|Year : 2016 | Volume
| Issue : 2 | Page : 188-193
|Detection of vancomycin resistance in enterococcus species isolated from clinical samples and feces of colonized patients by phenotypic and genotypic methods
Priyanka Paul Biswas1, S Dey1, L Adhikari2, Aninda Sen1
1 Department of Microbiology, Katihar Medical College, Katihar, Bihar, India
2 Department of Microbiology, Sikkim Manipal University, Gangtok, Sikkim, India
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|Date of Web Publication||9-May-2016|
| Abstract|| |
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.
Keywords: Van A, van B, vancomycin resistant Enterococci
|How to cite this article:|
Biswas PP, Dey S, Adhikari L, Sen A. Detection of vancomycin resistance in enterococcus species isolated from clinical samples and feces of colonized patients by phenotypic and genotypic methods. Indian J Pathol Microbiol 2016;59:188-93
|How to cite this URL:|
Biswas PP, Dey S, Adhikari L, Sen A. Detection of vancomycin resistance in enterococcus species isolated from clinical samples and feces of colonized patients by phenotypic and genotypic methods. Indian J Pathol Microbiol [serial online] 2016 [cited 2019 Dec 10];59:188-93. Available from: http://www.ijpmonline.org/text.asp?2016/59/2/188/182015
| Introduction|| |
Vancomycin resistant Enterococci (VRE) have caused hospital outbreaks worldwide, which has been dramatically amplified in recent years, because of widespread abuse and misuse of antibiotics, leading to increase in infections caused by these strains.
Promotion of vancomycin-resistant enterococcal colonization is due to rampant use of vancomycin, and also third generation cephalosporins, imipenem, metronidazole, and clindamycin with potent activity against anaerobes, which lead to VRE colonization of gastrointestinal tract (GIT) by competitive eradication of sensitive species. VRE colonization leads to cross-infection, dissemination, and endogenous infection. Hence, it has been proposed by many workers that attempts must be made to detect the rate of VRE isolation in diseased as well as colonized patients. Therefore, screening for the presence of VRE, in diseased persons only does not provide the total picture of this problem.
The errors associated with the phenotypic disc diffusion method leads to unwarranted use or elimination of this drug from a treatment regimen. Use of multiplex polymerase chain reaction (PCR) limits the errors arising out of phenotypic methods.
It is not always easy to assess the clinical significance of VRE isolation in routine culture or to differentiate colonization from infection. Therefore, the present study was undertaken to look for vancomycin resistance in Enterococci obtained in significant numbers from various clinical samples as well as fecal samples of colonized patients. The isolation of VRE is found to vary in different geographical regions, and this study was undertaken to determine the vancomycin resistant genotypes prevalent in Eastern Bihar, India.
| Materials and Methods|| |
The study population included patients of both sexes and all age groups attending the outpatient and inpatient departments of a tertiary care hospital in Eastern Bihar, India. A total of 500 strains of Enterococci were collected from samples submitted to the Microbiology Laboratory for culture and sensitivity. 250 enterococcal strains were collected from clinical samples of the patients, attending the hospital with infections of different types. The second group included another 300 patients (otherwise not suffering from any infections), who were admitted to the hospital and screened for gastrointestinal carriage of VRE. Another 250 enterococcal strains were isolated from this group. Clearance from Institutional Ethics Committee was obtained to carry out this study.
Isolation and identification
Two-hundred and fifty Enterococci were isolated from various clinical samples (urine, pus, blood, catheter tip, and tracheal aspirate). 300 fecal samples were collected from other patients, (as mentioned above) on three occasions, that is, at the time of admission, after 48 h, and after 5 days of admission to screen for VRE. The isolates were identified to species level using standard procedures.,,,,
Antimicrobial susceptibility testing
Antibiotic susceptibility test was done by Kirby-Bauer disc diffusion method on Muller-Hinton agar. Inoculum was prepared and adjusted to 0.5 McFarland's turbidity standard. Antibiotic disc was obtained from the Hi Media Laboratories (Mumbai).
Determination of minimum inhibitory concentration by agar dilution method
Agar dilution was used to determine minimum inhibitory concentration (MIC) of vancomycin. Brain-heart infusion agar (Hi Media, Mumbai) was supplemented with different concentrations of vancomycin. The test organism was grown in broth and the turbidity matched with McFarland's 0.5 standard. The bacterial strains were spot inoculated on the surface of agar medium using 10 μL bacterial culture. The plates were incubated at 37°C for 24 h. The minimum concentration of vancomycin, which inhibited bacterial growth, was considered MIC. Enterococci which had MIC ≥32 μg/mL were considered resistant; MIC of 8–16 μg/mL as intermediately resistant; and MIC of ≤4 μg/mL as susceptible to vancomycin.,
DNA extraction method
Genomic DNA used as template for PCR amplification was prepared using conventional phenol-chloroform DNA extraction method.
