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Phylogenetic Classification of Escherichia coli Isolated from Urinary Tract Infections in the Central Regions of Guilan Province

Escherichia coli is the normal flora of the gastrointestinal tract of humans and warm-blooded animals. Most E. coli strains are not pathogenic, but several strains have gained disease-causing genes that have enabled them to cause diseases in humans and animals. They can cause a variety of intestinal and extra-intestinal diseases such as meningitis, neonatal, gastroenteritis, septicemia, wound infections and urinary tract infections (UTIs) [1-3].

antibiotics, and new generations of antibiotics were introduced to the market. For this purpose, one of the most critical issues in the treatment of infectious diseases is the resistance of pathogenic bacteria to the antibiotics. The basis for appropriate treatment of UTIs is choosing high performance and inexpensive antibiotics. The indiscriminate use of antibiotics has resulted in high levels of antibiotic resistance of E. coli in many parts of the world.
The objective of this study was to develop a phylogenetic classification of the E. coli isolates obtained from the individuals with UTIs and to evaluate the antibiotic resistance among them.

MATERIAL AND METHODS
This cross-sectional study included 100 E. coli isolates from UTIs collected from the outpatients and hospitalized patients whom samples were sent to five laboratories, Al-Zahra Hospital, Dr. Ashtiyani, Razi, Afrah, and Dr. Afrayi in Rasht city during February 2015 through January 2016. To obtain single colonies, the samples were cultured on the EMB and Blood agar medium cultures and incubated for 24 h at 37°C.
Introduction: Urinary tract infection (UTI) is one of the most common infectious diseases, and Escherichia coli is known as the most dominant causative agent of this infection in 90-80% of patients. There is not much information about the phylogenetic groups, types, and virulence factors of E. coli, causing UTIs from Iran. The objective of this study was to evaluate the antimicrobial susceptibility of E. coli strains isolated from patients with UTI and to determine the phylogenetic classification of the strains. Methods: In this cross-sectional study, 100 E. coli isolates were collected from patients with UTI from five laboratories in Rasht city. The isolates were confirmed by using morphological and biochemical common tests. The frequency of virulence genes and patterns of phylogenetic groups were performed using Multiplex PCR. Additionally, antimicrobial susceptibility of all isolates was evaluated by disk diffusion method. Results: Distribution of phylogenetic B2, D, A and B1 groups in the isolates were 64%, 24%, 12%, and 0%, respectively. The highest antibiotic resistance was reported to cefotaxime (84%), piperacillin, and cefixime (80%), and the lowest resistance was demonstrated to imipenem (8%), chloramphenicol and gentamicin (12%). Conclusion: Our findings showed that the B2 was the most prevalent phylogenetic group and the most resistant strain to generally used antibiotics among patients with UTI. J Med Microbiol Infec Dis, 2017, 5 (1)(2): 17-20. Keywords: Escherichia coli, Multiplex PCR, Urinary Tract Infection.
Bacteria were cultured on the LB medium (Luriabrath), and for this purpose, a single clone of the bacteria was inoculated in LB medium according to instructions of the manufacturer (MIRMEDIA, Iran) and kept at 37°C for 12 to 16 h.
For molecular assessment of the isolated strains, the bacteria DNA was extracted using a DNA extraction kit (Cinnagen, Tehran, Iran). The quality of extracted DNA was evaluated by running 3µl of extracted DNA mixed with 1µl power load dye on 1% agarose gel. A multiplex PCR assay was used for typing and phylogenetic grouping of E. coli. The master PCR mix comprised 3μL of 10PCR buffer, 3μL of 25mM MgCl 2 , 3μL of 10mM dNTP mix, 0.5μL of Taq DNA Polymerase, 9.5μL of MilliQ water and 1μL of each of the forward and reverse primers. Finally, 4µL of each DNA template was added to the tubes to make up the final reaction volume of 25μL. In this study, specific primers for chuA, yjaA genes and TSPE4.C2 segment were used (Table 1). After PCR, the products were sequenced (Macrogen Company, Korea).

