The Open Biotechnology Journal




ISSN: 1874-0707 ― Volume 13, 2019
RESEARCH ARTICLE

High Frequency of Vancomycin-Resistant Enterococci in Sewage and Fecal Samples of Healthy Carriers



Fakhri Haghi1, Neda Shirmohammadlou2, Rabab Bagheri2, Sama Jamali2, Habib Zeighami1, *
1 Department of Microbiology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
2 Student Research Center, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran

Abstract

Objectives:

Enterococci are part of the microbial flora of the gastrointestinal tract of animals and human and can be released into the environment through fecal materials. These microorganisms play an important role in the dissemination of antibiotic resistance genes. Vancomycin-Resistant Enterococci (VRE) have been obtained in municipal sewage, hospital and agricultural wastes and healthy carriers. The aim of this study was to investigate the frequency of VRE in sewage and fecal samples of healthy carriers.

Methods:

This study was performed on fecal specimens of 100 healthy carriers and 100 samples of sewage in Zanjan Province. Fecal and sewage samples were cultured on Trypticase Soy Agar and biochemical tests were performed for Enterococci identification. Antimicrobial susceptibility testing was performed as CLSI guidelines and vancomycin resistance was determined using the agar dilution method.

Result:

Of 200 cultured samples, 141 isolates of Enterococci were detected. 64 isolates were detected from fecal and 77 were isolated from the sewage samples. Antibiotic resistance profile of fecal isolates was as follows: tetracycline (57.8%), ciprofloxacin (54.7%), phosphomycin (54.7%), erythromycin (51.5%), chloramphenicol (12.5%), amoxicillin (21.8%) and gatifloxacin (23.5%). Also for the sewage samples, the most antibiotic resistance was detected against ciprofloxacin (76.6%) followed by tetracycline (74%), erythromycin (68.8%), phosphomycin (61%). According to Agar dilution method, among 141 isolates of Enterococci, 15 (10.6%) isolates were vancomycin resistant: 11 of sewage isolates (14.3%) and 4 of the carrier isolates (6.2%).

Conclusion:

Our study describes the high frequency of VRE in municipal sewage and healthy carriers. Regarding the importance of VRE strains in the clinical and environment, it seems necessary to follow up on the issue.

Keywords: Enterococci, Healthy carrier, Sewage, Vancomycin resistant, Agricultural wastes, Antimicrobial susceptibility, Environment.


Article Information


Identifiers and Pagination:

Year: 2019
Volume: 13
First Page: 1
Last Page: 5
Publisher Id: TOBIOTJ-13-1
DOI: 10.2174/1874070701913010001

Article History:

Received Date: 17/12/2018
Revision Received Date: 25/01/2019
Acceptance Date: 09/02/2019
Electronic publication date: 28/02/2019
Collection year: 2019

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© 2019 Haghi et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Department of Microbiology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran; Tel:+98243314296; E-mail: zeighami@zums.ac.ir




1. INTRODUCTION

Enterococci are opportunistic and nosocomial human pathogens that can cause urinary tract infections, burn wound and surgical site infections, bacteremia and sepsis, endocarditis, cholecystitis, peritonitis, neonatal meningitis, and others [1Strateva T, Atanasova D, Savov E, Petrova G. Incidence of virulence determinants in clinical Enterococcus faecalis and Enterococcus faecium isolates collected in Bulgari. Braz J Infect Dis 2016; 20: 127-33., 2Soheili S, Ghafourian S, Sekawi Z, et al. Wide distribution of virulence genes among Enterococcus faecium and Enterococcus faecalis clinical isolates. Sci World J 2014; 2014: 623174.[http://dx.doi.org/10.1155/2014/623174] [PMID: 25147855] ]. Enterococcus faecalis is one of the most frequently isolated microbial flora in the human gastrointestinal tract, while Enterococcus faecium isolated with less frequency [3Karimi , Samarghandi , Shokohi , Godini , Arabestani. . Prevalence and removal efficiency of Enterococcal species and vancomycin-resistant Enterococci of a hospital wastewater treatment plant. Avicenna J Environ Health Eng [http://dx.doi.org/10.17795/ajehe-8623] ]. The emergence of Multidrug-Resistant (MDR) Enterococci has become a serious problem in healthcare settings worldwide [4Sharifi Y, Hasani A, Ghotaslou R, et al. Virulence and antimicrobial resistance in enterococci isolated from urinary tract infections. Adv Pharm Bull 2013; 3(1): 197-201.[PMID: 24312835] ]. The intrinsic resistance to various antimicrobials and dissemination of resistant genes by horizontal transfer are currently thought to play a major role in the development of MDR Enterococci. These MDR isolates are associated with increased mortality and costs due to prolonged hospitalization, need for surgery and prolonged treatment with antibiotics [5Freitas MCS, Pacheco-Silva A, Barbosa D, et al. Prevalence of vancomycin-resistant Enterococcus fecal colonization among kidney transplant patients. BMC Infect Dis 2006; 6: 133.[http://dx.doi.org/10.1186/1471-2334-6-133] [PMID: 16923193] ].

