Emergence of vanA gene among vancomycin-resistant enterococci in a tertiary care hospital of North - East India.

Background & objectives: Vancomycin-resistant enterococci (VRE) have become one of the most challenging nosocomial pathogens with the rapid spread of the multi-drug resistant strain with limited therapeutic options. It is a matter of concern due to its ability to transfer vancomycin resistant gene to other organisms. The present study was undertaken to determine the emergence of vancomycin-resistant enterococci and the vanA gene among the isolates in a tertiary care hospital of North-East India. Methods: A total of 67 consecutive enterococcal isolates from different clinical samples were collected and identified by using the standard methods. Antibiogram was done by disk diffusion method and VRE was screened by the disk diffusion and vancomycin supplement agar dilution method. The minimum inhibitory concentration (MIC) value for vancomycin was determined by E-test. The VRE isolates were analyzed by PCR for vanA gene. Results: A total of 54 (81%) Enterococcus faecalis and 13 (19%) E. faecium were detected among the clinical isolates and 16 (24%) were VRE. The VRE isolates were multidrug resistant and linezolid resistance was also found to be in three. MIC range to vancomycin was 16-32 μg/ml among the VRE. The vanA gene was found in nine of 16 VRE isolates. Interpretation & conclusions: Emergence of VRE and presence of vanA in a tertiary care hospital setting in North-East India indicate toward a need for implementing infection control policies and active surveillance.

Characterization of Vancomycin-Resistant Enterococci Isolated from Stools and Their Acquisition of Vancomycin Resistance

We have isolated 6 vancomycin resistant (VR) Enterococcus faecium and 5 VR-E. gallinarum. Vancomycin resistant enterococcus (VRE) isolates were resistant to multi-drugs, but susceptible to linezolid and quinupristin/dalfopristin. VRE isolates showed 10 VanA phenotypes and 1 VanB phenotype (E. gallinarum). However, all of them showed vanA genotype. vanA gene was detected on both genomic and plasmid DNA from all VRE isolates. Almost of VR-E. faecium had IS1216V which is worldwide type and almost of VR-E. gallinarum had IS1542 which is European type. IS1216V and IS1542 genes were not related with antibiotic types of VRE. Copy numbers of vanA were decreased in VRE with IS1216V or IS1542 but not in VRE with both ISs in broth without vancomycin. The copy numbers of vanA were significantly decreased in VanB phenotype of VRE with IS1542 in broth without vancomycin. Copy numbers of vanA were recovered in the presence of vancomycin. Growth time of reference E. faecium is faster than that of reference E. faecalis when cultured in the broth containing vancomycin. Reference strains cultured in the broth containing vancomycin showed intermediate resistance or resistance to antibiotics without acquisition of van genes. Naturally, multidrug-resistant E. faecium might be fast adapted in the presence of vancomycin compared to E. faecalis. Taken together, VanA phenotype E. gallinarum as well as E. feacium have been increasing in nosocomial infection and showed acquired inducible resistance. E. faecium and E. faecalis showed intermediate resistance in long exposure of vancomycin without acquisition of vanA.

Rapid Screening of Vancomycin Resistant Enterococci with Chromogenic Agar (ChromID VRE) / 병원감염관리

BACKGROUND: Rapid screening of vancomycin-resistant enterococci (VRE) is very important for controlling and preventing the spread of VRE in hospitals. We compared the performance characteristics of a chromogenic agar (ChromID VRE, bioMerieux, France: CA) to that of Enterococcosel agar (supplemented with 6 microgram/mL of vancomycin :EA) for direct detection of VRE from stool swabs. METHODS: Total 125 rectal swabs were collected from 57 patients in the intensive care units of an 850-bed university hospital over a period of 3 months. The samples were inoculated on EA, CA and into broth enrichment containing 6 microgram/mL of vancomycin (BE). BE was subcultured on CA after overnight incubation. RESULTS: Eighty two samples from 22 patients were positive for VRE by BE. At 24 h, the sensitivity/specificity of EA and CA were 89%/100% and 72%/100%, respectively. At 48 h, the sensitivity/specificity of EA and CA were 94%/89% and 89%/100%, respectively. CONCLUSION: CA provides equivalent sensitivity comparable to EA for the recovery of VRE at 48 h incubation, and has additional advantage of being able to differentiate between vancomycine resistant E. faecium and E. faecalis.

