Detection Of Vancomycin Resistance Research Articles

Detection of Vancomycin Resistance among Methicillin-Resistant Staphylococcus aureus Strains Recovered from Children with Invasive Diseases in a Reference Pediatric Hospital.

Vancomycin is the cornerstone in treating methicillin-resistant Staphylococcus aureus (MRSA) infections. However, therapeutic failures can occur when MRSA strains with decreased susceptibility to glycopeptides (DSG) are involved. The aim of this study was to detect and characterize DSG in MRSA recovered from children with invasive diseases at a reference pediatric hospital between 2009 and 2019. Fifty-two MRSA strains were screened using agar plates with vancomycin 3 and 4 mg/L (BHI-3 and BHI-4); the VITEK2 system; and standard and macro E-tests. Suspicious hVISA were studied by population analysis profiling-area under the curve (PAP-AUC), and wall thickness was analyzed by transmission electron microscopy. Neither VRSA nor VISA were detected in this set. As only three strains met the hVISA criteria, the PAP-AUC study included 12 additional MRSA strains that grew one colony on BHI-4 plates or showed minimum inhibitory concentrations of vancomycin and/or teicoplanin ≥ 1.5 mg/L. One strain was confirmed as hVISA by PAP-AUC. The wall thickness was greater than the vancomycin-susceptible control strain; it belonged to ST30 and carried SCCmec IV. As expected, a low frequency of hVISA was found (1.9%). The only hVISA confirmed by PAP-AUC was not detected by the screening methods, highlighting the challenge that its detection represents for microbiology laboratories.

Detection of Vancomycin Resistance among Hospital and Community-acquired Methicillin-resistant Staphylococcus aureus Isolates

Detection of Vancomycin Resistance among Hospital and Community-acquired Methicillin-resistant Staphylococcus aureus Isolates

Occult Vancomycin-Resistant Enterococcus faecium ST117 Displaying a Highly Mutated vanB2 Operon

Rare information is available on clinical Enterococcus faecium encountered in Sardinia, Italy. This study investigated the antimicrobial susceptibility profiles and genotypic characteristics of E. faecium isolated at the University Hospital of Sassari, Italy, using the Vitek2 system and PCR, MLST, or WGS. Vitek2 revealed two VanB-type vancomycin-resistant Enterococcus faecium (VREfm) isolates (MICs mg/L = 8 and ≥32) but failed to detect vancomycin resistance in one isolate (MIC mg/L ≤ 1) despite positive genotypic confirmation of vanB gene, which proved to be vancomycin resistant by additional phenotypic methods (MICs mg/L = 8). This vanB isolate was able to increase its vancomycin MIC after exposure to vancomycin, unlike the "classic" occult vanB-carrying E. faecium, becoming detectable by Vitek 2 (MICs mg/L ≥ 32). All three E. faecium had highly mutated vanB2 operons, as part of a chromosomally integrated Tn1549 transposon, with common missense mutations in VanH and VanB2 resistance proteins and specific missense mutations in the VanW accessory protein. There were additional missense mutations in VanS, VanH, and VanB proteins in the vanB2-carrying VREfm isolates compared to Vitek2. The molecular typing revealed a polyclonal hospital-associated E. faecium population from Clade A1, and that vanB2-VREfm, and nearly half of vancomycin-susceptible E. faecium (VSEfm) analyzed, belonged to ST117. Based on core genome-MLST, ST117 strains had different clonal types (CT), excluding nosocomial transmission of specific CT. Detecting vanB2-carrying VREfm isolates by Vitek2 may be problematic, and alternative methods are needed to prevent therapeutic failure and spread.

