Isolation of Vancomycin-Resistant Enterococci From Stool Specimens

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Background

Prevalence rates of vancomycin-resistant enterococci (VRE) associated with serious clinical infections has been on the rise worldwide for over the past 15 years. Control measures being taken primarily entail the detection of non-infected but gut-colonized patients that might suspected to be harboring the VRE. The use of improved cultural-based methods may lead to a possible alternative for cost-effective VRE surveillance. The performance characteristics of the chromed VRE medium and the CHROMagar TM VRE medium Paris are systematically compared in this study. Special emphasis is put on:

Selectivity
Permanence of color and growth characteristics within the colony
Their ability to recover VRE from clinical stool specimens

With the aim to determine selectivity all characteristics of the genus Enterococcus were investigated. The National Reference Center for Streptococci was the source of VRE reference strains used as positive controls. These media prevented growth of all but one Enterococcus strains monocultures. Only tiny bluish colonies of E. gallinarum were not suppressed and grew on CHR, though neither E. solitarius nor E. were observed on both media. Objective color evaluation of colony appearance was performed by means of analyzing pictures taken during the periods of incubation and use of light and electron microscope

4 Dilutions of VREfm (Enterococcus faecium; n = 7) and VREfs (Enterococcus faecalis; 47 n = 2) reference strains were streaked in pure and mixed culture on both media AC7075 (vancomycin MIC 48 ¼g/ml), and showed a rosy brown color. The CHR medium did not distinguish VREfm from VREfs. However, 24 h of incubation made color hues of the corresponding colonies more divergent than expected to be. For the colonies edges, seven and six color hues were found out for VREfm and VREfs, correspondingly. After 24 h of incubation, VREfs colonies could be recognized by their permanent green color, while there were difficulties in identifying VREfm, which color varied. The more intensive growth of colonies on C-ID compared with CHR succored early detection and distinction of VREfm and VREfs. In general, it can be concluded that C-ID conditions were more favourable for intensive growth of VRE, than CHR agar plates. For routine evaluation stool specimens obtained from suspected patients were used. The results of the preliminary experiment conducted using both direct and indirect plating were not clear due to bacteria and yeast reducing sensitivity. Therefore, before parallel inoculation of these chromogenic media an enrichment culture of the stool specimens was applied for comparative testing. The media were evaluated for growth of suspected VRE colonies after 24 and 48 h of incubation. Suspected VRE colonies were tested implementing using Gram staining and pyrrolidonyl arylamidase paper in order to confirm Entercocci. Further identification of species was done by susceptibility testing for 50 ¼g furazolidone and 10 ¼g mupirocin using Rosco Neo-Sensitabs on Mueller Hinton agar and a rapid D-xylose fermentation. The reaction pattern allowed confirmation of E. faecium or E. faecalis or identification of intrinsically low-level vancomycin resistant enterococci like E. casseliflavus and E. gallinarum. A confirmatory test consisted of two parts: antimicrobial resistance for vancomycin and Teicoplanin applying CLSI breakpoints. The presence of Vancomycin resistance genes were determined by multiplex PCR.

A total of 259 stool samples were screened simultaneously on both chromogenic media revealing 54 VREfm and two E. gallinarum isolates demonstrating 21% of vancomycin colonization rate. The genetic relatedness of the VREfm isolates was determined using multiple-locus variable-number tandem repeat analysis

Since each chromogenic medium failed to detect one VRE strain, which was successfully detected by the other, sensitivity, were 98% for both media. Although the specificity for both media was high, 95% for CHR and 96% for C-ID, there was a risk for false positive VRE identification when solely based on Gram staining and colony pigmentation on chromogenic media. Most strikingly, growth of Pediococcus species on both media was visually indistinguishable from. PYRase testing coupled with furazolidone and mupirocin, the two tests diagnosing agar diffusion susceptibility, aided detecting of Enterococci and thereby precluded Pediococcus. Cultivation of Pediococcus species requires human stool specimens, though patients with chronic underlying diseases or experienced abdominal surgery can have infections, from which the species can be derived too. The prompt xylose fermentation test aided the detecting of species in the case of two E. gallinarum isolates while misleading colony colors were growing on both media. Furthermore, both media created conditions for yeasts growth, resulting in white colour of colonies on CHR and dull green colour of colonies on C-ID; the latter could be confused with VREfs. Yeast cells were differentiated by gram staining. The vanA genotype was found in all but one VREfm isolates. The VanB phenotype was identified in the latter one, while vanB genotype was confirmed with PCR. Cases of isolation of Vancomycin susceptible Efm or Efs from chromogenic media with enriched stool specimens were rare. Overgrowth of other bacteria and following exhaustion of sample substrates might explain it. Our study has demonstrated that the results of C-ID and CHR media experiments contradict hypotheses of rapid VRE identification; direct plating of the stool specimens or plating them after dilution complicates the process of detection. Intensive growth of various bacteria and yeasts on both media prevents media evaluation if stools are plated directly and alters the innate color of stools. Consequently, stool specimens are inapplicable for direct plating onto C-ID and CHR media.

