Phylogenetic Grouping and Phenotypic Detection of Extended-Spectrum β-Lactamases Among Escherichia coli From Calves and Dairy Cows in Khuzestan , Iran

1PhD Student of Bacteriology, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 2Assistant Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 3Professor, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 4Professor, Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran 5Associate Professor, Department of Food Hygiene, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran Int J Enteric Pathog. 2017 February;5(1):24-29


Background
Escherichia coli strains are the major members of Enterobacteriaceae which are usually found in the digestive system of human and warm-blooded animals.Certain E. coli strains have been associated with neonatal diarrhea and extraintestinal infections in ruminants which cause appreciable economic losses in dairy industry all around the world. 1,21][12][13][14] Researchers have suggested that the study of antibiotic resistance in commensal microorganisms like E. coli from the healthy animals would be quite useful, because they can be used as gnomon organisms to measure the prevalence of antibiotic resistance.6][17][18][19] It has been shown that the resistant bacteria with animal origin can either colonize or cause infection in humans. 20

Objectives
Due to the importance and spread of ESBL-producing E. coli strains in the food-producing animals and possible relationship between the ESBL production and phylogenetic groups, the aim of this study was to evaluate the prevalence of ESBL-producing bacteria and the phylogenetic groups, on E. coli fecal isolates from calves and cows in dairy farms of Khuzestan province.

Sample Collection
A total of 237 fecal samples from healthy dairy cows (134 samples), healthy (90 samples) and diarrheic (13 samples) calves were aseptically collected from 8 dairy farms located in Khuzestan province, Iran.Samples were randomly obtained from the animals by recto-anal mucosal swab (RAMS) method and transported on ice to the microbiology laboratory.The samples were cultured on Mac Conkey agar and incubated at 37ºC for 24 hours.Three to 5 dark pink colonies (presumptive E. coli) were randomly selected and identified by subculturing on eosin methylene blue plate (EMB) and confirmed to be E. coli using the standard biochemical tests. 21,22The confirmed E. coli isolates were stored in skim milk at -70°C for further experiments.

DNA Extraction
DNA extraction from isolates was performed by boiling method as described previously. 23Briefly, a few colonies were suspended in 300 μL sterile distilled water and heated at 95°C for 10 minutes.Afterward, they were put on ice for 5 minutes.Then, they were centrifuged at 13 000 rpm for 10 minutes and the supernatant was used as the DNA template.

Phenotypic Detection of ESBL in Escherichia coli
Isolates An initial screen test was done using ceftriaxone (30 μg) disk (Mast diagnostic, UK) with the Kirby-Bauer method advised by NCCLS (formerly known as the National Committee for Clinical Laboratory Standards).On the initial screen test, the isolates that showed a zone of inhibition ≤25 mm were then confirmed for ESBL production by the phenotypic confirmatory test.The coli when a zone with increased diameter (≥5-mm) was observed for antimicrobial agent tested in combination with clavulanic acid compared to the zone diameter of the agent when tested alone. 25

Statistical Analysis
Analyses of data were performed by SPSS software (version 16.0) and proportions were compared using the Fisher exact tests with the significance level set at P < 0.05.

Results
Out of a total of 237 fecal samples, 205 E. coli isolates were obtained from the calves and dairy cows and confirmed.Phylogenetic grouping analyses showed that E. coli isolates were distributed among groups B1, A, D, and B2 with 67.8%, 21.4%, 6.8%, and 1.9% prevalence indexes, respectively.All phylogenetic groups and subgroups were observed among isolates but there was no isolate from the diarrheic calves belonging to the B2 group and A0 and D2 phylogenetic subgroups (Table 3).Furthermore, 4 isolates were remained untypable by Clermont method (Figure 1).
The results of initial screen test for the phenotypic detection of ESBL-producing E .colistrains showed that 35 isolates (20 isolates from cows and 15 isolates from calves) were ceftriaxone resistant.By the ESBL phenotypic confirmatory test, only 2 isolates from the dairy cows were confirmed for ESBL-producing E. coli (Table 4).All ESBL-producing isolates were found to belong to the phylogenetic group and subgroup A1.There was no statistically significant relationship between the phylogenetic group and the production of ESBLs (P = 0.11) and also no difference was observed between the calves and cows in the production of ESBLs (P = 0.5).