A common regimen for treatment of serious enterococcal infections is the synergistic combination of cell wall inhibitors as penicillin, ampicillin or vancomycin with aminoglycosides such as streptomycin or gentamycin. However, Vancomycin Resistant Enterococci (VRE) have caused significant problems for antibiotic therapy [6Li W, Li J, Wei Q, et al. Characterization of aminoglycoside resistance and virulence genes among Enterococcus spp. isolated from a hospital in China. Int J Environ Res Public Health 2015; 12(3): 3014-25.[http://dx.doi.org/10.3390/ijerph120303014] [PMID: 25768240] ]. VRE were first isolated in the UK and France in 1986. Since that time, hospital outbreaks of VRE have been reported from the UK, Ireland, Italy and Finland [7Simonsen GS, Andersen BM, Digranes A, et al. Low faecal carrier rate of vancomycin resistant enterococci in Norwegian hospital patients. Scand J Infect Dis 1998; 30(5): 465-8.[http://dx.doi.org/10.1080/00365549850161449] [PMID: 10066045] ]. VRE carriage has also been reported among out-patients in Germany and patients that newly admitted to hospital in France [8Klare I, Heier H, Claus H, et al. Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community. Microb Drug Resist 1995; 1(3): 265-72.[http://dx.doi.org/10.1089/mdr.1995.1.265] [PMID: 9158786] , 9Boisivon A, Thibault M, Leclercq R. Colonization by vancomycin-resistant enterococci of the intestinal tract of patients in intensive care units from French general hospitals. Clin Microbiol Infect 1997; 3(2): 175-9.[http://dx.doi.org/10.1111/j.1469-0691.1997.tb00594.x] [PMID: 1186 4101] ]. According to Van der Auwera et al., fecal cultures of 11 (28%) out of 40 healthy individuals who were not health care staff and who had not taken antibiotics in the previous year gain a heterogeneous collection of vancomycin-resistant E. faecium [10Van der Auwera P, Pensart N, Korten V, Murray BE, Leclercq R. Influence of oral glycopeptides on the fecal flora of human volunteers: Selection of highly glycopeptide-resistant enterococci. J Infect Dis 1996; 173(5): 1129-36.[http://dx.doi.org/10.1093/infdis/173.5.1129] [PMID: 8627064] ]. In the Netherlands, 5 to 10% of healthy people were colonized with VRE and a study in a cattle-rearing region in France revealed that 11.8% of non-hospitalized people and 37% of hospitalized patients were carriers of VRE [11van den Bogaard AE, Mertens P, London NH, Stobberingh EE. High prevalence of colonization with vancomycin- and pristinamycin-resistant enterococci in healthy humans and pigs in The Netherlands: is the addition of antibiotics to animal feeds to blame? J Antimicrob Chemother 1997; 40(3): 454-6.[http://dx.doi.org/10.1093/jac/40.3.454] [PMID: 9338505] -13Martinez JL. Environmental pollution by antibiotics and by antibiotic resistance determinants. Environ Pollut 2009; 157(11): 2893-902.[http://dx.doi.org/10.1016/j.envpol.2009.05.051] [PMID: 19560847] ].