Clinical Characteristics in Patients with Vancomycin-Resistant Enterococci Colonization or Infection during 5 years in a Private General Hospital / 대한구급학회지

BACKGROUND: Vancomycin-resistant enterococci (VRE) is increasing rapidly through the world and is now a major cause of nosocomial infection. The transmission dynamics and factors contributing their dissemination are complex. We conducted a study to investigate clinical characteristics in patients with VRE colonization or infection during recent 5 years. METHODS: 154 cases that had the VRE infection or colonization from January 1, 2000 to April, 2004, were reviewed. We analyzed the risk factors of VRE infection and colonization and also compared various parameters contributing their dissemination between burn and non-burn patients with VRE. RESULTS: Total 212 strains of VRE were isolated from 154 patients. Of 212 strains of VRE, Enterococcus faecium (178 strains, 83.9%) were most common and followed by E. casseliflavus (28 strains, 13.2%), E. faecalis (5 strains, 2.4%) and E. gallinaum (1 strains, 0.5%). The most common place of VRE isolation was in burn intensive care unit (ICU), 95 cases (61.7%); 27 cases (17.5%) in general wards; 17 cases (11.0%) in surgical ICU; 15 cases (9.7%) in medical ICU. Compared with patients with VRE colonization, patients with VRE infection had older age, higher APACHE II scores and high death rate significantly. Then, VRE colonization were more common in burn patients while VRE infection were more common in non-burn patients. CONCLUSIONS: The findings from this study suggest that VRE infection are not uncommon among hospitalized patients. More strict infection control, close surveillance and judicious use of antibiotics may be warranted to prevent infection and transmission of VRE.

Molecular Characterization of Vancomycin-Resistant Enterococci Isolated from Poultry in Korea / 대한임상미생물학회지

BACKGROUND: Vancomycin-resistant enterococci (VRE) have been increasingly isolated worldwide as a nosocomial pathogen. In Korea, because avoparcin has been used as a growth promoter in animal feed, vanA-containing enterococci have been found in animals. The aim of this study is to understand the epidimiology of VRE isolated from chicken of diverse geographic areas. METHODS: Thirty eight isolates of VanA VRE from chicken of diverse geographic areas were investigated. Multiplex PCR was used to confirm the genotype of VRE. Long PCR and restriction fragment length polymorphism (long PCR-RFLP) were performed to analyze the structure of Tn1546. If the RFLP pattern was different from the prototype, PCR amplification of internal regions of Tn1546 and subsequent sequencing analysis was performed. RESULTS: All 38 isolates harbored vanA gene and divided into 3 types by long PCR-RFLP. Thirty five isolates (92%) were classified as type I. Two isolates and one isolate were belonging to type II and type III, respectively. Type II revealed an insertion of IS1216V in vanX-vanY intergenic region. IS element integrated type III did not match any previously reqorted seguence. CONCLUSION: Structural analysis of vanA gene cluster from chicken revealed that the majority of isolates (type I) have indistinguishable structure to E. fecium BM4147. This results indicated horizontal spread of resistance gene among isolates from chicken. Genetic rearrangement of Tn1546 was infrequently detected in Korea.