The Investigation of the Efficacy of Some Antiseptics and Disinfectants to Vancomycin-Resistant Enterococci

Disinfection of contaminated material, surface and body parts with appropriate substances is one of the most effective practices in preventing the infection-colonization of Vancomycin-Resistant Enterococci (VRE). In this study, 1/10 and 1/100 dilutions of 5% sodium hypochlorite, 4% concentration of chlorhexidine gluconate and 0.1% and 0.5% concentrations of Akacid plus’s efficacy against VRE strains at 1, 5, 15 and 30 minutes was tested. Sixty-nine vancomycin-resistant E. faecium strains isolated from different body samples between 2012 and 2014 were included in the study. The BD Phoenix 100® fully automated system was used for the identification of strains and detection of vancomycin resistance. Quantitative Suspension Test method EN 1276 (March 2010) was used to evaluate the bactericidal activities of chemical disinfectants and antiseptics used in European Union countries in the investigation of disinfectant susceptibility of VRE strains. Five log10 decrease (105) in the number of bacteria in the initial test suspension between the number of bacteria after using the disinfectant indicates that the disinfectant is effective. In this study, it has been determined that a 1/10 dilution of 5% sodium hypochlorite (bleach) is the most effective disinfectant on VREs in all minutes and conditions. Effectiveness of 1/100 dilution of bleach has a significant decrease in dirty conditions. Akacid plus was not effective on all strains in any contact times we tested. It has been observed that Akacid plus is the disinfectant least affected by the changes in environmental conditions in all minutes. Chlorhexidine gluconate (4%) was ineffective in clean and dirty conditions in just 1 minute, while from the 5th minute, its effectiveness was found to be statistically at the same level as the 1/10 dilution of bleach. There was no decrease in the activities of chlorhexidine due to the changing environmental conditions. In conclusion, it has been determined that a 1/10 dilution of 5% sodium hypochlorite (bleach) is the most effective disinfectant on VREs in all minutes and conditions, but dirty environmental conditions cause a significant decrease in the effectiveness of sodium hypochlorite. In addition, it was observed that the effectiveness of all disinfectants and antiseptics on VREs increased as the contact time with the substances increased.

Two Screening Assays to Detect Vancomycin-Resistant Enterococcus spp.

Enterococci have become major nosocomial pathogens. An increasing number of these infections are as a result of vancomycin-resistant enterococci. Accurate detection of vancomycin-resistant enterococci (VRE) is important, so that appropriate therapy and infection control measures may be instituted, including veterinary surveillance. Two screening assays to detect vancomycin resistance in enterococci are proposed. Barnes Basal Medium agar (Ba) and Brain Heart Infusion (BHI) broth (plus 1% TTC-2,3,5-triphenyltetrazolium chloride) with several concentrations of vancomycin were used in this work. Five Enterococcus casseliflavus strains with low-level resistance to vancomycin (4 µg/mL) were used. Both media were able to quickly detect the breakpoint of the vancomycin-resistant strains used in this work, and also provided insight into the dynamics of the antibiotic effect at a low concentration on the tested bacterial suspensions.

Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (HVISA) at a Tertiary Hospital in Malaysia

Methicillin-resistant Staphylococcus aureus remains a global problem. The emergence of reduced susceptibility to Vancomycin in MRSA strains, leads to treatment failure and prolonged hospital stay. Therefore, we aimed to determine the strains with reduced susceptibility among MRSA clinical isolates. S. aureus isolates were collected from identified clinical samples. Antibiotic susceptibility was tested using the Kirby-Bauer method. MRSA strains were confirmed using PCR for mecA gene and subjected to the Epsilometer test (Etest®) for determination of Vancomycin minimum inhibitory concentration (MIC). Isolates with intermediate or reduced susceptibility were subjected to broth microdilution (BMD) and further confirmed by population analysis-area under curve (PAP-AUC) testing. Disc diffusion tests showed that all isolates (n = 105; 100%) were susceptible to Rifampicin, followed by Trimethoprim/Sulfamethoxazole. Meanwhile, a high resistance rate was demonstrated against Penicillin (n = 93; 88.6%). Among all isolates, only 26.0% (n = 27) were MRSA. According to the Vancomycin MIC value by Etest®, only two strains (A3, A106) had intermediate susceptibility, and one strain (A30) had reduced susceptibility to Vancomycin (MIC 3 μg/mL). No susceptibility to Vancomycin was identified among strains using the BMD method. According to the PAP-AUC method, it was confirmed that strain A3 was a heterogeneous VISA strain. Vancomycin Etest®, is a reliable screening test for VISA detection. Vancomycin BMD result was not in agreement with the Vancomycin Etest® result. PAP-AUC, the gold standard test used to detect Vancomycin resistance, should be conducted whenever possible for further confirmation and epidemiological record.

