Sangeeta Dey

Virulence markers of vancomycin resistant enterococci isolated from infected and colonized patients

Background: The aim of study was to find out the potential pathogenic role of virulence factors elaborated by strains of vancomycin resistant enterococci (VRE) isolated from clinical samples and VRE colonizing the gastrointestinal tract of hospitalized patients. Materials and Methods: 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. Various virulence determinants were detected by phenotypic tests. Vancomycin susceptibility in enterococci was detected by disc diffusion and agar screen method. Minimum inhibitory concentration was determined by agar dilution method. Results: Out of all the clinical and fecal samples processed, 12.0% isolates were either vancomycin resistant or vancomycin intermediate. Hemagglutinating activity against rabbit red blood cells was seen with 27.8% and 25.0% of clinical and fecal strains, respectively. Slime layer formation was seen with fecal VRE strains (37.5%) when compared to clinical VRE (27.8%). Among the clinical VRE strains the most prolific biofilm producers were Enterococcus. fecalis (92.9%) when compared to Enterococcus. faecium (52.9%). Biofilm formation/(presence of adhesions) was also seen in (29.2%) of the fecal VREs. In wound infection production of gelatinase, deoxyribonuclease (DNase), and caseinase (70.0% each) were the major virulence factors. The predominant virulence factors seen in the blood stream infection were adhesin, and hemolysin (44.4% each) and in catheter induced infection were DNase and adhesins (75.0% each). Adhesin (29.2%), slime layer (37.6%), DNAse (33.3%), gelatinase (25.0%), lipase (20.8%) and caseinase (16.6%) and hemolysin (8.3%) were produced the fecal isolates. Conclusion: An association between adhesin (as detected by biofilm formation) and urinary tract infection, adhesion and hemolysin with BSI, as also between DNase gelatinase & caseinase with wound infection was noted.

Molecular characterization of virulence genes in vancomycin-resistant and vancomycin-sensitive enterococci

Background: The aim of this study was to find out the correlation between presence of virulence (gelatinase [gel E], enterococcal surface protein [esp], cytolysin A [cyl A], hyaluronidase [hyl], and aggregation substance [asa1]) and vancomycin-resistant genes (van A and van B) in enterococci, with their phenotypic expression. Materials and Methods: A total of 500 isolates (250 each clinical and fecal) were processed. Enterococci were isolated from various clinical samples and from fecal specimens of colonized patients. Various virulence determinants namely asa1, esp, hyl, gel E, and cyl were detected by phenotypic methods. Minimum inhibitory concentration (MIC) of vancomycin was determined by agar dilution method. Multiplex polymerase chain reaction (PCR) was used to detect the presence of virulence and van genes. Results: Out of all the samples processed, 12.0% (60/500) isolates carried van A or van B genes as confirmed by MIC test and PCR methods. Genes responsible for virulence were detected by multiplex PCR and at least one of the five was detected in all the clinical vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE). gel E, esp, and hyl genes were found to be significantly higher in clinical VRE. Of the fecal isolates, presence of gel E, esp, and asa1 was significantly higher in VRE as compared to VSE. The presence of hyl gene in the clinical VRE was found to be statistically significant (P = 0.043) as against the fecal VRE. Correlation between the presence of virulence genes and their expression as detected by phenotypic tests showed that while biofilm production was seen in 61.1% (22/36) of clinical VRE, the corresponding genes, i.e., asa1 and esp were detected in 30.5% (11/36) and 27.8% (10/36) of strains only. Conclusion: Enterococcus faecium isolates were found to carry esp gene, a phenomenon that has been described previously only for Enterococcus faecalis, but we were unable to correlate the presence of esp with their capacity to form biofilms.

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

Background: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.