Identification Of Vancomycin-resistant Enterococci Research Articles

Phenotypic and Genotypic Identification of Vancomycin Resistant Enterococci from Different Sources

Phenotypic and Genotypic Identification of Vancomycin Resistant Enterococci from Different Sources

Evaluation of Chromogenic VRE Medium versus Conventional Vancomycin E Test in Detection of Vancomycin Resistant Enterococci

Background : Enterococci have become resistant to a wide range of antibiotics which include glycopeptides like vancomycin. The rapid increase of vancomycin resistance enterococci (VRE) compromises physicians to treat infections caused by these strains as the therapeutic options for VRE infections are very limited. Objectives : To evaluate the efficacy of chromogenic VRE medium in detection and identification of vancomycin resistant enterococci. Methodology : the present study was conducted in Al Abbassia Fever Hospital, El Sayed Galal Hospital, Tanta University Hospital and Kafr El Sheikh General Hospital. Sixty enterococcal isolates were collected (thirty vancomycin susceptible, eight vancomycin intermediate resistant and twenty two vancomycin resistant isolates). Antibiotic susceptibility pattern for enterococcal isolates was done using disc diffusion method, chromogenic medium and MIC of vancomycin for vancomycin resistant and intermediate resistant isolates was determined by E test. Results : The most common isolated vancomycin resistant and intermediate resistant species was E. faecium (53.1%) followed by E. faecalis (40.6%). The highest rate of isolation of VRE was from urine (50.0%). VRE isolates were mostly recovered from ICUs (45.5), 41.7%, of the collected isolates were vancomycin resistant by disc diffusion method. Regarding E-test, out of fourty two vancomycin resistant and intermediate resistant by disc diffusion method 52.4% were resistant and 19% were intermediate resistant. HiChrome VRE had 100.0% sensitivity and 83.3% specificity in identifying vancomycin resistant enterococcal isolates. Conclusion: We can depend on chromogenic media in both detection and identification of vancomycin resistant enterococci.

Phenotypic and Genotypic Identification of Vancomycin Resistant Enterococci from Different Sources

Enterococci are reservoirs for transmission of the most clinically important antimicrobial resistances such as vancomycin resistance. Therefore, this work aimed to determine the occurrence of enterococci and their respective vancomycine resistance genes ( van A and van B) from different sources. Two hundred and twenty-four samples from chickens, turkey, fish and human urine, as well as, two types of human food including milk (raw and milk from mastitic animals) and sausage were tested for isolation of Enterococcus species. The isolates were identified morphologically and biochemically using catalase test , sodium chloride tolerance and growth at pH 9.6 and 10- 45˚C. The vancomycin resistance profile of the isolates was verified by both disc diffusion and agar dilution methods. The genotypic enterococcal identification at both genus and species levels and their vancomycine resistance genes were also ascertained using PCR amplification of the respective genes for 28 isolates. Enterococci isolation rate was 70% of the examined samples with a higher percentage of vancomycine resistance (53.5%) and the minimum inhibitory concentrations (MICs) ranged from 16 to 512 µg/mL. Molecular identification of 28 enterococcal isolates revealed the dominance of E. faecalis (42.8%) and clarified a higher proportion of van A (78.5%) and van B (67.8%) genes. In conclusion, administration of the antimicrobials mainly vancomycin may be considered as a pronounced stress factor in the veterinary and human practices. In addition, VRE can act as a reservoir for vancomycin resistance.

Maldi in infection control (VRE)

The burden of multiresistant organisms in Australian hospitals continues to increase. In many facilities the optimum management of multi-resistant organism is limited by limitations on resources and budgetary constraints. Despite recent advances in molecular diagnostics, often cost is prohibitive or facilities are not available so many laboratories rely on phenotypic methods which take a considerable amount of time. Mass spectrometry by MALDI-TOF is now a well validated and widely implemented method for rapid and accurate organism identification. Although the number of organisms in the proprietary databases continues to expand, the determination of antibiotic resistance by mass spectrometry is still not widely implemented. Here I will report on a validated published method for the rapid accurate identification of vancomycin resistant enterococci (VRE) with MALDI-TOF MS and discuss other recent advances in the detection of antibiotic resistance by mass spectrometry and the possible infection control implications of this method.

P064: Direct identification of vancomycin-resistant enterococci from selective enrichment broth by mass spectrometry

It is mandatory in South Korea that patients and carriers, from whom vancomycin-resistant enterococci (VRE) are detected, should be isolated from the other patients. The rapid detection of VRE is essential to prevent the dissemination of VRE. We determined the possibilities of direct identification of VRE from selective enrichment broth by mass spectrometry for the shortening of turn-around-time.

Rapid identification of VRE with MALDI-TOF MS

Methods for the diagnosis of Vancomycin resistant Enterococci (VRE) include laborious time consuming phenotypic or costly molecular methods requiring appropriate facilities and staff. Mass spectrometry by matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) can identify organisms rapidly and has shown promise in the identification of antimicrobial resistance. We sought to establish whether MALDI-TOF MS could be used to identify VRE. Consecutive Enterococcus faecium isolates phenotypically suspected and subsequently confirmed to be vanB positive by PCR over 18 months were analysed using the Bruker mi-croflex. Control groups were similarly analysed and the ability of MALDI-TOF MS to identify VRE was calculated using statistical tools in the proprietary software program ClinProTools. The Support Vector Machine model generated the highest recognition capability and cross validation (99.24% and 88.45%, respectively). The peak statistics for the five most important peaks showed a statistically significantly difference when comparing the VREs with the controls. Internal validations produced an average sensitivity of 92.4% and specificity 85.2%. Once established the method was incorporated into routine laboratory use and validated prospectively with a specificity of 98.1% and sensitivity of 96.7%. MALDI-TOF MS provides a rapid and accurate method for the identification of VRE with significant implications for the clinical microbiology laboratory and hospital infection control.