Discussion
1][32][33][34] Moreover, some studies have shown that infections caused by ESBL-producing bacteria leads to the increase in the economic costs of infections treatment and the morbidity and mortality of diseases. 35urthermore, resistance to other antibiotic classes was observed in ESBL-producing E. coli more than non-ESBL-producing iso lates. 31,36It has been shown that the intestinal pathogenic E. coli strains mainly belong to the phylogenetic groups A, B1, and D and the commensal strains belong to the groups A and B1, while extraintestinal pathogenic strains usually belong to the groups B2 and D.
In our study, most of the isolates were found to belong to the phylogenetic groups B1 and A (A1).These results were almost similar to other studies which demonstrated that the isolates of phylogroup A were prevalent in the intestinal tracts of most animals 24,37 and phylogroup B1 was dominant among isolates from healthy cattle. 38n accordance with the previous reports in Iran, 23,39 this study showed that the phylogroup B1 was dominant in calves.The phylogroup B2 had also the least frequency, both in calves and dairy cows, and these outcomes confirmed the previous investigations. 23In contrast to a previous study 1 that reported subgroup B2 3 as only present in the human and B2 especially B2 3 as a good index of human fecal pollution, our results showed that subgroup B2 3 was found among isolates from both calves (healthy) and dairy cows.Similarly, another study showed that E. coli isolates of ruminants (sheep, goat, calves, and cattle) and dogs belonged to the subgroup B2 3 . 23,40Phylogenetic subgroups D2 and A0 and phylogroup B2 were not found among isolates from diarrheic calves and our results were similar to previous researches which showed that phylogenetic subgroups D2 and A0 and phylogroup B1 were not found among isolates from diarrhoeic calves. 23hough difficult to claim, the geographic variation, ecological conditions, environmental and health statuses enetic grouping and phenotypic detection riplex PCR profiles specific for E. coli phylogenetic groups and subgroups.Each combination of amplified 79 bp), yjaA (211 bp), and TSPE4.C2 (152 bp) genes allowed the phylogenetic group and subgroup ation of a strain.Lane 1: Marker 100 bp (SinaClon); lane 2: negative control, lane 3: positive control; lane play a significant role in E. coli phylogroup dispersion.Additionally, the prevalence of phylogenetic groups in animals is dependent on the body volume and host food diet. 41here are some reports on the ESBL-producing E. coli in sheep and poultry in Iran, [42][43][44] however based on a search in the databases we could not find any data about ESBLproducing E. coli in calves and dairy cows in Khuzestan province.In this study, the prevalence of ESBL-producing E. coli and their association with the phylogroups of E. coli isolates from calves and dairy cows were considered.First, the ESBL-producing isolates were determined by the combined disk method and by PCR, and then the plenty and distribution of phylogenetic groups in E. coli isolates were investigated.Afterward, the relative frequencies of four phylogenetic groups of ESBL-producing E. coli populations were compared.A low prevalence (2/205 isolates, 0.97%) of ESBL-producing E. coli was found only in the cow samples which belonged to the phylogenetic group A and phylogenetic subgroup A1.][49][50][51][52] Our results showed that there was no statistically significant relationship between the phylogenetic group and the production of ESBLs (P = 0.11).Previous studies showed that the use of antibiotics may strongly influence the phylogenetic group distribution of E. coli isolates. 53The resistance to different antibiotics was also particularly common for the phylogroups A and D and decreased in other groups. 54It is believed that alteration in antimicrobial agents' resistance occurs more easily in the phylogenetic groups of E. coli strains except for B2. 54uch communication between the phylogenetic groups and the antibiotic resistance of strain could describe why farm mammals undergo antibiotic pressure.The strains of phylogenetic groups of A and B1 are selected and B2 strains counter-selected.
In conclusion, food-producing animals are under suspicion for the reservoir and colonization of ESBLproducing bacteria especially Enterobacteriaceae and therefore infection of the humans with them.Further large scale investigations are necessary to control the antibiotic resistance in the human and animal foodstuff.

Table 2 .
6he Primer Sequence of Genes for Determination of Escherichia coli Phylogenetic Groups6

Table 3 .
Distribution of the Escherichia coli Phylogenetic Subgroups in Studied Animals