A paradigm shift in Enterococcal infections: A cause of concern

Introduction: Infections due to Gram-positive bacteria are on a rise, of which those due to Enterococcus spp. form a major part. The shifting pattern of infections caused by Enterococci from novel urinary tract infections to soft tissue and wound infections are increasingly reported. Objective: The aim of the present study is to isolate, speciate Enterococci, determine the antibiotic resistance pattern and to detect vancomycin resistance by E- test. Materials and Methods: All wound swabs, pus samples sent for bacterial culture, irrespective of the diagnosis of the patients which were received to the department of microbiology for a period of one year were included in the study. The samples were processed for isolation, identification and speciation of Enterococci. Antimicrobial susceptibility testing was done by Kirby-Bauer disk diffusion method. Vancomycin resistance was determined by E-test. Results: A total of 3000 pus samples were received over a period of one year. Out of which 43 isolates were detected as Enterococcus species. Among 43 isolates, 26 were E.faecalis, 17 were E.faecium. In present study, ampicillin resistance was seen in 88% followed by resistance to erythromycin (86%) and clindamycin (72%). High level gentamycin resistance was seen in 28% cases. Vancomycin resistance was not detected in any of the samples. Conclusion: The shifting spectrum of infections due to Enterococcus is gaining inquisitiveness among health care workers especially in the trauma care units. Thus there is a prerequisite to isolate speciate and determine the resistance that helps in holistic health care. Keywords: Enterococci, Soft tissue infection, Antibiotic resistance, Vancomycin resistance, E test.

Development of high-resolution melting curve analysis in rapid detection of vanA gene, Enterococcus faecalis, and Enterococcus faecium from clinical isolates

BackgroundHigh-resolution melting analysis (HRMA) is a novel molecular technique based on the real-time PCR that can be used to detect vancomycin resistance Enterococcus (VRE). The purpose of this study was to identify VRE species with HRMA in clinical isolates.ResultsOut of 49 Enterococcus isolates, 11 (22.44%) E. faecium isolates and 19 (38.77%) E. faecalis isolates were detected. Average melting temperatures for divIVA in E.faecalis, alanine racemase in E.faecium, and vanA in VRE strains were obtained as 79.9 ± 0.5 °C, 85.4 ± 0.5 °C, and 82.99 ± 0.5 °C, respectively. Furthermore, the data showed that the HRMA method was sensitive to detect 100 CFU/ml for the divIVA, alanine racemase, and vanA genes. Also, out of 49 Enterococcus spp., which were isolated by HRMA assay, 8 isolates (16.32%) of E. faecium and 18 isolates (36.73%) of E. faecalis were detected. The vanA gene was reported in 2 isolates (25%) of E. faecium and 9 isolates (50%) of E. faecalis.ConclusionsThis study demonstrated that using the HRMA method, we can detect E. faecium, E. faecalis, and the vanA gene with high sensitivity and specificity.

Occurrence of vancomycin-resistant Staphylococcus aureus in the oral cavity of patients with dental caries.