English

In South Africa, individuals who reside in most rural areas do not have access to portable water and they rely on untreated water from boreholes. This exposes them to disease causing microorganisms. The aim of the study was to isolate and determine the antibiotic susceptibility profiles of enterococcus in ground water from three rural communities in the Mmabatho area, North-West Province of South Africa. Enterococcus selective agar (ESA) was used for selective isolation of Enterococci species. The identities of the isolates were determined using preliminary (Gram staining, catalase test, oxidase test) and confirmatory (API 20 Strep and the ProlexTM Streptococcal Grouping Rapid Latex Agglutination test kit) test. Two hundred and twenty five presumptive isolates were screened and 139 were positively identified. Three enterococcus species were identified; Enterococcus faecium(71.2%), Enterococcus faecalis (23.0%) and Enterococcus avium (5.8%). A large proportion of the isolates (73.4%) belonged to the Lands field group D serotype when compared to the G group (26.6%). A large proportion of the isolates (75 to 100%) from Ramosadi and Seweding were resistant to vancomycin, penicillin G, erythromycin, chloramphenicol, oxytetracycline, amoxycilin and sulphamethoxazole. Isolates from Mothlabeng showed little resistance against these drugs. The identification of vancomycin-resistant enterococci was a cause for concern since they pose a severe challenge to the medical profession. The natural hosts of E. faecium and E. faecalis are humans and animals. From these results, it is proposed that the isolates could have originated from contamination of these water sources with faeces since most of the boreholes are constructed near the pit toilets. Key words: Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Vancomycin resistant enterococci (VRE), multiple antibiotic resistance.

Training costs and investment payback of implementing molecular diagnostics for identification of vancomycin resistant enterococci in a clinical microbiology laboratory

Molecular diagnostics may be a more efficient method to manage resources; but most Microbiology laboratories have not introduced them into routine use due to the specialized training required. Using vancomycin resistant enterococci (VRE) screening during a comparison of a multiplex PCR (MPCR) and conventional biochemical testing (CBT) we studied 3 objectives: 1) to develop a molecular diagnostics in-house training program, 2) to assess the training program outcomes for competency and confidence, and 3) to determine laboratory payback. A training program for 14 technologists using multiple adult learning methods was implemented. Methods to minimize technical errors were introduced and included: use of a calibrated loop to deliver sample; prealiquotting reagents; increasing volume of specimen; addition of gel loading dye directly into reaction tubes; and establishment of an equivocal zone. In our laboratory MPCR costs $7.06 less than CBT, therefore the payback period for training and implementation would be approximately 3 years.

Direct detection of vanA and vanB genes in clinical specimens for rapid identification of vancomycin resistant enterococci (VRE) using multiplex PCR

Surveillance for vancomycin resistant enterococci (VRE) by culture can be labour intensive and time consuming. We have developed a multiplex polymerase chain reaction (MPCR) which can be performed directly on the clinical specimen. The assay allows sensitive detection of enterococci with vanA - and vanB -mediated resistance to vancomycin. DNA was purified from stool and rectal specimens using the XTRAXTMDNA Extraction Kit (Gull Labs). Multiplex PCR amplifiedvanA and vanB targets were detected using a microtiter plate EIA. Two-hundred specimens were tested by routine culture and MPCR. Culture identified 44 VRE isolates and MPCR detected 38 of the 44 culture positives. Multiplex PCR detected three additional positive VRE specimens missed by culture for a sensitivity and specificity of 86·4 and 98·1%, respectively. When the presence of PCR inhibitors was addressed in the six culture positive/MPCR negative specimens, four additional VRE positive specimens were detected. Performing MPCR on the original specimens and on a 1:10 dilution of all specimens to minimize the effect of inhibitors gave a sensitivity and specificity of 95·5 and 98·1%, respectively. Multiplex PCR with confirmation by microtiter plate hybridization could be completed in 8 h compared with 24–48 h required for culture.

Detection of vancomycin-resistant enterococci in fecal samples by PCR.

Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtained by rectal or perirectal swabbing. Although PCR-inhibitory substances were present in DNA prepared directly from the swabs, the inhibitory substances could be reduced by processing the nucleic acid with two commercially available DNA preparation columns. Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. DNA was extracted from the fecal material and was analyzed by PCR. By using all four primer sets, only 59 (67.8%) of the samples were positive for vanA. However, after retesting the negative samples with only the vanA primer set, 77 (88.5%) of 87 specimens that were culture positive for Enterococcus faecium containing vanA were positive by PCR. One specimen was PCR positive for the vanA gene but culture negative for enterococci. The specificity of the vanA assay was 99.6%. PCR analysis of enrichment broth samples with all four primers sets after 15 to 18 h of incubation detected 74 (85.1%) of the 87 culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. No vanB-containing enterococci were recovered by culture. Since 16 samples can be tested by PCR in 4 h (including electrophoresis), identification of VRE is possible within 8 h of specimen submission at a cost of approximately $10.12/assay. Thus, PCR may be a cost-effective alternative to culture for surveillance of VRE in some hospitals.