Clinical Characteristics and Risk Factors of Death among Patients with Vancomycin-Resistant Enterococci (VRE) during 8 Years (1994-2001) in a University Hospital / 감염과화학요법

BACKGROUND: Vancomycin-resistant enterococci (VRE) infection is an emerging nosocomial problem. VRE usually multidrug-resistant, poses therapeutic dilemmas. The gene that encodes the resistance against vancomycin may spread the resistance to Staphylococcus aureus. However, there are no well-organized studies on the clinical manifestations and the factors that contribute to mortality in Korea. Herein, this study was focused on the clinical manifestations and mortality risks of patients with VRE infection during 8 years (1994-2001) in a university hospital. Understanding of the epidemiology and clinical manifestations of VRE would help develop control strategy of VRE outbreak in a hospital. METHOD: Sixty seven cases that had the VRE infection in Korea University Guro Hospital from January 1, 1994to December 12, 2001, were reviewed. We analyzed the risk factors of VRE infection and death by using univariable and multivariable statistic analyses. RESULTS: VRE infections have recently been increasing. Most of VRE infections were caused by Enterococcus faecium (85.1%) and Enterococcus faecalis (10.4%). Among 67 cases, 40 cases (59.7%) expressed VanA phenotype, 23 cases (34.3%) expressed VanB phenotype, and 3 cases expressed VanC phenotype (6%). The risk factors for death were renal dysfunction, central venous catheter insertion, and tracheostomy by using univariable analysis. The risk factor for death was renal dysfunction by using multivariable analysis. CONCLUSION: VRE has been increasing during the late 1990s in Korea. The VRE infection occurs especially in the patients who have renal dysfunction, long-term hospitalization, and ICU care. The implementation of careful isolation, infection control measures, prudent use of antibiotics, especially vancomycin, and periodic screening of patients populations are required to control VRE infection.

Clinical Characteristics and Risk Factors of Death among Patients with Vancomycin-Resistant Enterococci (VRE) during 8 Years (1994-2001) in a University Hospital / 감염과화학요법

BACKGROUND: Vancomycin-resistant enterococci (VRE) infection is an emerging nosocomial problem. VRE usually multidrug-resistant, poses therapeutic dilemmas. The gene that encodes the resistance against vancomycin may spread the resistance to Staphylococcus aureus. However, there are no well-organized studies on the clinical manifestations and the factors that contribute to mortality in Korea. Herein, this study was focused on the clinical manifestations and mortality risks of patients with VRE infection during 8 years (1994-2001) in a university hospital. Understanding of the epidemiology and clinical manifestations of VRE would help develop control strategy of VRE outbreak in a hospital. METHOD: Sixty seven cases that had the VRE infection in Korea University Guro Hospital from January 1, 1994to December 12, 2001, were reviewed. We analyzed the risk factors of VRE infection and death by using univariable and multivariable statistic analyses. RESULTS: VRE infections have recently been increasing. Most of VRE infections were caused by Enterococcus faecium (85.1%) and Enterococcus faecalis (10.4%). Among 67 cases, 40 cases (59.7%) expressed VanA phenotype, 23 cases (34.3%) expressed VanB phenotype, and 3 cases expressed VanC phenotype (6%). The risk factors for death were renal dysfunction, central venous catheter insertion, and tracheostomy by using univariable analysis. The risk factor for death was renal dysfunction by using multivariable analysis. CONCLUSION: VRE has been increasing during the late 1990s in Korea. The VRE infection occurs especially in the patients who have renal dysfunction, long-term hospitalization, and ICU care. The implementation of careful isolation, infection control measures, prudent use of antibiotics, especially vancomycin, and periodic screening of patients populations are required to control VRE infection.

