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Research Article

Vancomycin-Resistant Enterococcus faecalis from a Wastewater Treatment Plant in Tabriz, Iran

E.B. Gajan, R. Abashov, M. Aghazadeh, H. Eslami, S.G. Oskouei and D. Mohammadnejad
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The aim of this study was to determine the resistance pattern and the type of resistance genes of vancomycin-resistant Enterococcus faecalis from Gharamalek wastewater treatment plant in Tabriz, Iran. Following filtering of sewage samples, approximately 300 colonies grew on specific media, of which 53 were randomly selected and purified using 0.45 μm membranes. The membranes were placed on culture media containing antibiotics to isolate the vancomycin-resistant Enterococcus. Biochemical tests, antibiogram and determining minimum inhibitory concentration of antibiotics with E-test including vancomycin were performed. Polymerase Chain Reaction (PCR) was carried out to determine the type of resistance genes. All tested samples were found to be E. faecalis. Antimicrobial susceptibility tests indicated multidrug resistance in the sapmles, with 98% of them highly resistant to vancomycin. The highest frequency was of vanA (96%), followed by vanB (4%); vanC was not seen among the tested samples. The results confirmed that the risk of exposure to antibiotic-resistant pathogens from the evaluated urban wastewater is considerable.

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  How to cite this article:

E.B. Gajan, R. Abashov, M. Aghazadeh, H. Eslami, S.G. Oskouei and D. Mohammadnejad, 2008. Vancomycin-Resistant Enterococcus faecalis from a Wastewater Treatment Plant in Tabriz, Iran. Pakistan Journal of Biological Sciences, 11: 2443-2446.

DOI: 10.3923/pjbs.2008.2443.2446



Enterococcus sp., the fermentative gram-positive cocci, are part of human and animal intestine normal flora. Their clinical importance was neglected in the past; however, with gaining resistance to antibiotics, they have received attention (Oprea et al., 2004). They are regarded as the main reason for nosocomial infections as well as the second common cause of endocarditis, bacteremia and urinary system infections since 1970 (Flannagan et al., 2003). Glycopeptidic antibiotics such as vancomycin and teichoplanin are the front line of treatment for serious infections caused by gram-positive cocci in the US. However, the excessive use of vancomycin in the past two decades has resulted in Enterococcus sp., that are resistant to glycopeptides and therefore, has brought about major problems in the treatment of nosocomial infections caused by these microorganisms (Shankar et al., 2002). The importance of Vancomycin-Resistant Enterococcus (VRE) is emphasized when a patient with vancomycin-resistant Staphylococcus aureus infection has concurrent infection of vancomycin-resistant Enterococcus faecalis. Samples of S. aureus along with E. faecalis, resistant to high levels of vancomycin, have been isolated from wounds on diabetic feet, suggesting the transmission of resistance genes between these microorganisms.

Many studies have shown the spreading capability of these strains in different environments carrying their resistance genes. In vivo studies suggest that enterococcal sex pheromones play a role in transmission of vancomycin resistance from E. faecalis to S. aureus (Stosovic et al., 2004). Conjugated transposones of E. faecalis can also transmit the resistance to S. aureus (Flannagan et al., 2003). These microorganisms share vanA, the main genetic factor of resistance to vancomycin in S. aureus (Shankar et al., 2002). Vancomycin resistance genes of Enterococcus sp., include vanA, vanB, vanC1 and vanC2/C3. vanA and vanB are located on transposones Tn 1546 and Tn 1547 either on plasmids or chromosomes, where vanC1 and vanC2/C3 are located only on chromosomes. Polymerase Chain Reaction (PCR) has been used to detect the types of resistance genes.