Critical review

General

The paper is introduced by touching on the increased prevalence of the VRE and proceeds on to state how cost effective diagnosis can be achieved by use of improved culture methods. The thesis statement talks about comparison of two media. The introduction did not have anything on the role of sensitivity and specificity in the increased prevalence. The paper is not original; this comparison can be done using the already available laboratory data. The methodology is poor; there is no literature review to show what has been done and where this particular researcher(s) is picking from. The paper however can be important to microbiologist where by the results can be used to guide the choice between the two media investigated. The paper is mixed up with no clear methodology, results and discussion. The use of bacterial names and the elaboration of the procedures is okay but there are slight grammatical mistakes.

Title

The paper is too wide for the title. The title confines the paper to the two media but the researcher escalates out of the boundaries in a bid to confirm the results or identify the isolates. The use of PCR, electron microscopy makes the research to be too expensive; in fact the confirmation tests seem to be expensive than the main comparison of the two media. It is as if the researcher was investigating the appropriateness of these two methods. The researcher could have used gram staining and the basic biochemical tests to confirm the results.

Abstract

The abstract should be a summary of the study and should include all areas. The abstract in this paper is written like a conclusion, it does not give the aims of the study, main methods nor does it reflect the researchers point of view that is seen in the conclusion. In which the authors state that the two media are not sufficient for plating of stool specimens. (Heidrun 2009)

Introduction

The introduction includes the statement of the question to be addressed in the research; Comparison of Two Chromogenic Media for Selective Isolation of Vancomycin-Resistant Enterococci from Stool Specimens. However, the researchers do not provide sufficient literature review for readers to comprehend the question. He does not state the reason that prompted the comparison analysis; he/she only touches on cost effectiveness an issue that is never reflected in the rest of the paper. The authors fail to show how they developed their problem statement, what literature was reviewed and the missing links that prompted the research to be conducted.

Materials and methods

The methodology is not brought up well by the researcher, it is written like a discussion. However, it offers details for replication of the experiment conducted but does not really address the topic or research. For the methodology I think the topic should have something to do with the effectiveness of the two media (CHRC-ID) and not comparison of the two. The researcher repeatedly refers to the manufacturer in the methodology, it could have been better to write a note and state that all the procedures were carried out according to the manufacturers guide. The paper is mixed up and the author s might have done a quality investigation but he/she failed to right the report in the required format. The authors should have dedicated a section in the report for the procedures that were carried out in the study.

Results

The authors have given only the results they have observed in this study, from which they have drawn a very strong conclusion regarding the media C-ID CHR; there is no comparison with results conducted by other scientists. However, there are some instances of reference to the normally expected results as regards to the method and instrument used. The results re not organized, the authors give results after every test and so it is difficult to get a clear picture of the findings without having to go through the whole paper. Some results could have better been presented in tables, for instance the results of the determination of the isolates using the multiplex PCR. The photographs of the media plate to show the color changes should have been more for better differentiation. There is some repetition though the author clarifies that details are found in the table. There is no clear distinction between the result, discussion and conclusion parts in the paper.

Discussion

The authors interpretation of results is okay as per the methodology but he/she is a little off the mark in relation to the title of the research. The title states that the investigation seeks to compare the two chromogenic media for selective isolation of Vancomycin resistant Enterococci from stool. In the discussion the authors state that our study has clearly shown that C-ID and CHR media do not fulfill all expectations regarding rapid VRE detection, especially when plating stool specimens directly or predilution. (Heidrun 2009) The author puts a lot of emphasis on the shortcomings of the two media and this causes the deviation from the main topic of the research which is comparison of the two media. The authors havent distinguished their findings and the findings of others. The discussion is only based on the results of tests conducted in their study. The authors conclusions are minimally competent as regards to the topic of the study. On careful examination the research, one will realize that the research is okay only that the authors failed to articulate the issues appropriately.

Referencing

The referencing page is well done but the in text citations are lacking or poorly done. There are several technical terms in the paper that the author must have lifted from other sources which are not cited in the body of the text. All the references are important for the authors expanded investigation but these could have been better depicted in the literature review which is conspicuously absent.

Tables and figures

The tables are detailed and can be understood without reference to the main text. The table labeled the control table which has been sourced from culture collection of national reference center for streptococci Aachen, Germany shouldnt have been included as part of the report. (Heidrun 2009) The tables are well designed and informative.

Reference

Heidrun, P et al (2009) Comparison of Two Chromogenic Media for Selective Isolation of Vancomycin: Web.

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