RESULTS
The patients' age ranged from 2 month old children to 79-years-old elderlies. All 100 samples were tested using biochemistry tests, and results showed that the isolated bacteria were Gram-negative, catalase positive and oxidase negative. In TSI medium, the isolates were Acid/Acid (A/A) (yellow/yellow) and H2S negative. In SIM medium, the motility and indole were positive and H2S negative. Also, MR was positive, and VP, Simon citrate and urea results were negative. As a result, the identity of the isolated bacteria was confirmed as E. coli.
The PCR bands in 83 isolates (83%) revealed gene chuA, in 66 (66%) yjaA gene, and in 76 (76%), the TSPE4.C2 fragment. According to the results, Figure 2 shows the PCR bands for the three genes.   PCR results showed that 64 cases (64%) of strains belonged to the phylogenetic group B2, 24 (24%) to the phylogenetic group D, and 12 (12%) to the phylogenetic group A. the phylogenetic group B1 was not observed among the strains. According to the results, the isolates from group D showed the higher resistance to the antibiotics, whereas the highest susceptibility was noticed among the isolates belonging to group A, and a moderate level of multidrug resistance was observed among the strains of group B2.

DISCUSSION
At present, the clinical management of UTI is one of the primary concerns worldwide due to the increased resistance of E. coli infections to commonly used antimicrobial agents. The resistance of E. coli to antibiotics showed to be related to their phylogenetic grouping [9]. The objective of this study was to describe the phylogenetic groups of E. coli based on the Clermont et al. (2000) method and to determine the relationship between these phylogroups and antibiotic resistance patterns.
The results of the study showed that 64%, 24%, 12% and 0% of strains were in the phylogenetic groups of B2, D, A, and B1, respectively. Distribution of strains in our study in the phylogenetic groups is consistent with the results and pattern of Clermont et al. (2000). They showed that most extra-intestinal pathogenic strains were in group B2, and then group D. It should be noted that the strains belonging to the phylogenetic group B1 was not found among the studied strains in the present study, being similar with the research of Grude et al. (2009)  The findings are consistent with the results obtained in the study of Bashir et al. (2012) in Faisalabad, Pakistan on 59 UPEC isolates from patients, and one study by Abdi et al. (2014) on E. coli isolated from UTIs in Sistan region. These were also consistent with the study of Iranpour et al. (2015) who studied the phylogenetic typing of strains of E. coli isolated from UTIs, and with the study conducted by Zhao et al. (2009) in China on 202 strains of E. coli isolated from UTIs [12][13][14][15].
The present study results are consistent with those of the study of Moreno et al. (2006) and Johnson et al. (2005) in the United States in which groups A and D were dominant. Asadi et al. (2010) through phylotyping showed that the most common phylogenetic E. coli groups in southern Iran were D, A, and B1 with the frequencies of 70%, 23.3%, and 6.7%, respectively; they did not find group B2. The variation in prevalence of the phylogenetic groups in different studies might be attributed to the health status, diet and genetic factors of the host, and environmental, social, and geographic conditions of sampling areas [16][17][18][19].
Evaluation of antibiotic resistance profile of UPEC strains showed that the strains from phylogenetic group D were significantly resistant to the majority of antibiotics compared to other phylogenetic groups. In addition, a moderate level of multidrug resistance was observed among the strains from group B2, while a low frequency of multidrug resistance was noticed among the isolates belonging to group A. Our findings were in agreement with the results of two separate studies in India and Sweden [20][21], but not with the studies conducted in southern Iran in cities of Shiraz and Bushehr [19,14]. This issue might be due to bacterial characteristics in different geographic regions or use of antibiotics.
Our findings showed that group B2 was the most common phylogenetic group and the most resistant strain to generally used antibiotics among patients with UTI. Similar studies in other geographical regions are required to provide a better understanding of the prevalence and geographic distribution of E. coli phylogenetic groups. The routine monitoring of antibiotic resistance patterns will also help clinicians to prescribe the most effective antibiotic and to prevent further increase of antimicrobial drug resistance.