Transmission of resistance genes among Enterococcus sp., has been reported in urban wastewater systems. Samples of VRE in wastewater were first seen in the UK in 1993, followed by other cases including its detection in fecal samples of pigs and birds in a small town in Germany (Stosovic et al., 2004). Since vancomycin resistance genes are transmittable between microorganisms, environmental repositories like municipal wastewater system can have a significant role in the spreading of resistant Enterococci to healthcare facilities including hospitals. The purpose of this study was to determine the resistance pattern and the type of resistance genes of vancomycin-resistant Enterococcus faecalis from a wastewater treatment plant in Tabriz, Iran.


Sample collection: Sampling was carried out in September and October 2007 from Gharamalek municipal sewage treatment plant in Tabriz, Northwest Iran. Samples were collected from incoming sewage, sludge and outgoing sewage.

Isolation of bacteria: 1:10 dilutions of samples with phosphate-buffered saline were prepared and filtered using 0.45 μm membranes (Millipore Corporation, USA). Filters were then removed and samples were transferred to Brain Heart Infusion agar medium (DIFCO, USA) and kept at 37°C for 24 h. Filters were again placed in M Enterococcus agar plates (DIFCO, USA) containing 8 μg mL-1 vancomycin and incubated at 42°C for 48 h followed by another incubation on bile esculin agar (DIFCO, USA) at 45°C for 24 h. Finally, samples were purified from filters on blood agar (Shankar et al., 2002).

Determining biochemical identity: After purifying the cultures, the identity of species was determined by conventional biochemical tests including growth in presence of 6.5% NaCl, L-pyrrolidonyl-B-naphtylamide and growth in 45°C (Schouten et al., 2000).

Antimicrobial susceptibility testing: Antimicrobial resistance pattern was determined using vancomycin (30 μg), stereptomycin (10 μg), chloramphenicol (30 μg), gentamycin (120 μg), amikacin (30 μg), ampicillin (10 μg), ciprofloxacin (5 μg), tetracycline (30 μg) and erythromycin (15 μg) diffusion disks (BBL, SensiDisk, USA) as well as broth microdilution using vancomycin and teichoplanin disks (E-test, AB BIO DISK, Sweden) to determine the Minimum Inhibitory Concentration (MIC) according to the guidelines of the National Committee for Clinical Laboratory Standards (Schouten et al., 2000).

DNA extraction: Motanolysin method was used to extract the whole DNA content. Briefly, 2 mL of suspension from 24 h bacterial culture in BHI broth was centrifuged with 14000 rpm for 10 min. Then, 1 mL of lysis buffer (1 mmol NaCl, 10 μmol Tris HCl, 5 mmol EDTA, 0.5% Triton X-100) was added to the substrate. The followings were then added consecutively and the solution was incubated at each stage at 37°C for 1 h: 10 mg mL-1 lysosyme, 50 units of motanolysin, 20 mg mL-1 proteinase K, 20% sarcosil and 25% SDS. Finally, the obtained solution was extracted first with phenol and chloroform-isopropanol and then only with chloroform. Following centrifuge, the supernatant was collected (Van Horn et al., 1996).

PCR: PCR assay was performed with the specific primers to assess the vancomycin resistance genes including vanA, vanB, vanC1 and vanC2.3. Standard and positive controls included E. faecalis ATCC 51299 for vanA, E. faecium ATCC V583 for vanB and E. gallinarum ATCC BM4174 for vanC. PCR assay was perfomed separately for each primer. Then, electrophoresis of PCR products was carried out on 1% agarose gel and the results were evaluated after staining.


Sampling was performed for a total of three times from the sewage treatment plant. Following filtering of sewage samples, approximately 300 isolates grew on specific media, of which 53 were randomly selected and purified. Biochemical tests showed that all of the isolates were E. faecalis.

Ninety eight percent of species had high-level resistance to vancomycin (MIC ≥4); only one species had a MIC of ≤4. Antibiogram of the nine antibiotics evaluated demonstrated a high-level multidrug resistance in the tested samples (Table 1). Except for the cases of chloramphenicol (21%) and tetracycline (15%) which samples showed a considerably lower resistance to them compared to others, 94% of sample species were resistant to the tested antibiotics.