Enterococci are part of the microbial flora of the gastrointestinal tract of animals and human and can be released into the environment through fecal materials [14Iversen A, Kühn I, Franklin A, Möllby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002; 68(6): 2838-42.[http://dx.doi.org/10.1128/AEM.68.6.2838-2842.2002] [PMID: 1203 9740] ]. The sewage contains various materials such as antimicrobial agents, pathogenic and saprophytic microorganisms, organic materials, nutrients etc., that facilitate the Enterococci survival and result in Enterococcal release into the environment [15Araújo C, Torres C, Silva N, et al. Vancomycin-resistant enterococci from Portuguese wastewater treatment plants. J Basic Microbiol 2010; 50(6): 605-9.[http://dx.doi.org/10.1002/jobm.201000102] [PMID: 20806259] , 16Nuñez L, Tornello C, Puentes N, et al. Hospital effluent constitutes a source of vancomycin-resistant enterococci. Ars Pharmaceutica 2016; 57(3): 121-6.]. Antibiotics are extensively used for human therapy, animal farming and agricultural purposes. These applications made antibiotics to be released in large amounts in natural ecosystems and this is maybe the main reason for the high incidence of antibiotic resistance detected in environmental bacteria [13Martinez JL. Environmental pollution by antibiotics and by antibiotic resistance determinants. Environ Pollut 2009; 157(11): 2893-902.[http://dx.doi.org/10.1016/j.envpol.2009.05.051] [PMID: 19560847] , 17Baquero F, Martínez J-L, Cantón R. Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol 2008; 19(3): 260-5.[http://dx.doi.org/10.1016/j.copbio.2008.05.006] [PMID: 18534838] ]. Residues of antibiotics and antibiotic resistance genes were detected from human environments and farms and they must be considered as important pollutants [13Martinez JL. Environmental pollution by antibiotics and by antibiotic resistance determinants. Environ Pollut 2009; 157(11): 2893-902.[http://dx.doi.org/10.1016/j.envpol.2009.05.051] [PMID: 19560847] ]. Dissemination of resistant genes among microbial flora and pathogenic bacteria in sewage can play a major role in the development of MDR isolates [16Nuñez L, Tornello C, Puentes N, et al. Hospital effluent constitutes a source of vancomycin-resistant enterococci. Ars Pharmaceutica 2016; 57(3): 121-6.]. Methods to reduce the amount of antimicrobial agents and a load of resistant bacteria in wastewaters originated from hospitals and farms include the optimization of disinfection procedures and management of wastewater and manure [17Baquero F, Martínez J-L, Cantón R. Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol 2008; 19(3): 260-5.[http://dx.doi.org/10.1016/j.copbio.2008.05.006] [PMID: 18534838] ]. Vancomycin-resistant Enterococci were reported in sewage and wastewater treatment plants in previous studies [14Iversen A, Kühn I, Franklin A, Möllby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002; 68(6): 2838-42.[http://dx.doi.org/10.1128/AEM.68.6.2838-2842.2002] [PMID: 1203 9740] , 15Araújo C, Torres C, Silva N, et al. Vancomycin-resistant enterococci from Portuguese wastewater treatment plants. J Basic Microbiol 2010; 50(6): 605-9.[http://dx.doi.org/10.1002/jobm.201000102] [PMID: 20806259] ]. These resistant isolates may be transmitted to human through contaminated water, food or vegetables [14Iversen A, Kühn I, Franklin A, Möllby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002; 68(6): 2838-42.[http://dx.doi.org/10.1128/AEM.68.6.2838-2842.2002] [PMID: 1203 9740] , 17Baquero F, Martínez J-L, Cantón R. Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol 2008; 19(3): 260-5.[http://dx.doi.org/10.1016/j.copbio.2008.05.006] [PMID: 18534838] ].

Regarding the vancomycin resistance in Enterococci isolates may result in emerging and persistence of nosocomial infections, the present study aimed to investigate the frequency of VRE in sewage and fecal samples of healthy carriers.

