Antimicrobial Resistance Characteristics and Phylogenetic Groups of Escherichia coli Isolated From Diarrheic Calves in Southeast of Iran

Copyright © 2016 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Background Antimicrobial resistance (AR) in Escherichia coli is a significant issue in the treatment and control of diarrheal diseases1 and exposure to antimicrobial agents amplifies the resistance in E. coli population inhabited in animal and human intestinal tracts. Besides, the transmission of resistance genes is probable and also helps to spread out AR traits to other pathogenic and non-pathogenic organisms.2 Treatment with antibiotics such as amoxicillin, sulfonamides etc. is common in diarrheic calves.3 Consequently, calves are suggested as one of the major reservoirs for antimicrobial drug resistance amongst food-producing animals.4 Escherichia coli can be categorized into four recognized phylogenetic groups A, B1, B2, and D; this classification have been performed using a triplex polymerase chain reaction (PCR) based method defined by Clermont et al based on the detection of three genetic sequences called chuA, yjaA, and TSPE4.C2.5 It is now well accepted that achievement of evolutionary data through phylogenetic analysis of E. coli can help to determine the pathogenic and non-pathogenic nature of the bacterium.6

swabs were placed in Amies medium (BBL, USA) and transferred to laboratory within about 6 hours and immediately cultured onto MacConkey agar (Merck, Germany) and held in aerobic incubation condition at 37°C for 18 to 24 hours.Biochemical confirmations were performed on suspected colonies using IMViC (indole, methyl-red-Voges-Proskauer, citrate) tests 7 and finally confirmed E. coli isolates were subjected to next steps.
PCR for Antimicrobial Resistance Genes and Phylogenetic Grouping DNA extraction was carried out by NaOH method as described previously. 8Simplex and multiplex PCR methods were employed to detect the AR genes and three sequences for phylogenetic grouping.0][11][12][13][14] Determination of phylogenetic groups was done based on the presence or absence of chuA, yjaA, and TSPE4.C2 sequences in each E. coli isolate using a triplex PCR. 5 Positive and negative controls were used in each reaction; clinical E. coli strains 17DN for qnrS, sulI, and sulII; 21DN for qnrB; 25DN for tetA, tetB, aadA, aac(3)-I, floR, and cat1; 170DN for dhfrI and dhfrV all were kindly provided by Dr Reza Ghanbarpour from Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman.Distilled water was used as negative control.
For PCR, reaction mixtures were mostly prepared in a total volume of 25 μL containing 12.5 μL Hot Start 2x Master Mix (Ampliqon, Denmark), 0.5μM of each primer (Table 1), 3 μL of centrifuged (13 000 rpm for 1 min- A suspension of each organism with turbidity similar to a 0.5 McFarland standard was prepared in approximately 2 mL of sterile saline.A sterile swab was dipped into the inoculum tube and inoculation was performed on a Mueller-Hinton (MH) agar plate by streaking the swab 3 times over the entire agar surface.Antimicrobial disks were placed on the surface of the agar and plates were incubated at 37°C for 24 hours.
Finally, susceptibility to the antimicrobials was measured using Kirby-Bauer disc diffusion method according to the Clinical and Laboratory Standards Institute (CLSI). 15

Statistically Analysis
Proportions were compared through "N-1" chi-square test using MedCalc software (https://www.medcalc.org/).Calculations were performed in 95% confidence level and P value ≤ .05 was considered as marginal significance.For comparison of AR genotype with their phenotype in each antimicrobial group, the isolates were classified into 4 categories including phenotype + /genotype + , phenotype + /genotype -, phenotype -/genotype + , and phenotype -/ genotype -.Then, proportions were compared by chisquare test.