The following oligonucleotide primers were used for amplification of 1030 bp of the van A gene: F14-CATGAATAGAATAAAAGTTGCAATA and R14-CCCCTTTAACGCTAATACGATCAA and 433-bp of the van B gene: F15-GTGACAAACCGGAGGCGAGGA and R15-CCGCCATCCTCCTGCAAAAAA. Primers were obtained from Merck's, Lucknow, India. The total volume of PCR mix was 25 µl including: 2 µl dNTPs mix, 0.3 µl 5U Taq polymerase, 2.5 µl 10X buffer, 4 µl van A and 4 µl van B primer, and 12.17 µl double distilled water.
Polymerase chain reaction assay for van A and van B genes
The PCR amplification of the van genes were carried out as per standard protocol. Both positive control (positive VRE culture with known inherited vancomycin positive genes) and negative control, consisting solely of the PCR reaction mixture without DNA template, were included to check the validity of the technique. The amplified products were electrophoresed using 1.5% agarose gel. A 100 bp DNA ladder marker was included as the standard molecular weight marker. The electrophoresed gel was later subjected to ethidium bromide staining and photographed under ultraviolet transillumination  [Figure 1]. PCR was performed in PCR system, model number T1 Thermoblock.
|Figure 1: Bands corresponding to 1030 bp (van A) and 433 bp (van B) in polymerase chain reaction assay. Polymerase chain reaction was performed in polymerase chain reaction system, model number T1 Thermoblock|
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Statistical analysis was done using Chi-square test. P < 0.05 was considered significant and P < 0.001 was considered highly significant.
| Results|| |
A total of 500 Enterococci (250 from clinical samples and 250 from fecal samples of colonized patients) were processed, out of which 37 (7.4%) isolates were vancomycin resistant and 23 (4.6%) showed reduced susceptibility to vancomycin by phenotypic agar dilution method. However, by disc diffusion test (DDT), 34 (6.8%) strains were found to be resistant to vancomycin and 20 (4.0%) strains were found to show reduced susceptibility.
MIC of various clinical strains of enterococcus to vancomycin showed 11 strains to have reduced susceptibility to vancomycin, that is, MIC ranging from 8 to 16 μg/ml. Another 11 strains of E. faecalis, 9 strains of E. faecium, and 5 strains of E. gallinarum had MIC ≥64 μg/ml, and were considered as VRE. On the other hand, 12 fecal isolates showed reduced susceptibility and another 12 strains (6 strains each of E. faecalis and E. faecium showed resistance, that is, MIC ≥64 μg/ml [Table 1].
|Table 1: MIC range for vancomycin (μg/mL) in different clinical isolates|
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Thus, a total of 60 strains (36 clinical and 24 fecal) which were VRE/vancomycin intermediate Enterococci (VIE) by MIC tests were also confirmed to carry van A or van B genes by PCR methods. 17 strains of E. faecalis and 15 strains of E. faecium were found to carry van A gene and 10 strains of E. faecium and 9 strains each of E. faecalis and E. gallinarum were found to carry van B genes. The prevalence of van A gene was found to be significantly higher (P = 0.027) in the clinical strains [Table 2].
Out of 300 fecal samples collected, 50 (16.7%) samples were culture negative for enterococcus and 250 (83.3%) samples showed growth of enterococcus species. 70 strains each were isolated from gynecology, medicine, and surgery department and 20 strains each from Medical Intensive Care Unit (MICU) and pediatrics departments, respectively. 12 (50.0%), 7 (29.2%), and 1 (4.1%) VRE carriers were from surgery, medicine, and gynecology departments, respectively. On the other hand, 4 (16.6%) VRE carriers were from patients admitted in MICU. The VRE carriage rate was maximum in surgery followed by medicine department.
