The Antibiotic Resistance Profiles of Escherichia Coli Isolated from Pre and Post Treated Hospital Sewage

Background: The dissemination of resistant strains of bacteria into the environment through hospital sewage has been recognized as a public health concern. We investigated the antibiotic resistance profile of E. coli isolated from hospital sewage. Methods: E. coli strains were isolated from the hospital sewage system from both pre and post treatment phases in a general hospital of Kermanshah city (west of Iran). Resistance to antibiotics (clindamycin, ceftriaxone, Co-trimoxazole, penicillin, ciprofloxacin, amikacin, gentamicin, imipenem, and piperacillin) was determined by disc diffusion. Isolates were screened phenotypically for extended-spectrum beta-lactamases (ESBL) production. The frequency of common antibiotic resistance genes ( bla CTX-M, bla TEM, bla SHV, and qnr ) were detected by PCR and data was statistically analyzed. Results: Sixty E. coli strains (30 for pre treatment and 30 for post treatment sewage) were randomly selected from isolates. All ESBL-producing isolates showed resistance to three antibiotic classes and were MDR. For non-ESBL isolates, 70 and 90 percent were MDR for pre and post treatment sewage, respectively. Of isolates tested, 100% had at least one of resistance genes. The frequency of bla CTX-M-1 gene was significantly higher in isolates of post treatment sewage. The bla TEM gene was more common than other genes in ESBL-producing isolates. Conclusion: The high rate of antibiotic resistance and resistance genes in E. coli isolates of hospital sewage, especially in post treatment is alarming. These data suggest that despite the widespread use of active sludge system to treat hospital sewage, they may not capable to adequately eliminate or reduce the antibiotic resistance strains of E.coli .

strains or different species of bacteria (2). Escherichia coli is one of the common causes of nosocomial infections such as urinary tract, gastrointestinal and blood infections. The resistant strains of this bacterium can survive in the hospital sewage and enter into environment, consequently, disseminate the resistant strains contained antibiotic resistance genes (3). The E. coli strains resistant to various antibiotics have been isolated from hospital sewages (4).
The most common antibiotics for the treatment of E. coli infections include the broad spectrum cephalosporins, carbapenems and fluoroquinolones (5). In recent years, resistance to these antibiotics has dramatically increased. Acquired resistance to beta-lactams is mainly attained by the Extendedspectrum beta-lactamases (ESBL) in E.coli (6). The bla CTX-M, bla TEM, and bla SHV are the most common types of ESBLs that have been reported in bacteria isolated from hospital sewage (7). The TEM and SHV beta-lactamases, by hydrolysis the molecules of beta-lactam antibiotics, make them inactive. These types of enzymes tend to have preferred substrates, such as third and fourth cephalosporin generations (8). ESBL genes are often located on plasmids, and this phenomenon can cause the rapid spread of these genes among strains and even different species of bacteria, in particular inside hospital sewage (4). Due to the rapid spread, the plasmid-mediated quinolone resistance has played an important role in the quinolone resistance of enterobacteriaceae in recent years (9,10). The most common genes of this group are qnrA, qnrB and qnrS. The qnr genes code proteins that protect DNA by preventing the binding of quinolones to the DNA gyrase and tropoiosomerase IV enzymes (11).
Since the hospital sewage is an important source of spreading the resistant strains of E. coli to environment, the effective treatment of hospital sewage is important to eliminate these resistant strains (7). The aim of this study was to evaluate the antibiotic resistance and some common resistance genes of E. coli isolated from hospital sewage before and after treatment.

Bacteria isolation, identification, and Antibiotic susceptibility testing
This study was done on the isolates from hospital sewage treated by an activated sludge system in Imam Reza Hospital in Kermanshah city (West of Iran). The proposal of this study was approved by the Kermanshah University Ethical Committee. During 2 months, twenty times samplings were done to collect hospital sewage in sterile tubes (20 mL). Two sets of samples were collected simultaneously each time as pre and post treatment hospital sewage to find out the effect of sewage treatment process on antibiotic resistance profiles of E.coli strains. Samples were transferred to the microbiological laboratory of Infectious Diseases Research Center and Microbiology Department of Medical faculty.
The E.coli isolates were identified using bacteriological culture methods according to the standard procedures (12). The Eosin methylene blue (EMB) culture medium was used to grow both ESBL and non-ESBLs E. coli isolates. After confirming E. coli colonies, a sub-cultivation was performed on the Blood agar medium and two batches of growing colonies were stored at -70 °C for the rest of subsequent examinations. To determine the percentage of ESBL producing E. coli among total E. coli strains, 100 colonies of E.coli isolates grown and approved on antibiotic-free EMB were carefully picked up by toothpicks and cultured on EMB medium contained 1 mg/liter of each ceftazidime and cefotaxime (13). The ESBL producing E. coli colonies were subsequently confirmed by combined disk test as described (14). The average of three independent experiments was used to determine the percentage of ESBL producing E.coli.
Of the identified E.coli strains, 40 ESBL producing (20 for pre and 20 for post treatment) and 20 not ESBL producing isolates (10 for pre and 10 for post treatment) were randomly selected from all samples for further examinations. Antimicrobial susceptibility testing was done by the disc diffusion according to the standard method for a number of selected antibiotics classes, including Fluoroquinolone (ciprofloxacin), Clindamycin (clindamycin), Sulfonamides (Co-trimoxazole), Aminoglycosides (amikacin, gentamicin), Cephalosporin (ceftriaxone), Carbapenem (imipenem) and Penicillin (penicillin, piperacillin) (MAST, England)(14).