Phenotypic/Genotypic Antimicrobial Resistance
In this study, all (n = 170) isolates showed the phenotypic resistance to at least one of the β-lactam antimicrobials.Among them, 26 (15.2%) isolates carried one or more of related genes but remaining 144 isolates (84.8%) lacked these genes.In aminoglycoside family, 122 isolates demonstrated resistance to one or more of S, K, SPT, and GM antibiotics from which only 31 (25.4%)isolates had aadA and/or aac(3)-I; whereas three strains possessing the aminoglycoside genes did not show the correlated resistance phenotype.On the basis of phenotypic method, 125 isolates displayed to be resistant to tetracycline from which 19 (15.2%) isolates had each or both of the related genes.Two isolates were positive for tetracycline genes, but had not resistance phenotype to TE.In this research, phenotypic resistance to florfenicol was detected in low prevalence (n = 28/170).Although the catI and/or floR genes were amplified from 6 (21.4%)FF-resistant isolates, 9 positives for florfenicol genes did not show the resistance to FF in disk diffusion method.Totally, 56 enrofloxacin-resistant isolates were observed during antibiogram stage from which 7 (12.5%)strains carried at least one of the three associated AR genes, while 9 isolates having the enrofloxacin genes did not represent any resistance to NFX in plate.Results of sulfamethoxazole antibiotic were exactly similar to β-lactam family; 170 isolates were resistant phenotypically and among them, 26 (15.2%) isolates were sul1 and/or sul2-positve.These amounts were different for trimethoprim sulfamethoxazole-resistant isolates including 96 SXT-resistant strains among which 14 (14.6%)isolates were positive for dhfrI and/or dhfrV genes.
Relationships between AR genes and corresponding phenotypes were analyzed in each antimicrobial family.The statistical comparison of phenotype -/genotype -and phenotype + /genotype + isolates showed that there were significant differences among all antibiotic families except for aminoglycoside.The difference between phenotype -/ genotype + and phenotype + /genotype + isolates was considered not to be quite statistically significant in florfenicol and enrofloxacin, unlike other groups.And finally the phenotype + /genotype -isolates formed a notable proportion of strains (P < .0001;df = 1).

Phylogenetic Groups
Although four major E. coli phylo-groups (A, B1, B2, and D) and five sub phylo-groups (A 0 , A 1 , B1, B2 3 , and D 1 ) were recognized in this study, majority of strains were within A (65.8%; 95% CI: 58.7-73%) and B1 (30.6%; 95% CI: 23.6-37.5%)phylo-groups.Only 1 and 5 samples were detected as members of B2 and D, respectively, and none of the strains were identified for B2 2 and D 2 phylogenetic groups.A 1 and B1 were the predominant sub phylo-groups among AR-gene positive strains, but VG-positive isolates were mostly allocated to A 0 and B1 (Table 3).