Oral streptococci are the major group of microbes isolated from oral microflora. They represent frequent pathogens of infective endocarditis (IE), and it is assumed that in most of the cases oral streptococci are acquired via mucosa layer of oral cavity. Staphylococcus aureus is also frequently isolated from IE as it accounts for 20%-30% of all cases. Vancomycin has been the most reliable therapeutic agent against infections caused by methicillin-resistant S. aureus (MRSA). The main objective of this study was to examine the occurrence of S. aureus species in dental caries specimens. Antimicrobial susceptibility testing of S. aureus to four antibiotics namely vancomycin, linezolid, teicoplanin, and daptomycin was performed. Detection of vancomycin resistance was conducted using polymerase chain reaction. Among the tested 150 strains, 98 were MRSA and of that 54 were vancomycin sensitive and 27 were resistant. All 98 MRSA strains were positive for mecA and 36 yielded pvl, whereas 13 carried vanA and only 2 were positive for vanB. Majority of the isolates showed sensitivity toward daptomycin and linezolid. Strains of S. aureus exhibiting decreased susceptibility to different antibiotics like vancomycin, daptomycin, and linezolid severely compromise the therapeutic alternatives and require a considerable amount of time, public awareness, and integrative health-care strategies to prevent the emergence of resistance to these compounds.

Detection of vancomycin resistance among enterococcus faecalis isolated from dental caries population

Detection of vancomycin resistance among enterococcus faecalis isolated from dental caries population

Characterization of Vancomycin Resistant Enterococci in Hospitalized Patients and Role of Gut Colonization.

Enterococci are part of the normal intestinal flora and have been recognized as important human pathogens. Vancomycin Resistant Enterococci (VRE) are global threat as this resistance is transmissible and also poses a challenge for infection control. This study was undertaken to study phenotypic and genotypic characteristics of VRE from clinically significant infections among hospitalized patients and their association with gut colonization. Clinically significant isolates of enterococci (n=250) were studied. Species confirmation was done by Polymerase Chain Reaction (PCR). Minimum Inhibitory Concentration (MIC) for vancomycin was determined by E-test. PCR for VanA, VanB and VanC1 gene was done for genotypic characterization. MIC for teicoplanin, linezolid, tigecycline, daptomycin and quinupristin-dalfopristin was determined by E test. Patients with VRE infection were screened for gut colonization using vancomycin screen agar (6 μg/mL). Continuous data was analysed using the Student's t-test. Categorical data was assessed using Pearson's Chi-square test. A value of p ≤ 0.05 was considered statistically significant. There was good correlation between the phenotypic and genotypic methods used for species identification and detection of vancomycin resistance. E. faecium (162, 64.8%) was most common followed by E. faecalis (82, 32.84%) and E. gallinarum (6, 2.4%). Overall higher resistance was observed among E. faecium. Vancomycin MIC ≥ 2 μg/mL was noted in 63 (25.2%) isolates. Fifty seven isolates showed presence of vanA and vanC1 was detected in six isolates of E. gallinarum. Isolates with VanB genotype was not detected in the present study. MIC50 (μg/mL) for teicoplanin, linezolid, tigecycline, daptomycin and quinupristin-dalfopristrin was 24, 0.75, 0.064, 2 and 0.064 respectively. Resistance to linezolid (1, 1.6%) and tigecycline (2, 3.2%) was rare. Majority (33/47, 70.2%) patients with clinically significant VRE infection showed gut colonization. Vancomycin resistance among enterococci is emerging. Emergence of tigecycline and linezolid resistance is also posing a challenge for clinicians. Thus, further investigations are warranted to control vancomycin resistance among pathogens.

Antibiotic resistance profile of Enterococcus faecium andEnterococcus faecalis isolated from broiler cloacal samples

The present study was performed to isolate and identify Enterococcus spp. from broiler cloacal samples to species level, to determine their resistance patterns to various antibiotics, and to detect vancomycin resistance genes. Cloacal samples of broilers collected from slaughterhouses were inoculated in Slanetz and Bartley agars with and without vancomycin (6 μg/mL). Antibiotic resistance/susceptibility testing of the isolated and identified enterococci was performed by using the disk diffusion test. Multiplex PCR was used to identify the species and to detect vancomycin resistance genes. The majority of the isolated enterococci was Enterococcus faecium (60.43%, n = 142) and Enterococcus faecalis (33.62%, n = 79). E. casseliflavus and E. gallinarum were identified from 8 (3.42%) and 6 (2.56%) isolates, respectively. It was found that 88.9% of the enterococci were resistant to tetracycline and 83.4% of them were resistant to erythromycin. As a result, none of the strains isolated from cloacal samples of broilers carried the vanA and vanB genes. It was observed that 54.9% of E. faecium isolates and 78.4% of E. faecalis isolates were multidrug resistant (resistant to 3 or more antibiotic groups). The lack of vancomycin-resistant Enterococcus among the enterococci isolates was important for public health.