2. MATERIALS AND METHODS

2.1. Bacterial Isolation and Identification

From June to October 2017, a total of 200 individual samples of sewage and fecal including 100 fecal specimens of healthy carriers and 100 samples of urban sewage water were collected randomly in Zanjan Province. Stool specimens were collected from healthy people (carriers) with no history of diarrhea and antibiotic therapy for at least 1 month. The age of healthy people was between 15 to 50 years. Sewage samples were collected in sterile bottles from six different urban sewage waters and kept at 4°C until processing. All samples were cultured onto Trypticase Soy Agar (Merck, Germany) supplemented with 5% of sheep blood and incubated at 37°C for 24h. All isolates were phenotypically identified to the genus level using conventional biochemical tests as gram staining, catalase, growth on bile esculin agar, growth on 6.5% of NaCl, growth on 45°C and PYR. Confirmation of presumptive Enterococci isolates was performed using PCR, targeting the 16S rRNA gene with the following primers and amplicon size 733 bp: E1 (5'-TCAACC GGGGAGGGT-3') and E2 (5'-ATTACTAGCGATTCCGG-3') [18Deasy BM, Rea MC, Fitzgerald GF, Cogan TM, Beresford TP. A rapid PCR based method to distinguish between Lactococcus and Enterococcus. Syst Appl Microbiol 2000; 23(4): 510-22.[http://dx.doi.org/10.1016/S0723-2020(00)80025-9] [PMID: 112490 21] ]. Verified isolates of Enterococci were preserved at −70°C in Trypticase Soy broth (Merck, Germany) containing 20% (v/v) glycerol for further analysis.

2.2. Antimicrobial Susceptibility Testing

Susceptibility of isolates to the following antibiotics was examined using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines: Chloramphenicol (30 μg), Fosfomycin (200 μg), Erythromycin (15 μg), Gatifloxacin (5 μg), Ciprofloxacin (5 μg), Amoxicillin (10 μg) and Tetracyclin (10 μg). Multidrug resistance (MDR) was defined as resistance to three or more different classes of antibiotics.

2.3. Minimum Inhibitory Concentration Determination of Vancomycin

The MIC of vancomycin was determined using the Agar dilution method according to CLSI guidelines. Blood agar media with two-fold serial dilutions of vancomycin (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 µg/ml) were prepared. Then, the media were inoculated with Enterococci isolates overnight culture containing 5 × 106 CFU/mL and were incubated at 37°C for 24 h. The MIC was calculated as the lowest concentration that inhibited visible growth of the organism.

2.4. Statistical Analysis

The data were analyzed with SPSS version 17.0 software (SPSS, Inc., Chicago, IL). A chi-square test was used to determine the statistical significance of the data. A P-value of < 0.05 was considered significant.

3. RESULTS

3.1. Identification of Enterococci

From 200 cultured samples of sewage and fecal, 141 Enterococci were isolated. 64 (64%) isolates were detected from fecal samples of carriers and 77 (77%) were isolated from the sewage water samples. All Enterococci isolates were identified using biochemical tests and 16S rRNA. Of 100 healthy people participated in our study, 51 (51%) were younger than 30 years, 36 (36%) were 30-40 years and 13 (13%) were 40-50 years. The sex distribution was 56 (56%) female and 44 (44%) male.

3.2. Susceptibility to Antimicrobial Agents

Antibiotic resistance of 77 Enterococci isolated from urban sewage water samples and 64 Enterococci isolated from fecal samples of healthy carriers were determined by the disk diffusion method. Antimicrobial resistance patterns of isolates are presented in Tables 1 and 2. In all, 135 (135/141, 95.7%) isolates were resistant to one or more of tested antimicrobial agents. Among the sewage isolates, the most antibiotic resistance was detected against ciprofloxacin (76.6%) followed by tetracycline (74%), erythromycin (68.8%), and phosphomycin (61%). Chloramphenicol and amoxicillin showed the highest activity against isolates and 64.9% and 53.2% of isolates were susceptible to these antibiotics, respectively. The most antibiotic resistance in carrier samples was detected against tetracycline (57.8%), ciprofloxacin and phosphomycin (54.7%). Similar to sewage isolates, chloramphenicol and amoxicillin showed the highest activity against carrier isolates of Enterococci.

Table 1
Antimicrobial resistance of 77 Enterococci isolated from sewage samples.


Prevalence of antibiotic resistance varied among carrier and sewage isolates, and the frequency of resistance to amoxicillin, chloramphenicol and ciprofloxacin in sewage isolates was significantly higher than carriers (P<0.05).