Identification of antibiotic resistance genes
The whole genome of ESBL-producing isolates was extracted by boiling method and used as DNA template for polymerase chain reaction (PCR). Specific primers were used to identify antibiotic resistance genes using PCR ( Table 1). The PCR products were characterized on a 1% agarose gel electrophoresis contained gel-red stain along with a 100 bp DNA marker (SinaClon, Iran). The known strains of gram-negative bacteria from our Laboratory collection contained ESBL and qnr genes were used as controls (15,16).

ESBL production and Antibiotic susceptibility of isolates
On average, 15% percent of E. coli strains isolated from pre treatment and 19% from post treatment sewage were ESBL-producing, although this increase rate was not statistically significant (p = 0.367).
The antibiotic resistance for pre and post sewage treatment isolates were determined ( Table 2, 3). All the sewage treatment process were resistant to penicillin, ciprofloxacin and ceftriaxone. However, these isolates were mostly susceptible to Aminoglycosides. In general, in comparison to the pre treatment sewage isolates, in the post treatment isolates the resistance to gentamicin, clindamycin, imipenem and pipracillin was lower, but resistance to Co-trimoxazole and amikacin was higher (p > 0.05). In non-ESBL isolates, the rate of antibiotic resistance was higher for most antibiotics in post treatment isolates, but it was not statistically significant.   Table 3 Antibiotics susceptibility of non ESBL-producing E. coli isolates from pre and post sewages treatment phases.
Multidrug resistant strains (MDR) were determined by resistance to at least three antimicrobial classes. All ESBL-producing isolates (20 pre plus 20 post treatment) showed resistance to three antibiotic classes and were therefore regarded as MDR (Fig. 1). However, among non-ESBL isolates, 70 and 90 percent were MRD for pre and post treatment sewage, respectively.

Frequency of antibiotic resistance genes of isolates
The results of antibiotic resistance genes and their frequency for ESBL producing isolates were determined (Table 4). For post treatment sewage isolates, the frequency of the bla CTX-M gene was 25% less than pre treatment isolates. In contrast, the frequency of bla CTX-M-1 and bla CTX-M-2 was higher by 15% for post treatment sewage isolates. In case of qnr and bla SHV genes, there were no remarkable changes in their frequency rates for pre and post treatment sewage isolates.  (17). Since antibiotic resistance genes are often located on plasmids or transposons that capable to transfer to other microorganisms, the presence of these genes in hospital sewage can be a serious threat to public health (5). Therefore hospital wastewater should be properly collected, treated and returned to the natural environment (1).
In our study, all pre and post sewage treatment isolates were totally resistant to penicillin and highly resistant to other classes of antibiotics indicating the presence of high resistant strains of E. coli in hospital sewage. The both pre and post sewage treatment isolates was mostly susceptible to aminoglycosides (gentamicin and amikacin). The resistance rate of ESBL-producing isolates to most antibiotics tested was slightly lower in post sewage treatment isolates; however, the resistance rate in non-ESBL isolates for most antibiotics was higher in post treated isolates. It has been shown that in many cases, sewage treatment process cannot significantly eliminate resistant strains or resistance genes (7). Furthermore, there are some reports that showed the increase in both antibiotic resistance rates and antibiotic resistance genes for post treatment sewage isolates (18,19). For instance, research has shown that sewage treatment process caused an increase in antibiotic resistance of E.
coli to nalidixic acid, sulfamethoxazole, cephalexin and ceftriaxone (20). These results suggest that the effect of sewage treatment process on various classes of antibiotics may be different.
The rate of ESBL-producing E.coli strains have been reported higher than our results which may reflect difference in the regional frequencies of resistance genes (7,21). In the present study, the rate of ESBL-producing E. coli strains was slightly higher in the post treatment sewage isolates, which may reflect the transferring of EBSL among bacteria within sewage. Since the ESBL genes mostly located on plasmid in E.coli the horizontal transferring of ESBL genes between bacteria is reasonable. The bla SHV, including bla SHV-5 and bla SHV-12 have been found in hospital sewage isolates (8). A high incidence of SHV strains in untreated wastewaters in Australia has been reported, possibly due to the transmission of these genes in liquid environments (6). In our study, the frequency of bla SHV-5 and

Conclusions
In conclusion, the dissemination of antibiotic resistance strains and resistance genes of E. coli through hospital sewage is a serious concern for the efficacy of antibiotics globally. As it has been previously reported, despite the widespread use of active sludge system to treat hospital sewage, they are not able to adequately reduce the antibiotic resistance strains of bacteria (27

Consent to publish
Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article (Table 2,   Table 3, Table 4, Fig. 1)

Competing interests
The authors declare that they have no competing interests to disclose.

Funding
This study was supported by a grant from the Vice Chancellery for Research and Technology, Kermanshah University of Medical Sciences (grant number: 97297).

Authors' Contributions
AK participated in study design, sample collection, processing, bacterial culture, data analysis, acquisition of fund and preparing the manuscript. JSZ participated in sample collection, processing and bacterial identification. AB participated in data analysis and interpretation and assisted in manuscript preparation. FNZ participated in bacterial culture, identification. RC participated in bacterial identification and PCR. All authors read and approved the final manuscript.