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Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 337-341 Original Article Prevalence of virulence factors and antibiotic resistance in vancomycin-resistant Enterococcus faecium isolated from sewage and clinical samples in Iran S Jahangiri1, M Talebi2, G Eslami3, MR Pourshafie2 1 Department of Microbiology, Pasteur Institute of Iran, Tehran;Department of Microbiology, Faculty of Medical Science, Shahid Behenhti Medical University, Tehran, Iran Date of Submission: 26-May-2010 Code Number: mb10101 PMID: 20966565 Abstract Purpose: The purpose of the present study was to perform a molecular epidemiological survey by investigating the antibiotic resistance and the presence of known virulence factors in Enterococcus faecium isolates in Iran. The data collected from this study would allow us to control the spread and develop strategies for treatment of the enterococcal infections. Keywords: esp, Enterococcus faecium, vancomycin resistance, vancomycin sensitive, virulence factor Introduction Enterococci are normally considered as bacteria of low pathogenicity, which only infect persons with special immunodeficiency diseases. [1] Conversely, enterococci have emerged as nosocomial pathogens and cause serious disease in individuals with increasing antibiotic resistance. [2] This is further exacerbated by the presence of virulence factors which, in turn, could increase the pathogenicity of these organisms. [3] Enterococcus faecium has become difficult to be treated by glycopeptides and aminoglycosides, [4] which is the reason for the rapid emergence of E. faecium as an aetiological agent of nosocomial infections in the last two decades. [5] Furthermore, the presence of various virulence factors has been indicated for the widespread presence of the enterococci. The virulence factors such as aggregation substance (asal), cytolysin (cyl), hyaluronidase (hyl), the enterococcal surface protein (esp) and gelatinase (gelE) are often reported. Contrary to E. faecalis, no gelatinase, aggregation substance and cytolysin have been reported in E. faecium.[3],[6] Other virulence factors such as Esp and Hly have been described in both E. faecalis and E. faecium. [4],[7] In continuation with our previous reports, [8],[9] here we investigate the virulence factors in E. faecium isolated from clinical setting and sewage in Iran, where no such information is available. Materials and Methods Bacterial isolates A total of 156 enterococcal isolates were included in this study. All enterococcal isolates had been collected during 2006. Vancomycin-resistant and -sensitive enterococci had been isolated from clinical and sewage treatment plants (STPs). The clinical samples were collected from hospitalised patients in three major hospitals in Tehran, Iran. The samples were collected from urine, wound, blood, body fluids, respiratory tract and abscesses. The vancomycin-resistant E. faecium (VREF) isolated from sewage were collected from three urban STPs located at different parts of Tehran. The isolation of enterococci from different sources has been described in a previous publication. [9] A total of 98 VREF from clinical isolates (n = 49) and STP (n = 49) and also a total of 58 vancomycin-sensitive E. faecium (VSEF) from clinical isolates (n = 15) and STP (n = 43) were obtained in this study. This study was carried out from 2006 to 2008. Antibiotic susceptibility test The susceptibility of the isolates against the seven most commonly used antibiotics in Tehran was evaluated using disc diffusion method and interpreted according to the guidelines from the Clinical and Laboratory Standards Institute (CLSI, 2007). The antibiotics used for susceptibility tests obtained from Bio-Rad (Hercules, CA, USA) were vancomycin (30 μg), ampicillin (10 μg) tetracycline (30 μg), gentamicin (120 μg), erythromycin (15 μg), ciprofloxacin (5 μg) and chloramphenicol (30 μg). Minimal inhibitory concentration (MIC) of the Vancomycin Resistant Enterococcus (VRE) was determined using E test (AB Biodisk, Solna, Sweden). E. faecalis, ATCC 29212, and E. faecalis, ATCC 51299, were used as quality control strains. Genotyping of virulence genes Identification of virulence genes for each isolate was performed by separate polymerase chain reaction (PCR) as described previously. Primer sequences were derived from the published sequences of the genes. [3],[6] PCR assay was performed in a total volume of 25 μl containing 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 0.2 mM each of dNTPs, 0.5 U of Taq DNA polymerase (HT Biotechnology, Cambridge, UK) and each primer (40 pmol). Samples were amplified by heating for 2 minutes at 94ºC, followed by 30 cycles of 92ºC for 15 seconds, annealing at 49˚C for agg, 51˚C for gelE, 52˚C for hyl, 46˚C for cylA, cylB, cylM, 57˚C for esp for 2 minutes and at 72ºC for 2 minutes, then a final step of 72ºC for 15 seconds. PCR products were analysed by gel electrophoresis in 1.5% (w/v) agarose gel. Positive controls in the PCR reactions were E. faecalis MMH 594 (asa1, gelE and esp), E. faecalis DS16 (cylA, cylB, cylM) and E. faecium C68 (hyl). The negative control was performed for each set of PCRs containing all reagents but no DNA template. Phenotypic assays Cytolysin production was evaluated by observing β haemolysis surrounding colonies on Columbia agar supplemented with 5% (v/v) fresh human blood, rabbit or guinea pig blood, followed by incubation at 37ºC for 24 hours.