Acinetobacter is a nosocomial pathogen. Its ability to infect healthy hosts and its propensity to develop antimicrobial drug resistance is a cause for concern among infectious disease specialty. Although the ubiquitous existence of A. baumannii in hospital environment has been considered a routine delusion by several investigations [10, 12,13,14, 16, 17, 19, 21], different recent reports have undeniably highlighted the presence of the bacterium in different types of animal origins [11, 22, 23]. In recent years, nosocomial infections of A. baumannii, as an opportunistic pathogen, are increasing. Treatment of this bacteria especially multi-drug resistant and broad-spectrum beta-lactamases strains is of major concern [11, 22, 23]. These recent works have mainly been done only on the prevalence rate of A. baumannii and in some cases, on antibiotic susceptibility patterns. Reversely, to the best of our knowledge, the study presented here is the first report of the molecular typing, on the distribution of virulence factors and genotypic evaluation of antibiotic resistance of the A. baumannii strains isolated from different animal origins. A total of 22 strains of A. baumannii were assessed and 16 different genetic cluster were detected. Meat-derived A. baumannii strains may originate in the slaughterhouses, butchers and shopping centers especially due to the manipulation of the meat samples.
A. baumannii has previously been recognized as an animal colonizer with diverse distributions in different countries including in Scotland, 1.20% [22] and Senegal, 5.10% [24]. Rafei et al. (2015) [25] reported the high prevalence of A. baumannii strains in food samples with animal origins including raw meat, raw milk, and dairy products. The A. baumannii strains of the present research were frequently resistant to clinically relevant antibiotics. However, a few isolates displayed low levels of resistance against imipenem, azithromycin, meropenem, rifampin, levofloxacin, ceftazidime and tobramycin. This latter is still among the drugs of choice for the treatment of A. baumannii infections in humans and animals in Iran.
A. baumannii strains of the same molecular cluster (ERIC-type) had the same profile of the antibiotic resistance pattern. We also found that all of A. baumannii strains originated from chicken meat samples were susceptible to azithromycin and rifampin and resistant against tetracycline. These findings have been supported by several studies conducted on Lebanon [25], Switzerland [26], and France [27].
We found that molecular similarities of 19 and 22, 12 and 15, 6 and 14, 1 and 3, 4 and 5 and finally 2 and 10 A. baumannii strains were 84.60%, 100%, 87%, 80%, 83.30% and 80% with each other, respectively. All the other A. baumannii strains were classified as differing ERIC types. A. baumannii strains recovered from the bovine, camel, turkey and ovine meat samples didn’t show any similarity in their molecular typing and there were no similar antibiotic resistance patterns between them. Our findings showed that they also had similar phenotypic pattern of antibiotic resistance. One possible explanation for the similar molecular types of the A. baumannii strains recovered from different animal origins is their common source of infection. It may also be due to the close contact between bovine and chicken, bovine and turkey and between chicken and ovine species, facilitating transmission of the A. baumannii strains with same molecular type. Unfortunately, Iranian ranchers frequently maintain and breed different species of animals and especially bovine, ovine and caprine species in contact to each other. This may cause easy transmission of the A. baumannii strains between different species. The high genetic diversity of strains isolated from meat samples has also previously been reported by Lupo et al. (2014) [26] and Carvalheira et al. (2017) [28]. Recent research [21] revealed the similar antibiotic resistance pattern of the A. baumannii strains isolated from different types of raw meat samples. They showed that the prevalence of resistance against trimethoprim-sulfamethoxazole, tetracycline, amikacin, tobramycin, ampicillin-sulbactam, meropenem and imipenem were 23.20%, 23.20%, 14.30%, 12.50%, 12.50%, 8.30% and1.20%, respectively which was different to our findings. The prevalence of antibiotic resistant bacteria in meat samples has been attributed, at least partially, to the extensive use of antimicrobials for treatment, prevention and control of diseases and finally for growth stimulate in food-producing animals, since this enhances the antimicrobial selective pressure for strains present. Otherwise, using antibiotics for growth stimulation is allowed in Iran. Low prevalence rate of resistance against carbapenems is due to the fact that these antibiotics are not allowed to treat food-producing animals.