According to the results of PCR, the highest frequency was of vanA followed by vanB (Fig. 1). In two cases which did not contain vanA, both had a MIC of ≤8 for teichoplanin indicating vanB. The distribution of PCR results for the evaluated samples are presented in Table 2 and the same according to the source of sampling are presented in Table 3.

Fig. 1: The results of PCR indicate the presence of vanA and vanB, but no vanC, among the tested samples

Table 1: Antibiogram results in the tested samples with evaluated antibiotics

Table 2: Distribution of vanA and vanB in the tested samples (n = 53)

Table 3: Distribution of vanA and vanB according to the source of samples taken

In the present study, a selective method was used for the isolation of Enterococcus sp., with high resistance to vancomycin. This method provides suitable isolation of such species from an urban wastewater system and facilitates the phenotype and genotype assessments. All of the isolated species in the present study were E. faecalis. Earlier studies also showed a higher prevalence of E. faecalis in wastewater systems compared with other species (Harwood et al., 2001; Méndez-Alvarez et al., 2000).

Although multidrug resistance was seen to be high with the tested antibiotics, the samples showed a relatively lower resistance to chloramphenicol and tetracycline in comparison with other antibiotics such as ampicillin and gentamycin. The reason for this observation might be the lower clinical use of these two antibiotics, which results in a lower amount of resistance plasmids in the swage system (Murray, 2000; Lukášová and Šustáèková, 2003).

vanA phenotype is determined by acquired high-level resistance to both vancomycin and teichoplanin, where vanB phenotype is determined by resistance only to vancomycin. vanC phenotype is determined by low-level resistance to vancomycin and susceptibility to teichoplanin. In this study, only two species did not contain vanA and instead had vanB genotype; MIC of ≤8 for teichoplanin demonstrated the susceptibility of both samples confirming vanB phenotype as the range of MIC in species with vanB varies between 4-256. vanB is less frequent than vanA and is considered as a second factor for the spread of the phenotype (Murray, 2000). vanC1 and vanC2.3, the intrinsic resistance factors of E. gallinarum and E. calciliflavus, were not seen in the present study as neither of these species were found among the studied samples. Novais et al. (2005) in a similar study isolated 31 samples from municipal hospital and river wastewaters in Portugal and found that 25 cases were E. faecalis. Performing PCR revealed that 27 cases had vanA, four cases had vanB and only one case had vanC. In the study of Iversen et al. (2002) in Sweden, VRE samples from an urban wastewater treatment plant were isolated using M Enterococcus agar specific medium were containing 8 μg mL-1 vancomycin. From 35 VRE samples identified, 24 isolates (69%) were E. faecalis and most of the samples had multidrug resistance. PCR of the samples revealed that 30 isolates had vanA and five had vanB. Regarding the results of these studies and the present study, the use of membrane filtering and M Enterococcus agar medium containing 8 μg mL-1 of vancomycin seems to be a standard effective method for isolation of highly-resistant Enterococcus from an urban swage system.

Despite being differentiated by their metabolic activity, the two main E. faecalis and E. faecium species can cause similar diseases. Although E. faecium has steadily proved the most resistant species to various antibiotics to date among Enterococcus sp., E. faecalis yields the highest prevalence in the clinical settings, probably due to having the highest prevalence in the environment as well as containing more virulence factors. Regarding the frequency of resistance genes to vancomycin, many studies as well as the present study show that vanA owns the highest frequency, one reason being its high transferring capability of its transposone. vanB follows vanA as the second important gene responsible for the transmission of the disease. Present results confirmed that the risk of exposure to antibiotic-resistant pathogens from the evaluated urban wastewater is considerable. Therefore, measures for reducing or eliminating the microorganisms from the swage system in the wastewater treatment plants should be implemented in order to prevent the spread of the resistance species to healthcare facilities.


The authors wish to thank Ms. Zohreh Mousavi, Microbiology Laboratory Assistant, for her assistant in performing the laboratory procedures.

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