Among sewage and carrier isolates, a total of 89 (63.1%) isolates were resistant to at least three different classes of antimicrobial agents and considered as Multidrug Resistance (MDR): 57 of sewage isolates (74%) and 38 of the carrier isolates (59.4%). The most prevalent MDR pattern was resistance to tetracycline, ciprofloxacin and phosphomycin.

3.3. Determination of Minimum Inhibitory Concentration of Vancomycin

Vancomycin breakpoint was based on the CLSI cutoff for Enterococci (Resistant, MIC of ≥ 32µg/ml; Susceptible, MIC of ≤ 4; Intermediate, MIC of 8-16). Based on the Agar dilution method, the range of vancomycin MIC for Enterococci isolates varied from 0.25 to 256 µg/ml (Table 3). Among 141 isolates of Enterococci, 15 (10.6%) isolates were vancomycin resistant: 11 of sewage isolates (14.3%) and 4 of the carrier isolates (6.2%).

4. DISCUSSION

The emergence of vancomycin and multiresistant Enterococci, as an important nosocomial pathogen, has become a serious problem in healthcare settings worldwide [4Sharifi Y, Hasani A, Ghotaslou R, et al. Virulence and antimicrobial resistance in enterococci isolated from urinary tract infections. Adv Pharm Bull 2013; 3(1): 197-201.[PMID: 24312835] , 16Nuñez L, Tornello C, Puentes N, et al. Hospital effluent constitutes a source of vancomycin-resistant enterococci. Ars Pharmaceutica 2016; 57(3): 121-6.]. Enterococci are usually isolated from the environment, sewage, human and animals faeces. Treatment of VRE infections is complicated in USA, European and Asian countries such as Turkey, India and Iran [16Nuñez L, Tornello C, Puentes N, et al. Hospital effluent constitutes a source of vancomycin-resistant enterococci. Ars Pharmaceutica 2016; 57(3): 121-6., 19Banerjee T, Anupurba S. Prevalence of virulence factors and drug resistance in clinical isolates of Enterococci: A study from North India. J Pathog Volume 2015; 7.[PMID: 692612] , 20Naserpour Farivar T, Najafipour R, Johari P, et al. Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis, Enterococcus faecium and their vanA and vanB genotypes. Iran J Microbiol 2014; 6(5): 335-40.[PMID: 25848524] ]. Sewage contains bacterial pathogens causing risks to human health unless measures are taken to control the hazard. Enterococci as fecal bacteria have an excellent ability for adaption to adverse environmental conditions [21Borhani K, Ahmadi A, Rahimi F, Pourshafie MR, Talebi M. Determination of vancomycin resistant Enterococcus faecium diversity in Tehran sewage using plasmid profile, biochemical fingerprinting and antibiotic resistance. Jundishapur J Microbiol 2014; 7(2): e8951.[http://dx.doi.org/10.5812/jjm.8951] [PMID: 25147674] ]. In our study, 95.7% of sewage and fecal Enterococci were resistant to one or more antimicrobial agents and 63.1% were multidrug resistant. The most frequent resistance in sewage Enterococci was found against ciprofloxacin (76.6%) and tetracycline (74%). High-level resistance to tetracycline and ciprofloxacin have been reported in previous studies [14Iversen A, Kühn I, Franklin A, Möllby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002; 68(6): 2838-42.[http://dx.doi.org/10.1128/AEM.68.6.2838-2842.2002] [PMID: 1203 9740] , 22Talebi M, Rahimi F, Katouli M, et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185(1-4): 111.[http://dx.doi.org/10.1007/s11270-007-9435-8] ]. The high incidence of tetracycline resistance find in this survey is most probably due to the widespread use of tetracycline in animal feed in our country which eventually could end-up in sewage [22Talebi M, Rahimi F, Katouli M, et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185(1-4): 111.[http://dx.doi.org/10.1007/s11270-007-9435-8] ]. In a study conducted by Cupáková and Lukášová in Sweden, more than 95% of sewage isolated Enterococci were resistant to more than one antibiotic [23Cupáková Š, Lukášová J. Agricultural and municipal waste water as a source of antibiotic-resistant enterococci. Acta Vet Brno 2003; 72(1): 123-9.