Integron-Associated Antibiotic Resistance Patterns in Escherichia coli Strains Isolated from Human and Animal Sources in Two Provinces of Iran

Introduction

Escherichia coli is a cosmopolitan bacterium existing either as a commensal or pathogenic to humans and various animal species [1–5]. This organism has been reported to be responsible for significant veterinary, public health and socio-economic concerns in various countries worldwide [6–8].

The indiscriminate, unauthorized and unsupervised administration of antimicrobials in human and animal therapy has been suggested to be a predisposition for the dissemination of resistance genes among bacteria. This abuse of antibiotics during cattle breeding constitutes a serious threat to human and animal health because of the high risk of the selection of antibiotic resistance genes in the microorganisms [8–9].

Antimicrobial resistance determinants are carried mainly by genetic components such as plasmids, transposons, and integrons. Several authors have associated the integrons and conjugative plasmids with the spread of the resistance determinants from [9–12].

Integrons are genetic structures containing a site-specific recombination system that enables bacteria to acquire and express cassettes of genes that carry antibiotic resistance [13–15]. Integrons are transposition defective; however, they can be mobilized in association with functional transposons and/or conjugative plasmids [13]. They also contain a site-specific recombination system able to capture and express genes as gene cassettes [16, 17]. The essential components of class 1 integrons are a) the 5’conserved segment (5’-CS) that includes the integrase gene, intI, which encodes the site-specific recombinase, b) the adjacent site, attI, that is recognized by the integrase and acts as a receptor for gene cassettes, and c) a common promoter region(s), P_ant(P₁) and/or P_ant(P₂), from which the integrated gene cassettes are expressed [18, 19]. The 3’conserved segment (3’-CS) located downstream of the integrated gene cassettes, usually contains a combination of the three genes, qacE1 (responsible for antiseptic resistance), sulI (implicated in the resistance to sulfonamides), and the open reading frame (orf5) whose function is currently uncertain [20].

Several studies have demonstrated the mechanism of clonal spread of resistant strains, transfer of resistance genes between bacteria living in humans and animals and the exchange of phylogenetic and genotypic characteristics [21]. The exponential increase and spread of antimicrobial-resistant bacteria are of a great concern because of the difficulty in treating the bacteria-borne infections. Such complications often result from rapid expansion of antibiotic-resistant genes carried by plasmids, transposons, and integrons [17, 22–24]. Several studies have reported on widespread prevalence of integrons in clinical bacteria isolates [25–28]. Therefore, the increased drug resistance of clinical isolates may be explained by the selective pressure of antibiotic and the widespread presence of integrons.

To our knowledge, there is little information on the presence of integrons in E. coli isolates and the association between integrons and antimicrobial resistance. Therefore, the present study was performed to investigate the association between the existence of integrons and resistance to antimicrobial agents in E. coli strains isolated from human and animal sources in the Alborz and Isfahan provinces, Iran.

Materials and Methods

Study location and description. This study was conducted in two locations: the city of Karaj, located in the Alborz province, and the city of Zavareh, located in the Isfahan province of Iran. The Alborz province has 2.413 million populations, and the Isfahan province has 1.6 million populations.

The present study was approved by the Ethics Committee of the Karaj Branch, Islamic Azad University.

A total of 80 samples were collected from cattle (n=20), sheep (n=20), poultry (n=20), and humans (n=20). Faeces were collected from apparently healthy people who willingly submitted their samples to the Amini Medical Laboratory located at the Alborz province. Large intestinal swabs were collected from randomly selected chickens bred in a privately owned breeding farm in Karaj city. Faeces were collected per rectum from randomly selected cattle and sheep managed in a privately owned livestock facility located in Zavareh city. No animal was hurt during sample collection in this study.

Isolation of E. coli from samples. The faecal samples were inoculated into lauryl sulphate tryptose (LST) broth (Merck, Germany) followed by inoculation into EC medium (Merck) at 44.5°C, and then passaged on eosin methylene blue (EMB) agar (Merck). Colonies with metal shine were presumed to be E. coli isolates; these underwent IMViC test for confirmation [29].