[10] The production of gelatinase in E. faecium strains was scored using Todd Hewitt (TH) (Difco Laboratories, Detroit, Mich) agar plates containing 3% gelatin. After overnight incubation at 37ºC, colonies with opaque zone were considered positive for gelatinase. [11] Aggregation substance production was determined by bacterial clumping. Briefly, 200 μl (0.5%) of an 18-hour culture supernatant of the pheromone producing E. faecalis JH2-2 strain grown in TH Broth was added onto each of the enterococcal strain being tested. After incubation at 37ºC for 24 hours, bacterial clumping or no clumping were directly visualised and reported. [12] Adhesion experiments Briefly, Vero cells were cultivated in RPMI-1640 (Biosera-East Sussex, UK) medium supplemented with 15% fetal bovine serum and 4 mM l-glutamine. Vero cells were seeded at 5 Χ 10 4 per 24 well plate and incubated at 37ºC in 5% CO 2 . Once the cells reached semi-confluence, overnight grown E. faecium (10 6 ) were then added into each well and incubated at 37ºC in 5% CO 2 for 2 hours. Then, the wells were washed four times with phosphate buffer saline, fixed with methanol, stained with 5% Giemsa, and examined by an inverted microscope. The results were then compared to noninfected monolayer cells. [13],[14] Each sample was done in triplicate and at least 50 microscopic fields per sample were examined. Conjugation The esp-positive strains were tested for their ability to transfer this gene by conjugation. The recipient strain, E. faecalis JH2-2, is resistant to fusidic acid and rifampin. Filter mating was performed using a 1:1 donor-recipient mixture. After incubation at 37ºC for 48 hours, transconjugants were selected on Brain Heart Infusion (BHI) plates containing vancomycin (20 mg/ml) rifampin (20 mg/ml) and fusidic acid (20 mg/ml). The transconjugants were analysed by PCR for the presence of esp gene. [15] Pulse field gel electrophoresis PFGE was performed on a CHEF-DR III apparatus (Bio-Rad Laboratories, Richmond, CA, USA) as described previously. [16] After digestion with SmaI, genomic DNA was separated by electrophoresis, with ramped pulse times beginning with 5 seconds and ending with 35 seconds at 6 V/cm for 27 hours. The banding patterns were interpreted by Dice analysis and clustered by the unweighted pair group method with arithmetic averages with Gelcompar II version 4.0 (Applied Maths, Sint-Matens-Latem, Belgium). Statistical analysis Fisher's exact test was used to compare the significance of difference between samples. Results Antimicrobial susceptibility results All the clinical VREF and STP VREF isolates were resistant to at least three different antibiotics. Also, all of them were resistant to ampicillin. Among the VSEF isolates from clinical and STPs, 73 and 5%, respectively, were found to be resistant to ampicillin. Majority of clinical VREF and VSEF (92 and 73%, respectively), and STP VREF and VSEF (100 and 95%, respectively) were resistant to ciprofloxacin. The highest (90%) and lowest (5.25%) resistance amongst the E. faecium isolates were found against ciprofloxacin and chloramphenicol, respectively. The percentage of isolates resistant to the drugs were the following: erythromycin - 92%, 80%, 100%, 53%; gentamicin - 96%, 53%, 92%, 0%; and chloramphenicol - 4%, 0%, 10%, 7% for clinical VREF, VSEF, and STP VREF, VSEF isolates, respectively. The MIC of vancomycin-resistant isolates was ≥128 ΅g/ml. The prevalence of virulence genes The cytolysin (cylA, cylB, cylM), asa1 and gelE genes were not detected in any of 156 isolates from clinical and STP samples. The results showed significant variations in the presence of hyl and esp genes among isolates obtained from different sources. The hyl gene was obtained in 80%, 28.5%, 7% and 0% of clinical VSEF, VREF, STP VSEF and VREF isolates, respectively. The prevalence of esp was found to be as follows: 82 and 53% for the clinical VREF and VSEF isolates, respectively, and 73.5 and 4.6% for STP VREF and VSEF isolates, respectively [Table - 1]. Concurrent presence of esp + and hyl + was found in clinical VSEF (33%) and VREF (18%) isolates, and none in the STP VREF or VSEF isolates [Table - 1]. Adhesion analysis Bacterial adhesion to Vero cell line was examined. Among the 156 E. faecium strains examined, adhesion to Vero cell line was exhibited by 40%, 20%, 53% and 47% of the isolates obtained from STP VSEF, VREF, and clinical VSEF and VREF, respectively [Figure - 1]. Conjugation results Filter-mating conjugation revealed that none of the virulence genes from the positive donor isolates (clinical or STP) could be transferred to E. faecalis JH2-2 recipient. Pulse field gel electrophoresis The 156 isolates were found to be heterogeneous as determined by PFGE. Vancomycin-resistant isolates obtained from patients (n = 49) were classified into 29 distinct PFGE types including 9 common (59% of the total isolates) and 20 single types (41%). All sewage