Molecular Relationship of vanA Glycopeptide Resistance Gene in Enterococci from Hospitalized Patients and Poultry in Korea / 감염

BACKGROUND: Vancomycin-resistant enterococci (VRE) with vanA gene have been reported as a significant nosocomial pathogen. The vanA gene cluster (Tn1546) located on mobile DNA elements is known to be transferable from VRE to other enterococci. The purpose of this study was to investigate the genetic relationship between the vanA VRE strains isolated from hospitalizd patients and poultry. METHODS: Total 145 isolates, including 58 E. faecium, 12 E. faecalis, 3 E. casseliflavus, and 4 E. gallinarum from humans and 68 E. faecium from poultry, were studied. Antimicrobial susceptibility tests were done by disk diffusion or agar dilution methods and molecular epidemiological analysis was performed by pulsed-field gel electrophoresis (PFGE). The internal and structural regions of vanA gene cluster were analyzed by PCR fragment length polymorphism, restriction enzyme, and sequencing of Orf2D region and vanXY intergenic region. The point mutation at Tn1546 nucleotide position 8234 (G->T) within the vanX gene was screened with DdeI restriction enzyme. RESULTS: The antibiotic resistance patterns of human isolates were different from those of poultry. PFGE patterns revealed high heterogeneity. Three PCR fragment length patterns in the vanA gene cluster were found : (I) PCR amplicon of the same size as prototype (E. faecium BM4147) in 17% of human isolates and 100% of poultry ones; (II) PCR amplicon for vanXY intergenic region due to an insertion between vanX and vanY genes in 2.5% of human isolates; (III) the insertions in vanX-vanY intergenic and Orf2 regions in 81% of human isolates. The T type in vanX gene of human and poultry isolates was not found. CONCLUSION: Despite the diverse PFGE patterns, 81% of human and all of poultry isolates belonged to vanA gene cluster type III and I, respectively. These results indicate that the horizontal spread of vanA gene is occurring among genetically diverse strains of VRE in Korea.

Susceptibility of Fosfomycin against Vancomycin Resistant Enterococci / 감염

BACKGROUND: Vancomycin-resistant enterococci (VRE) were first recovered from clinical isolates in Korea in 1992, and the incidence has been steadily increasing. Alternatives to vancomycin are few because VRE are frequently resistant to commonly used antimicrobial agents. The present study was designed to assess the in-vitro activity of fosfomycin to clinical isolates of VRE. METHODS: For 199 VRE isolates from 1995 to 2000, and 91 enterococcal isolates that were consecutively isolated during the January of 2001 at Wonju Christian Hospital, fosfomycin (200 microgram) disk diffusion test was done by NCCLS method. The number of enterococcal isolates tested for fosfomycin were as follows:58 E. faecalis (42 vancomycin susceptible isolates, 16 vancomycin resistant isolates, and 1 vancomycin intermediate resistance isolate); 210 E. faecium (185 vancomycin resistant and 25 vancomycin susceptible isolates); 15 E. gallinarum, and 6 E. casseliflavus isolates. RESULTS: Among the VRE isolates, the resistance rates of fosfomycin according to enterococcal species were 6.3% in E. faecalis, 4.9% in E. faecium, 0% in E. casseliflavus, and 16.7% in E. gallinarum. CONCLUSION: Fosfomycin could be a potentially useful drug for the treatment of infections caused by VRE.

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.

Investigation of Risk Factors for Vancomycin-Resistant Enterococci(VRE) Infection and Colonization / 대한임상병리학회지

BACKGROUND: The emergence of vancomycin-resistant enterococci(VRE) is increasingly serious problem throughout the world and is likely to increase in Korea. However, a few epidemiologic studies and risk factors of VRE infection have been reported in Korea. We investigated risk factors for VRE infection and colonization. METHODS: We analyzed 48 patients with VRE(24 infection, 24 fecal colonization) and 62 vancomycin-sensitive enterococci(VSE) in Ewha Womans university hospital from January 1997 to December 1998 and we performed case-control study to assess the risk factors for VRE. RESULTS: The incidence of VRE infection was 7.3% of all enterococcal isolates and the incidence of VRE colonization from surveillance cultures was increased 0.1% to 1.5% from 1997 to 1998. Compared with patients with VSE, patients with VRE had significantly longer hospital stays. They also had more frequent stays in intensive care unit(ICU) and oncology wards. They had more frequent invasive procedures such as central lines, urinary catheters, nasogastric tubes, ventilators and were more likely to have received vancomycin or teicoplanin or aztreonam or aminoglycosides or cephalosporins therapy(P<0.05). Compared with the patients infected with VRE, the patients colonized with VRE had significantly more frequent stays in the ICU(P <0.05). CONCLUSIONS: To prevent the VRE infection and colonization, appropriate antibiotic therapy according to the guidelines and cautious handling of medical devices may be necessary.

