Iranian Journal of Medical Microbiology

 
Table 1. Sampling sites' names and locations.

^* UTM: Universal Transverse Mercator
 
Table 2. The number of samples collected from different sites in different seasons.

Bacterial Isolation and Detection

Following the sampling, the most probable number (MPN) method was used to estimate the concentration of viable Enterococci in water samples utilizing replicate growth in Azide Dextrose Broth (Merck, Germany) (14). The grown bacteria were transferred to Pfizer Selective Enterococcus (PSE) Agar for confirmatory testing. After 24 hours of incubation at 35℃, the formation of blackish-brown colonies was considered E. faecalis. Then, bioche-mical tests, including esculin hydrolysis test, growth in Trypticase Soy Broth (TSB) medium containing 6.5% salt (NaCl), bile tolerance test, heat resistance test, growth at temperatures of 10°C and 45°C, growth at pH=9.6, tellurite reduction, motility test, H2S production, and sugar fermentation tests.

 Antimicrobial Susceptibility Testing

For antibiotic susceptibility determination of the isolates, the Kirby-Bauer disk diffusion method on Mueller-Hinton agar was performed based on the guide-lines of the Clinical and Laboratory Standards Institute (CLSI) (15, 16). Standard antimicrobial drugs (Mast, UK), including vancomycin (30 µg), kanamycin (30 µg), streptomycin (30 µg), erythromycin (15 µg), amikacin (30 µg), ampicillin (10 µg), gentamicin (120 µg), ciprofloxacin (5 µg), and chloramphenicol (30 µg) were used for this aim. E. faecalis (ATCC 10541) was used as a control to standardize antibiotic susceptibility testing.

Molecular Confirmation of VREs

Using ExiPrep Plus Bacteria Genomic DNA Kit, genomic DNA was extracted according to the manufacturers' instructions. Then, the quality of the extracted DNA was determined using agarose gel electrophoresis.
For molecular identification of the E. faecalis isolates, a 475-bp fragment corresponding to the ddlE gene was amplified using specific primers. The forward and reverse primers sequence was: 5'-CACCTGAAGAAACAGGC-3', and 5'-ATGGCTACTTCAATTTCACG-3', respectively (17). E. faecalis JH2-2 reference strain was used as the control. The PCR reactions were set up according to the manufacturer's recommendations. Each reaction conta-ined 1X PCR buffer, 1.5 mM of MgCl2, 1 μL of the isolated bacterial DNA, 0.2 mM of each dNTPs, and 0.25 μM of each primer. The cycling conditions were set to 95°C for 2 min, followed by 45 cycles at 95°C for 5 s, 50°C for 30 s, and 72°C for 1 min.

Enterococci are common pathogens that cause severe nosocomial infections (18-20). In the present study, Enterococci isolates were obtained from environmental samples from recreational waters, mainly swimming places in Guilan Province, Iran. We chose to investigate these places because many people, both travelers, and natives, go there for swimming, and therefore, monitoring the microbial load of these places is very important.
We observed a relatively high resistance level to different antibiotics, including vancomycin, kanam-ycin, streptomycin, erythromycin, amikacin, ampic-illin, gentamicin, ciprofloxacin, and chloramphenicol, among these isolates. Of the isolates, 46.1% were resistant to vancomycin, which is relatively high. Almost all strains (̴ 98%) were resistant to strep-tomycin. Among the studied antibiotics, ampicillin and chloramphenicol had the highest antibacterial effect on the isolates, ̴18%, and 19%, respectively.
The prevalence of the VREs and their antibiotic-resistant patterns among clinical as well as enviro-nmental samples is variable in different regions (21-24). Same as the present study, Alipour et al. exam-ined the presence of Enterococcus spp. as well as their antibiotic resistance patterns in samples from a river and coastal waters in Mazandaran Province, Iran. Of 70 isolated Enterococci, 68.6% and 20% belonged to E. faecalis and E. faecium, respectively. They reported a high resistance rate to chloramphenicol, ciprofloxacin, and tetracycline (25). In 2019, Mazaheri et al., investigated the prevalence of VREs among dried vegetable samples in Tehran, Iran, which they found that 48% of the isolates were VREs (26). In a study by Roberts et al. on the prevalence of VREs in crows and their environment, 24.5% and 55% of the crows and environmental samples, respectively, were VRE positive (27).
Rezvani et al. studied the prevalence of Entero-coccus spp. and their antibiotic-resistant patterns in gastroenteritis patients. They found Enterococcus spp. in 37% of samples; most of them belonged to E. faecalis (91 %), and 9 % belonged to E. faecium. The prevalence of VREs among Enterococci isolates was relatively low (6 %), all of which belonged to E. faecalis (28). Zavaryani et al. assessed the susceptibility of the clinical Enterococcus isolates to five antibiotics, includ-ing vancomycin, gentamicin, teicoplanin, fosfomycin trometamol, and quinupristin/dalfopristin among 400 Enterococcus species. In their study, teicoplanin and vancomycin were the most effective antibiotics, while Quinupristin/dalfopristin was the least effective against the clinical samples (29).
Khanmohammadi et al. investigated the prevalence of VREs among two different sets of samples, fecal and clinical samples. The rate of VREs among fecal samples (52%) was higher than in clinical isolates (32%) (30). As can be seen, different results are being reported from different samples. This variation can be related to the types of samples (clinical or environmental), geog-raphical location of sample collection, the treatment strategies exploited in each region (high rate of antibiotics consumption), sewage disposal, etc. However, due to the high spread rate of antibiotic-resistant bacteria and the horizontal transfer of antibiotic-resistant genes, it is a real public health concern.
In the present study, the E. faecalis-specific ddl gene (ddlE. faecalis) was used as the specific gene to identify E. faecalis strains (31). ddl encodes D-alanine:D-alanine ligases and related glycopeptide resistance proteins (32). D-alanine--D-alanine ligase is an essential enzyme for the peptidoglycan biosyn-thesis. This enzyme dimerizes D-Ala before its incorpo-ration in peptidoglycan precursors (33). Mutation in this gene results in the deficiency of bacterial growth. This gene is widely used for the identification of different bacterial species, including various species of Enterococci as well as other species (34-40).

 

A relatively high prevalence of VREs in the studied places poses a serious epidemiological threat and a risk to public health. The possibility of horizontal gene transfer among the bacteria may transmit resistant genes from VREs to other bacteria; therefore, it is necessary to think about the necessary measures to determine the source of the pollution and at the same time prevent swimming in places with a high level of bacterial pollution until the problem is addressed.

 

The authors acknowledge the staff and students of I.A. University. Indeed, we appreciate the warm help of the staff of the B.R.C.

 

Author Contribution

H.K. and M.F.G. carried out the experiment. H.K. wrote the manuscript with support from A.H. K.I and A.H. supervised the project.

 

None.

 

Conflicts of Interest

The authors declared that there is no conflict of interest regarding this article.