vancomycin-resistant isolates (n = 49) were classified into 23 types, 27% of isolates in 13 single and 73% in common types. In addition, vancomycin-sensitive isolates (58) were highly diverse belonging to 35 clonal types, 8 in clinical and 27 types in STP isolates. Discussion High prevalence of virulence factors among enterococci was found in the present study. Out of the total isolates, 94 and 22% of the clinical and 44 and 0% of the STP isolates carried one or simultaneously two virulence factors, respectively. Several conclusions were drawn from these results: i) the prevalence of virulence factors was more common in the isolates from clinical than sewage; ii) presence of the virulence factor(s) in the sewage isolates suggests that these isolates can be potentially harmful for humans and iii) the strains possessing two virulence determinants (esp and hyl) were only observed in the clinical isolates, suggesting the loss of the virulence genes in the sewage milieu. Some investigators have shown the association of esp with increased virulence, colonisation and persistence in the urinary tract and biofilm formation, suggesting the possible role of esp in the pathogenicity of enterococci. [4] We determined that the prevalence of esp was significantly higher (P = 0.0001) in the vancomycin-sensitive clinical isolates (53%) than in the STP isolates (5%). Contrary to this, the prevalence of the esp was not significantly different (P > 0.05) in the vancomycin-resistant clinical (82%) than in the STP isolates (74%). The results may suggest the following: i) the loss or mutation of esp gene which may occur more frequently in the vancomycin-sensitive E. faecium than in STP; ii) decreased survival rate of these isolates due to sensitivity to antibiotics present in the STP milieu and iii) vancomycin-resistant isolates carrying esp are naturally selected, regardless of the environmental surroundings which cause them to have a sustainable existence. It has been reported [4] that the esp gene has been restricted to vancomycin-resistant strains. Although we found the presence of esp in the vancomycin-resistant isolates to be significantly different than the sensitive isolates, it was not exclusively restricted to VRE isolates as suggested by others. In agreement with the results of other investigators, [13],[17] the majority of the esp-positive isolates were also resistant to more than three antibiotics. As Lund and colleagues suggested, this can be the result of increasing of conjugation frequencies in the presence of esp in E. faecium.[18] Although conjugative transfer of the esp gene could not be confirmed in this study, Oancea and colleagues [19] demonstrated that the esp gene is transferable by conjugation among the enterococcal isolates. The adherence tests of E. faecium isolates to Vero cell line indicate no correlation between the cell adhesion phenotype and the presence of esp gene. These results demonstrated the lack of adherence in 58% of esp + E. faecium isolates. Moreover, 24% of the esp− E. faecium did not show any adherence to the Vero cell line. Our data are in accordance with a study which showed that bacterial adherence was not associated significantly with the presence of esp gene. [13] The results may suggest the following: i) Vero cell line probably is not an appropriate cell line for the adherence study, whereas it was suggested to be so by Dupre et al; [13] ii) other virulence factors might be more important in this assay and iii) esp gene may not have been expressed. PFGE results confirmed that most of the isolates were clonally unrelated. Cluster analysis of the PFGE patterns showed that out of 76 vancomycin-resistant esp + E. faecium isolates, 56 (57%) showed common PFGE patterns. Out of these, 19 isolates (19%) in the clinical and STP shared common types. Contrary to these data, we did not find any significant number of vancomycin-sensitive isolates to share common PFGE types. Therefore, this supports the hypothesis that the presence of vancomycin along with Esp are the major factors in endurance of certain E. faecium clones in the clinical setting and eventually the tribulations associated with this nosocomial infection. We assessed different patterns of PFGE for the presence of esp, hyl and adhesion to Vero cell lines. There was no significant difference in the prevalence of esp- and hyl-positive isolates with respect to PFGE clusters. This result is in contrast with those which reported differences in the frequency of esp gene between unique isolates and those belonging to clusters of genetically related. [20],[21] Furthermore, comparing isolates within the same cluster revealed no association between the presence of esp gene and adhesion to Vero cell line which is in accordance with other studies. [20],[21] In this study, the presence of the virulence determinants in E. faecium strains may be significant in the presence of more pathogenic E. faecium strains, with more antibiotic resistance making these pathogens excellent survivors in hospital environments. References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10101t1.jpg] [mb10101f1.jpg] |
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