[http://dx.doi.org/10.2754/avb200372010123] ]. Borhani and colleagues also reported that all or most of sewage Enterococci were resistant to 5 or 6 antibiotics. They have isolated VRE from sewage on different occasions in Tehran [21Borhani K, Ahmadi A, Rahimi F, Pourshafie MR, Talebi M. Determination of vancomycin resistant Enterococcus faecium diversity in Tehran sewage using plasmid profile, biochemical fingerprinting and antibiotic resistance. Jundishapur J Microbiol 2014; 7(2): e8951.[http://dx.doi.org/10.5812/jjm.8951] [PMID: 25147674] ]. Recovery of sewage VRE has been shown in other studies. High prevalence of vancomycin-resistant Enterococci (60%) in untreated sewage samples was reported in Swedish [14Iversen A, Kühn I, Franklin A, Möllby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002; 68(6): 2838-42.[http://dx.doi.org/10.1128/AEM.68.6.2838-2842.2002] [PMID: 1203 9740] ]. The use of avoparcin glycopeptide as a feed additive is considered to have created a pool of VRE in Europe [14Iversen A, Kühn I, Franklin A, Möllby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002; 68(6): 2838-42.[http://dx.doi.org/10.1128/AEM.68.6.2838-2842.2002] [PMID: 1203 9740] ]. In the USA and Portugal, the rate of VRE isolation from farm wastewater and hospital sewage samples has been reported 6% and 20%, respectively [24Novais C, Coque TM, Ferreira H, Sousa JC, Peixe L. Environmental contamination with vancomycin-resistant enterococci from hospital sewage in Portugal. Appl Environ Microbiol 2005; 71(6): 3364-8.[http://dx.doi.org/10.1128/AEM.71.6.3364-3368.2005] [PMID: 1593 3043] , 25Harwood VJ, Brownell M, Perusek W, Whitlock JE. Vancomycin-resistant Enterococcus spp. isolated from wastewater and chicken feces in the United States. Appl Environ Microbiol 2001; 67(10): 4930-3.[http://dx.doi.org/10.1128/AEM.67.10.4930-4933.2001] [PMID: 11571 206] ]. In our study, vancomycin-resistant Enterococci in sewage samples (14.3%) were detected with higher frequency than some previous studies in Iran [3Karimi , Samarghandi , Shokohi , Godini , Arabestani. . Prevalence and removal efficiency of Enterococcal species and vancomycin-resistant Enterococci of a hospital wastewater treatment plant. Avicenna J Environ Health Eng [http://dx.doi.org/10.17795/ajehe-8623] , 22Talebi M, Rahimi F, Katouli M, et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185(1-4): 111.[http://dx.doi.org/10.1007/s11270-007-9435-8] ]. Talebi and colleagues were investigated the occurrence, stability and antibiotic resistance of 593 Enterococci in six samples collected from three urban Sewage Treatment Plants (STPs) in Tehran, Iran. According to their results, the most prevalent species in all three STPs were E. faecium followed by E. hirae and E. faecalis and 45% of isolates were resistant to 1 or more tested antibiotics [22Talebi M, Rahimi F, Katouli M, et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185(1-4): 111.[http://dx.doi.org/10.1007/s11270-007-9435-8] ]. These resistant Enterococci could pass through STPs to surface waters. As reported in previous studies, antibiotic resistant enterococci in the environment could serve as a resistance genes reservoir and can be transferred to other bacterial pathogens [22Talebi M, Rahimi F, Katouli M, et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185(1-4): 111.[http://dx.doi.org/10.1007/s11270-007-9435-8] ]. Furthermore, the presence of vancomycin-resistant Enterococci in sewage, farm animals, municipal and hospital STPs, suggesting that VRE can be transmitted to humans through the food chain and water [14Iversen A, Kühn I, Franklin A, Möllby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002; 68(6): 2838-42.[http://dx.doi.org/10.1128/AEM.68.6.2838-2842.2002] [PMID: 1203 9740] , 22Talebi M, Rahimi F, Katouli M, et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185(1-4): 111.[http://dx.doi.org/10.1007/s11270-007-9435-8] , 24Novais C, Coque TM, Ferreira H, Sousa JC, Peixe L. Environmental contamination with vancomycin-resistant enterococci from hospital sewage in Portugal. Appl Environ Microbiol 2005; 71(6): 3364-8.[http://dx.doi.org/10.1128/AEM.71.6.3364-3368.2005] [PMID: 1593 3043] ].