Out of a total of 250 fecal isolates, VRE were detected in 24 (9.6%) patients, out of which 16 (6.4%) were males and 8 (3.2%) were females. The male to female ratio of patients colonized with VRE was 2:1. The difference in the colonization rate of VRE in both sexes was insignificant (P = 0.09) [Table 3]. VRE carriage was maximum in middle age group 41–50 years, 11 (45.8%) followed by 10 (41.7%) in 51–60 years, and 3 (12.5%) in 31–40 years of the 24 VRE patients, rate of colonization was 87.5% (21/24) after 5 days as compared to 12.5% (3/24) after 48 h.
|Table 3: Age and sex--wise distribution of the patients harboring VRE in the GIT|
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Eighteen out of the 24 patients with VRE colonization had received various antibiotics viz., 41.7% received cephalosporins, 12.5% each received fluroquinolones and metronidazole, and 8.3% received gentamicin. Prior administration of antibiotics had significant effect (P = 0.001) on VRE carriage [Table 4]. None of the patients with VRE colonization had preceding Gram-positive bacteremia or had undergone GIT surgery or received vancomycin.
|Table 4: Administration of antibiotics in patients colonized with VRE and VSE|
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VRE from clinical samples were isolated from patients in the age group 51–60 years, 9 (25.0%); 31–40 years, 8 (22.2%); 41–50 years, and 7 (19.4%); 6 (16.6%) each in 61–70 years and 21–30 years.
Out of the 36 clinical VRE, 13 strains were from the cases of urinary tract infection (UTI) among which the predominant isolate was E. faecium 7 (53.8%); 10 strains were from wound infection of which E. gallinarum 4 (40.0%) was the major isolate. 5 out of the 9 VRE that caused blood stream infection (BSI) were E. faecium (55.5%). Another 4 (50.0%) strains were found to cause catheter induced infection (CII), of which two each were E. faecalis and E. faecium [Table 5]. Out of the 214 vancomycin sensitive isolates, 105 isolates were from UTI, 69 from wound infection, 25 from BSI, and 15 from catheter related infection. Most of the nonfaecalis, nonfaecium strains were isolated from urine followed by wound infection [Table 5]. No significant difference (P = 0.112) in the distribution of VRE and vancomycin susceptible Enterococci (VSE) in different infection types was seen.
|Table 5: Distribution of VSE and VRE (clinical) in different infection types|
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[Table 6] shows the antibiotic resistance pattern of VSE and VRE (clinical) by disk diffusion test. For VSE, maximum resistance was seen with penicillin 84.1%, followed by ampicillin 70.0%, and piperacillin 67.8%. For VRE, maximum resistance was seen with the same antibiotics viz.: penicillin 100%, ampicillin 91.7%, and piperacillin 75.0%. Least resistance was seen with linezolid for both VSE and VRE being 0% and 2.8%, respectively. For fecal VSE, maximum resistance was seen with penicillin 88.5%, followed by piperacillin 66.4%, and ampicillin 64.2%. All strains were sensitive to linezolid. VRE also showed maximum resistance to these 3 antibiotics being 100% for penicillin, 87.5% for ampicillin, and 79.2% for piperacillin. None of the strains was resistant to linezolid.
|Table 6: Antibiotic resistance pattern of VSE and VRE (clinical and fecal) by DDT|
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| Discussion|| |
VRE has become an important nosocomial pathogen because of its rapid spread, high mortality rates associated with infections, limited option for treatment, and the possibility of transferring vancomycin resistance genes to other more virulent and more prevalent pathogens such as Staphylococcus aureus. We investigated the prevalence of vancomycin resistance in both clinical and fecal isolates (250 each) by phenotypic and genotypic methods. Our study showed correlation between the presence of van genes and their expression as detected by MIC tests by agar dilution method. However, the DDT failed to detect 6 strains of VRE/VIE. Overall, 12.0% of the 500 strains isolated were found to be VRE/VIE. Other authors reported the VRE positivity rate to be low as 6.9%. This differences in the prevalence of VRE in different regions are governed by various factors including the use of glycopeptides in humans and animals (growth promoters).
In the present study, 54 (10.8%) isolates were vancomycin intermediate or resistant by DDT. However, by agar dilution method 23 (4.6%), isolates showed reduced susceptibility to vancomycin, that is, MIC ranging from 8 to 16 μg/ml and another 37 (7.4%) isolates showed resistance, that is, MIC ≥ 64 μg/ml, that is, total 60 (12.0%). This observation clearly indicates that DDT may fail to recognize as resistant those enterococcal strains that have reduced susceptibility to vancomycin. Similar observation was made in another study, where all the enterococcal strains were susceptible to vancomycin by DDT but showed intermediate resistance to vancomycin (MIC, 8 µg/ml).
Many species of Enterococci may be vancomycin resistant but majority are E. faecium. In this study, multiplex PCR proved helpful in detecting the van genotypes present in this geographic region. In this study, expression of van A gene was seen with E. faecalis (28.3%) isolates followed by Enterococus faecium (25.0%) in both clinical and fecal isolates. Few E. gallinarum (15.0%) strains were found to carry van B gene. In clinical isolates, prevalence of van A was more than van B, a finding that was found to be statistically significant (P = 0.027). PCR test result of other studies showed that 13 strains of E. faecium carried van A gene and 1 strain of E. gallinarum carried van C1 gene. This finding is quite different from the findings of this study in which E. faecalis, which is a more virulent strain, was predominantly found to carry van A gene. As compared with van B, the van A gene is known to have increased transferability, which may explain the rapid increase in the number of van A isolates. Strains of E. gallinarum carrying van B gene also has important role in the dissemination of antibiotic resistant genes as compared to E. gallinarum carrying van C genes which is nontransferable and an inherent characteristic.