Antibiotic susceptibility testing. Phenotypic antibiotic susceptibility was tested for by the Kirby–Bauer disk diffusion method. Padtan-Teb disks (Tehran, Iran) were placed on Mueller–Hinton agar plates according to the guidelines of the Clinical and Laboratory Standards Institute. The 11 antibiotic discs included ampicillin (AM) 10 µg, piperacillin (PIP) 100 µg, cefazolin (CZ) 30 µg, streptomycin (SM) 10 µg, kanamycin (K) 30 µg, gentamicin (GM) 10 µg, neomycin (N) 30 µg, tobramycin (TOB) 10 µg, amikacin (AN) 30 µg, nalidixic acid (NA) 30 µg, and sulfamethoxazole/trimethoprim (SXT) 23.75/1.25 μg. For antimicrobial susceptibility testing, the inoculum of E. coli was homogenized with a sterile swab in sterile saline solution (0.85% NaCl) to adjust turbidity to match the 0.5 McFarland standards. These were then placed evenly on Mueller–Hinton agar plates.

The plates were inverted and then incubated at 35°C for 18 h; the diameters of the growth inhibition zones were measured and compared with the standard chart and with the E. coli ATCC 25922 and Staphylococcus aureus ATCC 29213 as positive controls. Isolates with intermediate resistance were defined as susceptible; the isolates were considered multi-drug-resistant if they were resistant to at least three classes of antibiotics [30–32].

Amplification of integrons by PCR

DNA extraction. Two colonies of each bacteria isolates were placed into a tube containing 100 µl of double-distilled water. Tubes were heated at 100°C for 10 min, and then the cells were pelleted by centrifugation. The supernatant containing DNA was taken out and stored at –20ºC [29].

Duplex PCR reaction for E. coli isolates. All E. coli isolates were tested by multiplex PCR using previously described conditions and protocols [33]. Two sets of primers were used to amplify the 287 and 789 bp fragments of the int1 and int2 genes respectively (Table 1). Duplex PCR reaction was performed in a 25 µl reaction mixture, containing PCR buffer (10 mM Tris-HCl, 50 mM KCl, and 1.5 mM MgCl₂, pH 8.7), dNTP (200 μM), a primer (0.4 μM), Taq DNA polymerase (1 U), and template DNA (2 µl). The PCR reaction was performed in a DNA thermocycler (Model CP2-003; Corbett, Australia) as follows: initial denaturation — at 94°C for 4 min, 30 cycles of denaturation — at 94°C for 5 s, annealing — at 59°C for 10 s, elongation — at 72ºC for 30 s and the final extension step — at 72°C for 5 min, followed by cooling at 4°C. PCR products were electrophoresed on 1.5% agarose gel containing ethidium bromide at 80 volts for 1 h.

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Table 1. Two sets of primers used in the multiplex PCR reaction

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Results

In this study, 33 of 80 E. coli isolates (41.25%) contained integron-associated genes. Among these, 25 isolates (31.25%) harbored class 1 integrons. Class 1 integrons were identified in the strains recovered from sheep (n=4), chickens (n=12), cows (n=1), and humans (n=8). Eight isolates (10.0%) contained class 2 integrons which were identified in the strains recovered from sheep (n=1), chickens (n=6), and humans (n=1) (see Figure).

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Detection of int1 and int2 genes in E. coli strains. Lane M, 100 bp marker scale; lanes 1–6, positive strains

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Most of the E. coli strains isolated from sheep, chickens, cows, and humans were resistant to piperacillin, tobramycin, amikacin, and gentamicin while a lower percentage showed resistance to cefazolin and nalidixic acid (Table 2).