Table 2
Antimicrobial resistance of 64 Enterococci isolated from fecal samples.


Table 3
The range of vancomycin MIC for sewage and carrier isolates.


VRE carriage has been reported among out-patients in Germany and patients that newly admitted to hospital in France [8Klare I, Heier H, Claus H, et al. Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community. Microb Drug Resist 1995; 1(3): 265-72.[http://dx.doi.org/10.1089/mdr.1995.1.265] [PMID: 9158786] , 9Boisivon A, Thibault M, Leclercq R. Colonization by vancomycin-resistant enterococci of the intestinal tract of patients in intensive care units from French general hospitals. Clin Microbiol Infect 1997; 3(2): 175-9.[http://dx.doi.org/10.1111/j.1469-0691.1997.tb00594.x] [PMID: 1186 4101] ]. We detected VRE in 6.2% of fecal samples of healthy carriers. In a study conducted by Tabatabaei and colleagues in Iran, fecal VRE colonization in hospitalized children was 16.9% [26Tabatabaei SR, Karimi A, Navidinia M, Fallah F, Fard AT, Rahbar M. A study on prevalence of vancomycin-resistant enterococci carriers admitted in a children hospital in Iran. Ann Biol Res 2012; 3(12): 5441-5.]. The prevalence of fecal VRE colonization among kidney transplant patients was reported 13.6% in Brazil [5Freitas MCS, Pacheco-Silva A, Barbosa D, et al. Prevalence of vancomycin-resistant Enterococcus fecal colonization among kidney transplant patients. BMC Infect Dis 2006; 6: 133.[http://dx.doi.org/10.1186/1471-2334-6-133] [PMID: 16923193] ]. Some previous studies have also reported a high frequency of VRE in carriers: 11% in French [12Gambarotto K, Ploy M-C, Turlure P, et al. Prevalence of vancomycin-resistant enterococci in fecal samples from hospitalized patients and nonhospitalized controls in a cattle-rearing area of France. J Clin Microbiol 2000; 38(2): 620-4.[PMID: 10655356] ], 12% in Germany [8Klare I, Heier H, Claus H, et al. Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community. Microb Drug Resist 1995; 1(3): 265-72.[http://dx.doi.org/10.1089/mdr.1995.1.265] [PMID: 9158786] ], and 28% in a Belgium [10Van der Auwera P, Pensart N, Korten V, Murray BE, Leclercq R. Influence of oral glycopeptides on the fecal flora of human volunteers: Selection of highly glycopeptide-resistant enterococci. J Infect Dis 1996; 173(5): 1129-36.[http://dx.doi.org/10.1093/infdis/173.5.1129] [PMID: 8627064] ]. According to Gambarotto and colleagues study, a total of 37% of the hospitalized patients and 11.8% of the subjects from the community were VRE carriers [12Gambarotto K, Ploy M-C, Turlure P, et al. Prevalence of vancomycin-resistant enterococci in fecal samples from hospitalized patients and nonhospitalized controls in a cattle-rearing area of France. J Clin Microbiol 2000; 38(2): 620-4.[PMID: 10655356] ]. National survey data have indicated the prevalence of VRE in 0-59% of isolates in 126 adult ICUs from 60 US hospitals [27Fridkin SK, Edwards JR, Courval JM, et al. The effect of vancomycin and third-generation cephalosporins on prevalence of vancomycin-resistant enterococci in 126 U.S. adult intensive care units. Ann Intern Med 2001; 135(3): 175-83.[http://dx.doi.org/10.7326/0003-4819-135-3-200108070-00009] [PMID: 11487484] ]. As confirmed by several previous studies, the presence of VRE in the fecal samples of humans in the community suggests that VRE are part of the gut microbial flora and can be acquired in the community [8Klare I, Heier H, Claus H, et al. Enterococcus faecium strains with vanA-mediated high-level glycopeptide resistance isolated from animal foodstuffs and fecal samples of humans in the community. Microb Drug Resist 1995; 1(3): 265-72.[http://dx.doi.org/10.1089/mdr.1995.1.265] [PMID: 9158786] , 10Van der Auwera P, Pensart N, Korten V, Murray BE, Leclercq R. Influence of oral glycopeptides on the fecal flora of human volunteers: Selection of highly glycopeptide-resistant enterococci. J Infect Dis 1996; 173(5): 1129-36.[http://dx.doi.org/10.1093/infdis/173.5.1129] [PMID: 8627064] -12Gambarotto K, Ploy M-C, Turlure P, et al. Prevalence of vancomycin-resistant enterococci in fecal samples from hospitalized patients and nonhospitalized controls in a cattle-rearing area of France. J Clin Microbiol 2000; 38(2): 620-4.[PMID: 10655356] ]. A possible source of VRE in fecal carriers could be the consumption of contaminated food, as VRE has been reported in farm animals and animal product-based foodstuffs. Furthermore, previous use of vancomycin or broad-spectrum cephalosporins, prolonged hospitalization and chronic dialysis were reported as important risk factors for VRE colonization [5Freitas MCS, Pacheco-Silva A, Barbosa D, et al. Prevalence of vancomycin-resistant Enterococcus fecal colonization among kidney transplant patients. BMC Infect Dis 2006; 6: 133.[http://dx.doi.org/10.1186/1471-2334-6-133] [PMID: 16923193] , 11van den Bogaard AE, Mertens P, London NH, Stobberingh EE. High prevalence of colonization with vancomycin- and pristinamycin-resistant enterococci in healthy humans and pigs in The Netherlands: is the addition of antibiotics to animal feeds to blame? J Antimicrob Chemother 1997; 40(3): 454-6.[http://dx.doi.org/10.1093/jac/40.3.454] [PMID: 9338505] , 12Gambarotto K, Ploy M-C, Turlure P, et al. Prevalence of vancomycin-resistant enterococci in fecal samples from hospitalized patients and nonhospitalized controls in a cattle-rearing area of France. J Clin Microbiol 2000; 38(2): 620-4.[PMID: 10655356] ].

