VanC phenotype / 대한임상미생물학회지

BACKGROUND: Pigment production and acidification of ribose are most frequently used biochemical tests for the differentiation of three enterococcal species carrying vanC genes such as Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens. However, pigment production may occasionally be negative in E. casseliflavus, and some of E. casseliflavus may be negative or delayed reaction with ribose fermentation test. So, we performed this study to find out biochemical tests capable of distinguishing the strains possessing vanC genotypes. METHOD: A total of 17 enterococci composed of 14 clinical isolates with motility or pigment positive strains and three ATCC strains(E. gallinarum ATCC 49573, E. casseliflavus ATCC 25788, and E. flavescens ATCC 49997) Were tested by multiplex PCR of the vanC genes(vanC-1, vanC-2 and vanC-3)and various biochemical tests. RESULTS: Among the 17 isolates including three ATCC control strains, four were genotyped as VanC-1, 11 were VanC-2, one were vanC-2/3, and any of vanC genes were not detected in one clinical isolate, respectively, Among the enterococci with vanC genotype, acid production from alphaD-cyclodextrin and hippurate hydrolysis were positive only in VanC-1 gneotype(E. gallinarum), acid production from glycerol and methyl-alpha-D-mannopyranoside were positive only in vanC-2 genotype(E. casseliflavus), and acid production from rhamnose and pigment production were negative only in VanC-1 genotype. Acid production from alphaD-cyclodextrin was negative only in vanC-2 genotype. The positive rate of ribose fermentation of VanC-1, VanC-2, and VanC-2/3(E. flavescens) genotype were 100%, 82%, and 0%, respectively. CONCLUSION: Acid production from rhamnose, alphaD-cyclodextrin, betaD-cyclodextrin, glycerol and methly-alphaD-mannopyranoside, pigment production, and hippurate hydrolysis test were useful biochemical tests for differentitating E. gallinarum form E. casseliflavus. The production of acid from alphaD-cyclodextrin, glycerol, methyl-alpha-D-mannopyranoside and were suitable biochemical tests for differentiating E. casseliflavus from E. flavescens.

Use of Methyl-alpha-D-glucopyranoside Test for Species Identification of Vancomycin Resistant Enterococci / 대한임상미생물학회지

BACKGROUND: The precise identification of Enterococcus gallinarum and E. casseliflavus has assumed additional importance in clinical microbiology due to the intrinsic low-level resistance to vancomycin and the difficulty in differentiating them from E. faecium or E. faecalis, which are frequently found to be clinically significant vancomycin resistant enterococci(VRE). We evaluated the usefulness of Methyl-alpha-D-glucopyranoside(MDG) test for accurate species identification among them. METHODS: A total of 23 enterococci isolates including 18 clinical isolates of VRE from Nov 1997 to Aug 1998 and 5 VRE strains which had previously been reported as E. faecalis (2), E. faecium(2), E. avium(1) carrying vanC were tested for acidification of MDG. MDG test was done using 1% MDG in phenol red broth base and yellow coloration was interpreted as positive after 1 and 2 days of incubation at 35 degrees C. MDG results were compared with species identification by MicroScan Pos Combo type 6 (Dade, US A), motility test, pigment production, and PCR results of vanA, vanB, vanC1, vanC2/C3. RESULTS: Vancomycin resistance of 23 strains were genotyped as 7 strains of vanA, 12 strains of vanC1, 4 strains of vanC2/C3. MicroScan identified 7 vanA VRE as E. faecalis(1) and E. faecium(6), 12 VRE carrying vanC1 as E. faecalis(3), E. faecium(8) and E. avium(1), and 4 VRE carrying vanC2/C3 as E faecalis(3) and E. avium(1). Sixteen vanC VRE strains were all positive for MDG test and only 8(50%) of the 16 strains were motile. Yellow pigment were detected in all 4 vanC2/C3 VRE but only after a careful examination with a prolonged incubation. Seven vanA VRE were all negative in MDG tests, motility test and pigment production. CONCLUSIONS: MicroScan system plus motility and pigment production test was not able to differentiate reliably E. gallinarum and E. casseliflavus from E. faecalis and E. faecium. The MDG test was shown to be superior to motility test in differentiating those from E. faecalis and E. faecium. We conclude that the MDG test should be included for identifcation of VRE.

Vancomycin-resistant Enterococci: Incidence, Antimicrobial Susceptibility, and Resistance Genotypes / 대한임상병리학회지

BACKGROUND: Infections caused by vancomycin-resistant enterococci (VRE) are becoming increasingly prevalent throughout the world. VRE can spread by direct patient-to-patient contact as well as on the hands of personnel and contaminated environmental surfaces. The purpose of this study was to examine the incidence of VRE among total enterococci from clinical specimen and investigate the antimicrobial characteristics and resistance genotypes of isolated VRE. METHODS: A total of 790 enterococcal isolates from patients over a period of 12 months were screened for vancomycin resistance using brain heart infusion agar plates supplemented with 6 g/mL of vancomycin. The incidence of VRE among enterococcal isolates was calculated from microbiology statistics program. Twenty three isolates of VRE were tested for minimal inhibitory concentrations (MIC) of vancomycin, penicillin, and gentamicin and resistance genotypes. RESULTS: In the first half period, the incidence of VRE was 1.9%, and in the second half, the incidence increased to 7.7%. Thirteen strains were found to be highly resistant to vancomycin, penicillin and gentamicin (MIC, >128 g/mL). According to the direct PCR analyses, the frequency of vanB, vanC1, and vanC2 types was 13, 7, and 3 strains, respectively. CONCLUSIONS: Continued vigilance, strict enforcement of infection control, and curtailment of vancomycin use seem to be our best approaches to controlling this increasingly important problem. For this purposes, accurate and timely detection of vancomycin-resistance and periodic investigation for incidence are essential.