Detection of vancomycin resistance in enterococcus species isolated from clinical samples and feces of colonized patients by phenotypic and genotypic methods.

The aim of this study was to find out the clinical correlation between the presence of vancomycin-resistant genes (van A and van B) and their expression as detected by phenotypic tests in colonized patients and in clinical isolates. Enterococci were isolated from various clinical samples and also from fecal specimens of colonized patients at the time of admission, after 48 h and after 5 days of admission. Identification to species level was done using standard methods. Vancomycin susceptibility in Enterococci was detected by disc diffusion test. Minimum inhibitory concentration was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of van genes. Out of all the clinical and fecal samples processed, 12.0% isolates were either vancomycin resistant or vancomycin intermediate. Further, these isolates carried van A or van B genes as confirmed by PCR methods. Expression of van A gene was found to be more in Enterococcus faecalis (28.3%) as compared to Enterococcus faecium (25.0%) in both clinical and fecal isolates. 16.6% strains of E. faecium and 15.0% strains each of E. faecalis and Enterococcus gallinarum were found to carry van B genes. The overall prevalence of vancomycin resistant Enterococci (VRE) in colonized patients was about 9.6%. Prior administration of antibiotics had significant effect (P = 0.001) on VRE carriage. Urinary tract infection was the most common infection caused by vancomycin susceptible Enterococci (VSE), 105/214 (49.0%) and VRE, 13/36 (36.1%). There was no significant difference (P = 0.112) in the distribution of VRE and VSE in different infection types. Both clinical and fecal VRE showed maximum resistance to penicillin, ampicillin, and piperacillin. Resistance to linezolid was 2.8% in clinically isolated VRE. VRE in our study were found to be resistant to a number of commonly used antibiotics. The frequency of isolation of vancomycin resistant E. faecalis (VRE.fs), which is highly virulent, and the number of strains harboring van A gene in our hospital setup is high and needs to be addressed.

Detection of vancomycin resistance among clinical isolates of enterococci

Vancomycin -resistant Enterococci (VRE), are the bacterial strains of the genus Enterococcus that are resistant to vancomycin. Enterococci bacteria live in our intestines and on our skin, usually without causing problems. But if they become resistant to antibiotics they can cause serious infections, especially in people who are ill or weak. This study is aims to detect the vancomycin resistance in 20 clinical isolates of Enterococcus spp. We found varied resistance pattern by disc diffusion method and none of the isolates was found to be vancomycin resistance. As Vancomycin Resistant Enterococci (VRE) is emerging to be a threat in nosocomial settings, it is very important to check the same for prompt and accurate therapy.

Detection of Vancomycin Resistance among Enterococcus faecalis and Staphylococcus aureus.

Vancomycin remains the drug of choice for resistant gram positive infections caused by Enterococcus spp and Methicillin resistant Staphylococcus aureus (MRSA). Increased use of vancomycin has led to frank resistance and increase in MIC (MIC creep). Vancomycin intermediate Staphylococcus aureus (VISA), Vancomycin resistant Staphylococcus aureus (VRSA) & Vancomycin resistant Enterococci (VRE) are important emerging nosocomial pathogens resulting in treatment failures. This study was undertaken to detect vancomycin resistance among clinical isolates of Staphylococcus aureus and Enterococcus faecalis by phenotypic and genotypic methods. The study was conducted in a 1850 bedded university teaching hospital from November 2013 to April 2014. Non-repetitive, consecutive clinically significant Staphylococcus aureus (109) and Enterococcus faecalis (124) were included in this study. They were identified up to species level by conventional and automated methods. Susceptibility to various antibiotics was tested by disc diffusion method. MIC of vancomycin was determined by agar dilution method. Inducible resistance to clindamycin was detected by the D test. Methicillin resistance in Staphylococcus aureus (MRSA) was screened using cefoxitin disc. All isolates were subjected to polymerase chain reaction (PCR) to detect van A and van B genes. Out of 109 Staphylococcus aureus isolates, 54 were MRSA. By MIC there was no resistance observed to vancomycin.MIC50 was 1μg/ml. None of the isolates harboured van A and van B. Among Enterococcus faecalis, sixteen isolates (12.9%) and four isolates (3.2%) exhibited resistance to vancomycin and teicoplanin by disc diffusion respectively. All isolates were susceptible to linezolid. Van A was detected in 2, van B in 7 and one had both van A and van B. PCR remains the gold standard for diagnosis of vancomycin resistance. There was no resistance observed to vancomycin among Staphylococci though the MIC creep detected is a cause for concern. Eight percent of Enterococci were vancomycin resistant.