Phenotypic characterization of antibiotic resistance was also confirmed by genotyping analysis of antibiotic resistance. We identified the genes for resistances to aminoglycosides (aadA1 and aac(3)-IV), beta-lactams (blaSHV and blaCTM), chloramphenicol (cat1 and cmlA), tetracyclines (tetA and tetB), sulfonamides (sul1 and dfrA1), carbenicilins (imp, sim and vim) and to fluoroquinolones (qnr). Furthermore, A. baumannii strains harbored their own specific antibiotic resistance genes (Table 2). This part of our study was in agreement with previous researches [21, 29]. Additionally, we found that A. baumannii strains of the same molecular cluster (ERIC-type) had the same profile of the antibiotic resistance genes., as follows: strains no 1 and 3 of the same ERIC-type (type XIII) were positive for CITM,dfrA1, tetA, tetB, aac(3)-IV and sul1 which showed their same genetic pattern of antibiotic resistance. Strains no 12 and 15 of the same ERIC-type (type IV) were positive for tetA, aadA1, aac(3)-IV, blaSHV, dfrA1 and sul1. Strains no 19 and 22 of the same ERIC-type (type I) were positive for tetB, aadA1, sim, oxa-58-like and sul1. Despite of the different animal origin of the A. baumannii strains no 6 and 14 of the same ERIC-type (type IX), were positive for tetA, tetB, aac(3)-IV, sul1, dfrA1 and blaSHV antibiotic resistance genes. Additionally, A. baumannii strains no 4 and 5 of the same ERIC-type (type XV) had different origins but they harbored tetA, tetB, sul1 and aac(3)-IV antibiotic resistance genes. Finally, A. baumannii strains no 2 and 10 of the same ERIC-type (type XVI) were positive for tetA, aac(3)-IV, dfrA1 and oxa-58-like antibiotic resistance. A. baumannii strains of other ERIC types had unique pattern of antibiotic resistance.
Virulence of A. baumannii is dependent on several other attributes (i.e. “slime”, LPS production and etc.) beside the selected virulence genes listed here [30, 31].
Another notable finding is the high prevalence of certain virulence factors in the A. baumannii strains isolated from meat of different animals. The most commonly detected virulence genes amongst these A. baumannii strains were fimH, afa/draBC, csgA, cnf1, cnf2 and iutA. Prevalence of non-adhesive virulence factors including traT (serum resistance), cvaC (colicin V), ibeA (invasion), fyuA (yersiniabactin) and PAI (indicator gene) were low. This finding is in agreement with those of previous researches [30, 31]. Non-adhesive virulence factors play an important role in bacterial survival in special conditions such as in human blood and exposure to serum and poor iron environments contributing to the pathogenesis of extra instestinal diseases. However, presence of these non-adhesive virulence factors may not be essential for pathogenicity of food-borne enteric diseases. The gene iutA (aerobactin) has a high prevalence (50.00%) in our strains. Darvishi (2016) [32] reported that the prevalence of cnf1, csgA, cvaC and iutA virulence factors amongst the A. baumannii strains isolated from hospitalized patients were 50%, 70%, 10% and 25%, respectively. Daryanavard and Safaei (2015) [33] reported that the total prevalence of csga, cnf1, cvaC and iutA virulence genes among the samples of UTIs were 55%, 40%, 10% and 30%, respectively which was similar to our findings. Momtaz et al. (2015) [13] reported that the prevalence of csga, cnf1, cvaC and iutA virulence genes among the A. baumannii strains of clinical infections in Iran were 12.39%, 35.53%, 21.48% and 19%, respectively which was lower than our results.
We also found that the A. baumannii strains of the same molecular cluster (ERIC-type) had the same virulence factors. Despite of the different origin of the A. baumannii strains no 6 and 14 of the same ERIC-type (type IX), were both positive for fimH, fyuA, cvaC, cnf1, cnf2, papGII, papGIII, and afa/draBC virulence factors. Furthermore, A. baumannii strains no 4 and 5 of the same ERIC-type (molecular type XV) had different origins but they both harbored fimH, cvaC, csgA, cnf1, afa/draBC and cnf2 virulence factors. Finally, A. baumannii strains no 2 and 10 of the same ERIC-type (type XVI) were positive for fimH, fyuA, csgA, afa/draBC, cvaC, iutA, cnf1 and sfa/focDE virulence factors. These high similarities in the pattern of virulence factors of the A. baumannii strains of same ERIC types recovered from different origins showed that genetic cluster of bacterial strains is closely related to their virulence determinants.
A. baumannii strains isolated from the chicken meat samples had similar molecular type. A. baumannii strains isolated from other meat samples did not fall into common molecular types. Two A. baumannii strains of bovine origins had similar molecular type with turkey and chicken. A camel and ovine A. baumannii strains had also similar molecular type.