Discussion
One of the critical issues and concerns in public health is increasing AR among bacterial populations.AR decreases therapeutic impact of antibiotics and may be transmitted to other microorganisms by related genes.In this study, prevalence of genetic and phenotypic AR was evaluated among the E. coli isolates to 6 antibiotic groups.The most prevalent AR genes and phenotypes were correlated to β-lactam, sulphonamide, aminoglycoside, and tetracycline families.Moreover, the specific AR genes of aadA (streptomycin and spectinomycin), bla TEM (penicillins and first generation of cephalosporins), and sulII (sulphonamides) were determined as the most common genes, which is in agreement with a study on ruminant E. coli strains in Spain in that predominant resistance genes were bla TEM in β-lactam, tetA in tetracycline, aadA in ami-noglycosides, catI in chloramphenicol, and sulI and sulII in sulfamethoxazole groups. 16In the Spanish study, dhfrI was predominant gene in trimethoprim group, unlike to our results.Furthermore, in agreement with previous studies on diarrheic neonatal calves, prevalence of genetic AR compared to ceftiofur (bla CTX-15 ), apramycin (aac3-I), and florfenicol (floR) were found in low rate. 17Frequency of AR gene was different in diverse researches which may be similar or dissimilar to our results.
In phenotypic level, isolates had resistance to two (penicillin and sulphonamide) or more of tested antibiotics; 84.11% of isolates were defined as multi (3 ≤ )-drug resistant.Regardless of P and SMZ, resistance to TE, S, SXT, and K were observed in high frequency, while the isolates represented a considerable susceptibility to five antibiotics including CTX, CZA, GM, FF and NFX, respectively.Our results was comparable with a study in Iran that showed the extensive resistance to penicillin and sulphonamide.Our study was in agreement with the Iranian study in prevalence of SXT and NFX, but there were significant differences about S, TE, and GM.Although there is agreement between our results and previous studies in family level, there are some differences in frequency of specific antibiotics. 18ur isolates were significantly resistant to the antibiotics streptomycin, tetracycline, and sulphonamide, having prophylactic and therapeutic usages in calf diarrhea.Other choices for above-mentioned applications are amoxicillin, neomycin, ampicillin, and chloramphenicol, 2 and evaluation of phenotypic resistance to them should not be neglected.Irregular consumption of antibiotics and nourishment of calves with AR contaminated milk are the important risk factors that amplify the selection of resistant strains in gut. 19Horizontal transferring of resistant bacteria and genes to environment, foods, and other hosts is completely probable. 20Therefore, tracing AR in commensal microorganisms can help us to adopt strategies for controlling resistance.
In this research, E. coli isolates possessing β-lactamase and sulphonamide AR genes showed the phenotypic resistance to corresponding antibiotics, but approximately 60% of gene-positives for amphenicol and quinolone and 10% of gene-positive strains for aminoglycoside and tetracycline isolates did not show the related AR phenotype.Additionally, only 12%-25% of isolates displaying AR phenotype had the correlated genes.Based on AR phenotype/genotype analysis, all AR genes but floR and qnr had been expressed.Attendance of isolates with AR pheno- type without genotype may also be due to resistance genes that have not been studied during research.Nevertheless, some studies have pointed to association between prevalence of AR genes and phenotypes, while there are some reports about isolates without the phenotypic expression of AR genes. 21Various forms of resistance to antimicrobial drugs can be correlated to some factors such as oxidative stress, iron depletion, and retained motility which change expression of resistant genes. 22n phylogenetic analysis, all isolates were classified into phylo-groups and their subsets; majority of our positives for AR and multi-drug resistance were allocated to A, followed by B1 phylogenetic group.Totally, the three groups A, B1, and D contain the major phylogenetic groups of antibiotic-resistant E. coli. 23In a report, B1 was the most virulent phylo-group, but A and D were the predominant groups in non-virulent isolates. 23It seems that phylo-groups A and B1 are more common in animals than in humans and B1 is predominant in herbivorous animals. 24elatedness of pathogenic E. coli with B2 and D phylo-groups is documented in some researches 24 and A and B1 phylogenetic groups are predominantly distributed in commensal E. coli populations. 25his work shows that diarrheic calves are reservoirs for non-pathogenic phylogenetic groups (A and B1) and important AR.Resistance to β-lactam, streptomycin, tetracycline, and sulphonamide groups is significantly common.Because of transmission probability of resistance traits to other microorganisms and hosts, these forms of resistance may lead to a series of consequences such as complications in antimicrobial treatment process in animals and human.Thus, for management of resistant strains, screening of AR among gut commensals should be continued and also evaluation of further different AR genes is required for each antimicrobial category.

Table 1 .
Primers Used for Detection of Antimicrobial Resistance Genes and Phylogenetic Sequences

Table 2 .
Prevalence of Each Antimicrobial Resistance Gene and Phenotype Among E. coli Isolates a Name of antimicrobial family/group.b Name of antimicrobial resistance genes in abbreviated and italic form.c Name of antibiotics in abbreviated form.

Table 3 .
Prevalence of Phylogenetic Subgroups Among Isolates Based on Presence or Absence of AR Genes (%)