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Table 2. Patterns of E. coli resistance to antibiotics

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In sheep, class 1 integrons were detected in four sulfamethoxazole/trimethoprim-resistant E. coli isolates and class 2 integrons were detected in one sulfamethoxazole/trimethoprim-resistant E. coli isolate (Table 3). On the other hand, class 1 and class 2 integrons were detected in twelve and six multiple drug resistance E. coli isolates from chickens, respectively (Table 4). In cows, only class 1 integron was detected in an E. coli isolate with streptomycin and sulfamethoxazole/trimethoprim resistance (Table 5). In one of eight E. coli isolates from humans, class 1 integrons and class 2 integrons were detected. Multiple drug resistance was also detected in majority E. coli isolates from humans (Table 6).

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Table 3. Types of drug resistance in E. coli from sheep

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Table 4. Comparison of antibiotic resistance patterns in E. coli from chickens

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Table 5. Comparison of antibiotic resistance patterns in E. coli from cows

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Table 6. Comparison of antibiotic resistance patterns in E. coli from humans

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Resistance to more than six antimicrobial agents was observed among integron-positive strains (Table 7). Our findings showed that integrons were common among E. coli isolated in the Alborz province. Class 1 integrons prevailed over class 2 integrons.

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Table 7. Comparison of antibiotic resistance patterns between isolates with and without integrons (%)

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Discussion

Resistance to antibiotics in enterobacteriaceae can be caused by mutation or action of mobile DNA elements such as plasmids, transposons, and integrons [34]. Integrons have the ability to capture antibiotic resistance genes by site-specific recombination. Based on the type of integrase gene, five integron classes have been described to date [35, 36].

There are few reports on the occurrence and activity of integrons in microorganisms. In the present study, 41.25% of the E. coli strains isolated from sheep, chickens, cows, and humans harbored one or two integrin-associated genes. This number (41.25%) was within the range of prevalence (22 to 59%) reported in clinical E. coli isolates by others [27, 37]. In some of these isolates, only one integron class was detected while others had multiple integrons. This observation suggests that integrons commonly exist in the genome of enterobacteriaceae and may be responsible for the rapid development of antibiotic resistance.

The results of our study also indicated that 31.25% of E. coli isolates carried class 1 integrons; this number was higher than that reported by Tennstedt et al. [38], who detected the presence of class 1 integrons in 12.4% of resistance plasmids obtained from urban waste water. This figure (12.4%) was however lower than those reported from Norway [39], Western and Central Europe [27], Netherlands [40], France [26], Korea [41], and China [42].

Antibiotic resistance patterns found in our study showed that streptomycin-resistant bacteria could be isolated from animals, probably as a result of streptomycin and spectinomycin use in animal husbandry [43–47]. Furthermore, coliform bacteria isolated from humans became colonized with streptomycin- resistant bacteria via the food chain in a contaminated environment [48–50]. It is also possible that integrons are transferred from animal E. coli to human E. coli while transiently passing through the human intestine.

Antibiotic resistance patterns observed in the animal and human E. coli isolates in the present study are in line with a study from Ireland that multi-drug resistance is associated with class 1 integrons in E. coli serotypes isolated from soil samples and cattle faeces [51]. Several studies have also reported the presence of integrons in uropathogenic E. coli and have established a strong association between the presence of integrons and antimicrobial resistance in multi-drug- and single-drug-resistant E. coli strains [52, 53].

In the present study, class 1 and class 2 integrons were detected in twelve and six multiple drug resistance E. coli isolates from chicken samples, respectively. These results are similar to a recent report that found antimicrobial resistance in uropathogenic E. coli from Europe and Canada [52].

Conclusion

Integrons were widely disseminated among E. coli isolates from the Alborz province. Increased surveillance and the development of adequate prevention strategies are warranted to elucidate the diversity of factors occurring in these environments.

Authors’ contributions. RR carried out the molecular genetic studies and participated in their design. HM carried out the biochemical study. NH participated in designing the study and performed the statistical analysis. RK performed the sampling and carried out the molecular genetic studies. FK conceived and coordinated the study and helped in drafting the manuscript. All authors contributed equally to this study. All authors have read and approved the final manuscript.

Study funding. This work was supported by a grant from Karaj Branch, Islamic Azad University to Hamed Moradi for obtaining her MSc degree.

Conflict of interests. The authors declare that they have no conflict of interests.