English

The increasing occurrence of Enterococcus species , worldwide, since late 1980s, is of particular concern due to the emergence of Vancomycin Resistant Enterococci (VRE). The appearance of VRE has limited the therapeutic options available for clinicians. VRE infection is the most common type of infection acquired by patients while hospitalized. Patients at risk for VRE are those who are already ill, and hospitalized, including individuals with diabetes, elderly, ICU patients, kidney failure patients, or patients requiring catheters. Present study was undertaken to detect vancomycin resistance in enterococcal isolates from hospitalized patients and the comparison of Kirby-Bauer disc diffusion method and vancomycin agar screen method to screen for vancomycin resistance. A total of 45 enterococcal isolates from various samples of hospitalized patients were speciated by standard biochemical reactions and screened to detect vancomycin resistance by Kirby-Bauer disc diffusion method and vancomycin agar screen method. Out of 45 enterococcal isolates, 26 (57.77%) were Enterococcus faecalis, 18 (40%) were Enrerococcus faecium and 1(3.33%) was Enterococcus avium. Resistance to vancomycin was 29% (13 isolates) where as 71% (32 isolates) were sensitive. By Kirby-Bauer disc diffusion method, 1 isolate (2%) showed resistance to vancomycin whereas by vancomycin screen agar, 13 (29%) showed resistance. The study highlights that vancomycin screen agar method is more sensitive than Kirby- Bauer disc diffusion method for detecting vancomycin resistance in enterococcal isolates. This study also signals the emergence of VRE in the hospital and highlights the importance of screening for VRE in enterococcal isolates from various samples.

Glycopeptide Resistance in Gram-Positive Cocci: A Review

Vancomycin-resistant enterococci (VRE) have emerged as important nosocomial pathogens in the past two decades all over the world and have seriously limited the choices available to clinicians for treating infections caused by these agents. Methicillin-resistant Staphylococcus aureus, perhaps the most notorious among the nosocomial pathogens, was till recently susceptible to vancomycin and the other glycopeptides. Emergence of vancomycin nonsusceptible strains of S. aureus has led to a worrisome scenario where the options available for treating serious infections due to these organisms are very limited and not well evaluated. Vancomycin resistance in clinically significant isolates of coagulase-negative staphylococci is also on the rise in many setups. This paper aims to highlight the genetic basis of vancomycin resistance in Enterococcus species and S. aureus. It also focuses on important considerations in detection of vancomycin resistance in these gram-positive bacteria. The problem of glycopeptide resistance in clinical isolates of coagulase-negative staphylococci and the phenomenon of vancomycin tolerance seen in some strains of Streptococcus pneumoniae has also been discussed. Finally, therapeutic options available and being developed against these pathogens have also found a mention.