Majority of the VRE isolated from the colonized patients were from surgery department 50.0% (12/24) followed by medicine 29.2% (7/24), MICU 16.6% (4/20), and gynecology 1.4% (1/70) departments. No VRE was isolated from pediatrics department. Other authors have reported that 30.5% (39/128) patients were colonized with VRE strains: 7.8% (10/39) in the ICU, 10.9% (14/39) in surgery, and 11.7% (15/39) in medicine wards. No VRE strain was isolated from pediatric ward, a finding similar to our study. The authors explain this finding may be due to physical isolation, intrinsic differences in bowel mileu, and lack of exposure to food or other environmental sources.
Majority of the colonized patients with VRE infections were males 16 (6.4%) and 8 (3.2%) were females. The reason for the predominance of male patients colonized with VRE infections could be due to the fact that, especially in rural settings, it is generally the males who seek medical attention as compared to females who tend to ignore their sufferings.
The rate of colonization with VRE was 12.5% (3/24) after 48 h which increased to 87.5% (21/24) after 5 days. Thus, our study reveals that the rate of colonization with VRE increased proportionately with increase in length of stay in the hospital, which is probably due to longer exposure to the hospital mileu which harbor these organisms.
Eighteen out of the 24 VRE colonized patients were found to receive various antibiotics, which had significant effect (P = 0.001) on VRE carriage. In another study, significant relation between previous administration of antibiotics and VRE carriage was reported (P = 0.02).
Majority of the VRE positive isolates from clinical samples were from the age group 51–60 years, 9 (25.0%), which is expected as most VRE infections are found in immunosuppressed and debilitated patients. Some authors reported 12.7% VRE positive cases from the middle age group 46–60 years and 5.2% cases in the age group 61–75 years. In yet another study, the minimum age of the patients with highly resistant Enterococci was 18 years and the maximum age was 71 years. No significant relation between age and VRE colonization was noted by the authors. These differences may be due to different sample size.
Majority of the clinical isolates (both VRE and VSE) were recovered from urine 47.2% (118/250), followed by wound infection 31.6% (79/250), blood 13.6% (34/250), and catheter samples 7.6% (19/250), which is consistent with reports that Enterococci have become the leading cause of UTI, surgical wound infection, bacteramia, and catheter induced infection. The bladder, prostate, and kidney are commonly infected by Enterococci, especially in patients with structural abnormalities of the urinary tract, indwelling catheters, or following instrumentation.
Our results were concordant with many Indian studies showing a gradual increase in resistance to penicillins and ampicillin over the years. For clinical VSE, maximum resistance was seen with penicillin 84.1% (180/214), followed by ampicillin 70.0% (150/214), and piperacillin 67.8% (145/214). For clinical VRE, maximum resistance was also seen with the same antibiotics viz.: Penicillin 100% (36/36), ampicillin 91.6% (33/36), and piperacillin 75.0% (27/36). Least resistance was seen with linezolid for both VSE and VRE, being 0%. Other authors have reported that clinical isolates of Enterococci showed maximum resistance to gentamicin (58.0%), followed by tetracycline (47.1%), and ampicillin (43.0%), with 0% resistance to vancomycin and linezolid. In another study from Bangalore, maximum resistance was seen with erythromycin and least with teicoplanin and linezolid.
| Conclusion|| |
This study reveals the emergence of vancomycin resistant E. faecalis isolates carrying van A gene and vancomycin resistant E. gallinarum with van B gene from this geographic region. This finding is alarming since it suggests the possibility of transfer of these plasmid borne van A and van B genes to other Gram-positive bacteria as well as to other plasmid free Enterococci both in the GIT and in hospital environment. It is, therefore, imperative to maintain a strict vigil on the spread of these organisms in the hospital and also from the hospital to the community.
The use of molecular methods reduces the errors associated with phenotypic disc diffusion methods. Standardization of the PCR results with phenotypic tests will enable a laboratory to choose an array of tests that needs to be performed in a particular laboratory in a particular area based on the requirement and infrastructure of the laboratory concerned.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Priyanka Paul Biswas
Department of Microbiology, Katihar Medical College, Katihar, Bihar
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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