A Molecular Epidemiologic Study on Vancomycin-Resistant Enterococci Isolated from Clinical Specimens over a 5-year Period / 감염

BACKGROUND: Vancomycin-resistant enterococci (VRE) infection is an emerging nosocomial problem. In Chonnam National University Hospital, 64 vanA VRE isolates were recovered from clinical specimens of 21 patients from January 1995 through December 1999. It is important to understand the epidemiology of VRE within a hospital for implementing appropriate infection control measures. METHODS: Twenty-one vanA VRE isolates obtained from 21 patients during over a 5-year period were analyzed by pulsed-field gel electroporesis (PFGE) fingerprinting. Medical records of the patients with VRE isolates were reviewed. RESULTS: By PFGE analysis for 21 VRE isolates, eleven genotypes were determined; 4 genotypes were shared by more than 2 patients. This four small clusters of VRE involved patients from hemato-oncologic units and ICU (intensive care unit) over 3 days to 1 year period. CONCLUSION: Although the VRE epidemic at our hospital is polyclonal, some spread of VRE from a clonal origin were found in this hospital. The implementation of careful isolation and infection control measures, prudent use of antibiotics, especially vancomycin, and periodic screening of patients populations, are required.

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.

Investigation of Prevalence of Vancomycin-resistant Enterococci and Genotypes of Glycopeptide Resistance Using Polymerase Chain Reaction / 대한임상병리학회지

BACKGROUND: Enterococci are a leading cause of nosocomial infection and the emergence of resistant strain to various antibiotics including vancomycin is increasingly serious problems among enterococci. And the risk of spread of glycopeptide genes to other Gram-positive cocci makes the problems more serious. To evaluate the presence of vancomycin-resistant enterococci (VRE) in Ewha Womans University Hospital (EWUH), we screened hospitalized patients for fecal colonization and clinical isolates. METHODS: We screened VRE in 574 stool specimens requested for routine cultures and 91 perirectal swabs or stool specimens from patients who reside in intensive care unit and hemato-oncologic ward in Mookdong and Tongdaemoon EWUH from December 1996 through April 1997. And 295 enterococcal species isolated from various clinical specimens were also included. To detect VRE, specimens were cultured in BHI agar medium including 6 g/mL of vancomycin and to determine the antibiotic susceptibility pattern, broth microdilution test using VITEK GPS-IZ, disk diffusion test and standard agar dilution test were performed. Multiplex PCR was done to determine the genotypes of VRE. RESULTS: Nine enterococci (0.9%) were interpreted as VRE in standard agar dilution method. Two (0.3%) out of 665 were from stool speciemens for surveillence cuture and 7 (2.3%) out of 295 were from various clinical specimens for ordinary cultures including 5 E. casseliflavus, 2 E. gallinarum, 1 E. flavescens and 1 Enterococcus species. All isolates showed low-level resistance against vancomycin (8-16 g/mL) by standard agar dilution. But both disk diffusion method and VITEK system demonstrated difficulties in detecting low-level resistance. The genotypes of VRE were classified as van C-1 in 2 isolates and as van C-2 in 6 isolates except 1 isolates, which was unclassifiable in our study. CONCLUSIONS: Even though VRE with high- or medium-level resistance against glycopeptide was not detected in EWUH from this period of investigation, the possibility of presence of VRE is impanding because several teaching hospitals already reported the presence of VRE in clinical isolates and fecal colonization. So continuous surveillence and strict infection control measures must be implemented to detect and prevent transmission of VRE infection.

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.