Vancomycin-resistant Enterococcus spp.: validation of susceptibility testing and in vitro activity of vancomycin, linezolid, tigecycline and daptomycin

Vancomycin-resistant enterococci (VRE) have emerged to become a significant nosocomial pathogen. However, detection may be challenging and treatment possibilities are limited. Reports of resistance to linezolide, daptomycin and tigecycline underline the need for reliable susceptibility testing with respect to these compounds. We evaluated the in vitro activity of vancomycin, linezolid, daptomycin and tigecycline against a panel of VRE and vancomycin-susceptible enterococci by broth microdilution (BMD). Etest for determination of minimum inhibitory concentration of these four antibiotics and two disc diffusion assays for detecting VRE and for susceptibility testing against tigecycline and linezolid were evaluated. Before susceptibility testing, all isolates were classified by polymerase chain reaction as vanA or vanB gene positive or vanA/B gene negative. Linezolid, daptomycin and tigecycline had excellent in vitro activity towards all isolates. For daptomycin and tigecycline, the overall agreement between BMD and Etest was suboptimal. For both disc diffusion assays, use of current break points was inadequate to detect vancomycin resistance for isolates carrying the vanB gene. Inspection of the inhibition zone for a diffuse edge, as recommended, accurately predicted presence of the vanB gene.

Improved Detection of vanB2 -Containing Enterococcus faecium with Vancomycin Susceptibility by Etest Using Oxgall Supplementation

We have isolated a number of vanB-containing Enterococcus faecium isolates on bile esculin screening agar containing 6 mg/liter vancomycin, which on subsequent susceptibility testing using Etest have repeatedly demonstrated vancomycin MICs of <or=4 mg/liter. To investigate this genotype-phenotype incongruence of "low-MIC vancomycin-resistant enterococci" (LM-VRE), we examined the molecular characteristics of these isolates, including the presence of the vanB operon, using PCR amplification and DNA sequencing. All LM-VRE isolates contained vanB associated with the transposon Tn1549 and were polyclonal. Sequencing of the vanB ligase gene showed no differences from the prototype vanB2. In addition, we examined supplemented media to improve phenotypic detection of these isolates. Etest detection of LM-VRE improved when Mueller-Hinton agar (MHA) and brain heart infusion agar (BHIA) were supplemented with 10 g/liter oxgall (MHA-Oxg and BHIA-Oxg, respectively). We assessed the sensitivity and specificity of these media to detect vancomycin resistance using vancomycin-resistant vanB-containing E. faecium (n = 11), vancomycin-susceptible (van negative) E. faecium (n = 11), vancomycin-susceptible (van negative) E. faecalis (n = 11), and our LM-VRE (n = 23) isolates. After 48 h of incubation, both MHA-Oxg and BHIA-Oxg were 100% (34/34) sensitive and 100% (22/22) specific in the identification of vancomycin resistance. These findings suggest that supplementation of MHA or BHIA with 10 g/liter oxgall should be considered in laboratories where VRE detection protocols rely primarily on strain phenotype rather than early vanB gene detection by PCR.

The Resazurin Microplate Method for Rapid Detection of Vancomycin Resistance in Enterococci

Vancomycin-resistant enterococci (VRE) are a serious challenge for physicians because of the limited treatment options for infections caused by this organism. Prevention of VRE transmission in hospitals requires early detection of infected or colonized patients. Therefore rapid and correct detection of vancomycin resistance is essential. In this study, we use the resazurin microplate method (RMM), which is a modification of the NCCLS and BSAC broth microdilution methods to rapidly determine the susceptibilities of clinical enterococci isolates to vancomycin. The alteration in the RMM was relevant to the final bacterial count. In this method, inoculum that was 10-fold higher than standard methods was used. A total of 80 enterococci, including 11 VRE isolates and 6 vancomycin intermediate isolates, were screened with this modified colorimetric broth microdilution method. After 4 h of incubation 30 μl of 0.01% resazurin solution were added to each well and the plates were reincubated for color change for 5-10 min. The MICs were obtained at the 4th h. The results were in exact agreement with the NCCLS and the BSAC microdilution methods. Absolute and essential agreements were 100% and there were no minor, major or very major errors. In conclusion, this modified colorimetric broth microdilution method can be used as a reliable, easy, cheap and rapid method for early detection of VRE. Moreover, this method has the potential of being used to test the susceptibilities of different bacteria to other antibiotics.