Chloramphenicol showed the highest activity against isolates and 64.9% of sewage and 82.8% of fecal isolates were susceptible to chloramphenicol. Similar results have been reported by Talebi and colleagues from Iran [22Talebi M, Rahimi F, Katouli M, et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185(1-4): 111.[http://dx.doi.org/10.1007/s11270-007-9435-8] ]. The low incidence of chloramphenicol resistance most probably due to chloramphenicol is not prescribed as commonly as other antibiotics in Iran [22Talebi M, Rahimi F, Katouli M, et al. Prevalence and antimicrobial resistance of enterococcal species in sewage treatment plants in Iran. Water Air Soil Pollut 2007; 185(1-4): 111.[http://dx.doi.org/10.1007/s11270-007-9435-8] ]. Chloramphenicol resistance was also reported with low frequency in Switzerland and New Zealand [28Liassine N, Frei R, Jan I, Auckenthaler R. Characterization of glycopeptide-resistant enterococci from a Swiss hospital. J Clin Microbiol 1998; 36(7): 1853-8.[PMID: 9650924] , 29Manson JM, Keis S, Smith JM, Cook GM. A clonal lineage of VanA-type Enterococcus faecalis predominates in vancomycin-resistant Enterococci isolated in New Zealand. Antimicrob Agents Chemother 2003; 47(1): 204-10.[http://dx.doi.org/10.1128/AAC.47.1.204-210.2003] [PMID: 124991 92] ].

CONCLUSION

According to our results, 14.3% and 6.2% of sewage and fecal samples were carried vancomycin-resistant Enterococci, respectively. Due to increasing resistance rate of Enterococci to vancomycin and most common antibiotics, appropriate surveillance and control measures are essential to reduce the amount of antimicrobial agents and a load of resistant bacteria in wastewaters.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

Not applicable.

HUMAN AND ANIMAL RIGHTS

No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION

Not applicable.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

This work was supported by Student Research Center, Zanjan University of Medical Sciences, Zanjan, Iran (A-12-392-24 (UMS.REC.1396.11), A-12-392-26 (ZUMSREC.1396.29).

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