The subtherapeutic application of antibiotics as prophylaxis or for growth promotion in farmed animals is described as a major contributor to the clinical problem of resistant disease in human medicine. There is evidence that food producing animals are likely contributor to the recent emergence of clinically important bacteria such as
mcr-positive colistin resistant Enterobacteriaceae in human [
22]. Colistin is among the most commonly used veterinary antibiotics in food animals worldwide and it is widely used in Iran for prevention/treatment of digestive tract diseases in chickens. In the current work we studied the prevalence and genetic background of ColR enteric bacteria colonizing the gut of poultry from 131 chicken farms located in 29 cities of East Azerbaijan province. We introduced here an effective screening method using selective broth media for accurate identification of ColR isolates from the polymicrobial samples such as cloacal swabs. It is important to note that alongside the screening using MHB supplemented with colistin, we performed the screening using the colistin containing agar plates (supplemented with 2 and 4 mg/L colistin) as well. However, since the agar dilution method has been found to produce higher colistin MIC values compared to broth dilution method [
23,
24], we confronted with large number of false resistant cases. Moreover, there were some ColR isolate cases which were missed by agar screening method but detected by broth screening method (data not shown). From the 931 screened enteric bacterial isolates, 9 (0.96%) were found to be colistin resistant which were identified as
K. pneumoniae. In this study we did not find any ColR
E. coli isolate. ST11 was one of the most frequent Sequence types being found in 22.2% of ColR isolates originated from different farms and characterized with diverse resistance mechanisms. This sequence type has been previously reported by several other studies as a common ST among CRKP with human origin [
11,
25,
26] indicating the successful circulation of this hypervirulent CRKP among the human and animal sources. One CRKP isolate obtained from turkey was characterized with sequence type 37 which has been frequently reported from CRKP of animal origin and is found to be associated with resistance to the last-hope anti-XDR-GNB antibiotics, colistin and tigecycline [
27,
28]. In a search to unravel the colistin resistant mechanisms, we found that resistance in the studied CRKP isolates was not related to plasmid-encoded colistin resistance genes
mcr-
1 to
mcr-
4. This was not a surprise for us as CRKP isolates studied here characterized with high MIC values (≥ 64 mg/L) which have been found to be caused by chromosomal alterations. On the other hand,
mcr-type genes are suggested to confer a low level of colistin resistance. In 88.8% of the studied isolates, MgrB was inactivated by either insertion of IS elements or premature termination due to nonsense mutations. In four isolates belonging to sequence types ST3380, ST525 and ST726 a premature stop codon was identified in codons 8, 16 and 30 leading to production of truncated protein with 7, 15 and 29 amino acids respectively compared to wild-type MgrB (47aa). These amino acid substitutions were probably leading to production of a non-functional MgrB protein, and therefore were probably the source of colistin resistance. While Q30STOP substitution in CRKP had been previously reported by several other studies [
10,
29], C16STOP and L8STOP were identified in our study as novel MgrB substitutions. In 4 isolates assigned to sequence types ST485, ST37, ST11 and ST74
mgrB was disrupted by insertion of mobile DNA belonging to three different IS
1, IS
3 and IS
5-like families. Surprisingly, all IS elements targeted 4 different sites within the
mgrB gene. Several other studies have reported interruption of
mgrB by IS
5, and it was found that this mobile DNA always targeted the same location, between nucleotides 74 and 75 of the
mgrB sequence [
10,
18]. By comparing our results to other published data, it can be concluded that nucleotide positions + 74/+ 75 and + 88 (codon 30) in
mgrB might constitute a hot spot of integration for ISs belonging to the IS
5 family or nonsense mutations respectively. Sequence analysis of PmrB revealed some variants including T246A and R256G in ColR isolates with the latter being also found in one ColS isolate. Since these alterations have been found in numerous ColS isolates [
28,
29], they can’t contribute to colistin resistance alone and most likely correspond to polymorphisms as demonstrated previously [
11,
17]. Overall, five amino acid substitutions were identified in CrrB, among which S195N had been previously described by Cheng et al. as contributing to colistin resistance [
9]. Among the remaining 4 novel substitutions, only F303S and T150R were predicted by the PROVEAN tool to have deleterious impact on protein structure. We also suggest that amino acid change K325R in CrrB cannot contribute to colistin resistance alone since this mutation was also found in a ColS isolate. This substitution was also categorized as neutral change by the PROVEAN tool. One ColR isolate with high level colistin resistance (MIC > 128 mg/L) assigning to ST11 carried a wild type MgrB, CrrB, PmrAB, PhoPQ systems, but A83V substitution was detected in CrrA which might be harmful for protein as predicted by PROVEAN tool. In the three laboratory induced ColR mutants resistance was developed in a very short time period (6 or 7 selection cycles) indicating that colistin exposure might act as an important risk factor for resistance emersion. Sequence analysis of the studied genes revealed no alteration in the case of laboratory induced ColR mutant K79R2, implying that molecular factors other than what were studied here might mediate resistance in this isolate. PmrB T93N substitution and complete deletion of
mgrB locus were identified in the other two laboratory induced ColR mutants. Cannatelli et al. previously reported CRKP isolates characterized with complete
mgrB locus deletion which did not yield any PCR product when amplification was performed using different primers designed for flanking or internal sequences of
mgrB [
18]. Overall, whether novel changes recognized here influence colistin resistance is not known, as some have never been described before. The exact role of these novel variants in resistance conferring, require to be further studied by confirmatory tests, such as complementation assays with wild-type counterparts. However, according to previous studies demonstrating the key role of MgrB alterations in conferring colistin resistance, and observations regarding inactivation of
mgrB gene in 88.8% of the avain CRKP isolates and induction of high level colistin resistance following
mgrB locus deletion it can be concluded that colistin resistance in the studied avian CRKP isolates was mostly linked to alterations identified within the
mgrB gene. We had previously reported MgrB inactivation as the major mechanism mediating colistin resistance in Iranian KP isolates of clinical origin indicating the fact that colistin resistance in CRKP isolates of human or animal sources is mediated by a common mechanism [
17].