The emergence of MDR Enterobacteriaceae and the lack of new antibiotics coming to market to combat them bring us perilously to the end of the “Antibiotic era.” Among Enterobacteriaceae, Klebsiella pneumoniae, is found to be associated with the highest rates of carbapenem resistance (Marinelli and Genilloud, 2013). Polymyxin antibiotics such as colistin are among the few antimicrobial agents that retain activity against MDR-GNB in particular CRE and often considered as the last-line therapy to treat infections caused by these superbugs (Li et al., 2006; Olaitan et al., 2014b). Polymyxins (polymyxin B and colistin) are cationic antimicrobial peptides that target anionic lipid A phosphate moiety of bacterial LPS causing membrane leakage and eventually cell death (Gupta et al., 2009; Deris et al., 2014). Bacteria employ several means to protect themselves from antimicrobial activity of polymyxins. The most common resistance mechanism in Enterobacteriaceae is attributed to the covalent modifications of the lipid A moiety of LPS through the incorporation of positively charged groups such as PEtN and L-Ara4N. These modifications neutralize the negative charges of LPS and subsequently reduce binding affinity of colistin to its target (Tamayo et al., 2005). LPS modification in K. pneumoniae is mediated by the activation of PmrA/PmrB two-component regulatory system (TCRS) which regulates expression of the genes located in pmrCAB and pmrHFIJKLM operons encoding for enzymes implicated in the LPS modifications. Moreover, a second TCRS, PhoP/PhoQ is known to contribute to polymyxin resistance by indirectly activating the PmrA/PmrB TCRS via PmrD linker protein. Mutations in these TCRSs can cause constitutive expression of the pmrCAB and pmrHFIJKLM operons and consequently transfer of PEtN and L-Ara4N, respectively, to lipid A (Tamayo et al., 2005; Olaitan et al., 2014b). Moreover, inactivation of the PhoQ/PhoP negative regulator encoded by mgrB gene has been identified to play a prominent role in polymyxin resistance in K. pneumoniae isolates (Cannatelli et al., 2014b; Olaitan et al., 2014b; Poirel et al., 2015). Genetic alterations such as Leu26Pro in PhoQ (Cheng et al., 2015), Leu82Arg (Cannatelli et al., 2014a), and Thr157Pro (Jayol et al., 2014) in PmrB, Q30stop, C28stop and insertion of IS elements in MgrB (Poirel et al., 2015) have been found to contribute to colistin resistance in K. pneumoniae isolates. In addition to these chromosomally mediated mechanisms, plasmid-encoded colistin resistance genes, mcr-1, mcr-2, mcr-3, and mcr-4 encoding a PEtN transferase have been recently reported as a transmissible resistance mechanism in Enterobacteriaceae (Gu et al., 2016; Liu et al., 2016; Xavier et al., 2016; Carattoli et al., 2017; Yin et al., 2017). Due to lack of information about the molecular mechanisms of colistin resistance in K. pneumoniae isolates in Iran, we aimed to explore these mechanisms in colistin resistant K. pneumoniae strains from this geographic region.

Colistin has recently regained significant interest constituting the major part of therapeutic regimen for management of severe infections caused by CRE (Morrill et al., 2015). Despite the scarcity of colistin resistance, this anti-CRE magic bullet has begun to wane as a consequence of frequent use in clinical settings and subsequent resistance development. In our study, plasmid encoded mcr-1, mcr-2, mcr-3, and mcr-4 genes were not detected in any of the col-R KP isolates demonstrating that resistance is mediated by chromosomally encoded mechanisms. Of 20 col-R KP isolates, 15 (75%) were identified as having an inactivated MgrB mediated by insertion of IS elements or nonsense mutations. MgrB, a small transmembrane protein with 47 amino acids mediates potent negative feedback on the PhoQ/PhoP regulatory system. It is hypothesized that critical alterations within mgrB found in the current study including disruption of promoter or coding region result in gene silencing or production of truncated MgrB. In fact, MgrB inactivation by any of these events results in PhoP/PhoQ activation which in turn activates the PmrA response regulator. Complementation with wild type MgrB protein restored colistin susceptibility in all col-R KP isolates with mgrB modifications. This verifies the resistance conferring role of mgrB alterations in the studied isolates. Poirel et al. (2015) demonstrated that insertion of IS elements (IS5-like, ISKpn13 ISKpn14, IS10R) and Q30stop and C28stop substitutions in mgrB were involved in resistance to colistin in K. pneumoniae isolates determined by complementation assays. The Q30stop substitution has been found by several other studies (Olaitan et al., 2014a; Aires et al., 2016) reinforcing the hypothesis that position +88 in the mgrB (codon 30 in protein) is a critical region which is prone to mutate upon resistance emergence. Also, Cannatelli et al. (2014b) reported that mgrB alterations, including insertion of IS5-like, IS1-like, or ISKpn14 elements, non-silent point mutations, and small intragenic deletions were mediating colistin resistance in 53% of studied col-R KP isolates. All together three substitutions were found in the pmrB including L213M, A246T, and R256G. Complementation with wild type pmrB gene was not able to restore colistin susceptibility in isolates characterized with these pmrB alterations. Similarly, Cheng et al. (2015) showed that R256G substitution found in col-R KP isolates did not contribute to colistin resistance determined by site-directed mutagenesis method. Also, Aires et al. (2016) suggested that A246T mutation found in the pmrB was not related to colistin resistance. This was because they had found this alteration in colistin susceptible isolates as well. Therefore, mechanisms of colistin resistance were not fully unraveled in five isolates characterized with pmrB mutations alone. A recent study showed that alterations in the newly described TCRS, CrrAB mediate colistin resistance in K. pneumoniae (Wright et al., 2015). Cheng et al. (2015) found R256G replacement in the pmrB as the only genetic alteration among six studied genes including mgrB, pmrA, pmrB, phoP, phoQ, and pmrD in 8 of 26 col-R KP isolates. All of these eight isolates had overexpressed pmrHFIJKLM operon. In an attempt to determine the exact mechanisms of decreased susceptibility to colistin in these isolates, the same authors found mutations in CrrAB TCRS. The mutations were found to be associated with overexpression of pmrHFIJKLM and pmrCAB operons in these isolates (Cheng et al., 2016). Therefore, it is tempting to speculate that genetic modifications in systems other than PmrA/PmrB and PhoP/PhoQ such as CrrAB might contribute to colistin resistance in these five isolates.

Colistin resistance has been found to be associated with upregulation of pmrHFIJKLM and pmrCAB operons which result in addition of L-Ara4N and PEtN, to LPS, respectively. Generally, either L-Ara4N and/or PEtN can be added to the 4-phosphate and 1-phosphate of lipid A and both modifications decrease the anionic charge of LPS (Zhou et al., 2001; Lee et al., 2004; Tamayo et al., 2005). However, it has been demonstrated that the LPS modification by L-Ara4N confers a higher level of polymyxin resistance compared to PEtN modification (Tamayo et al., 2005). We found a significant association between colistin resistance and upregulation of pmrHFIJKLM and pmrCAB operons. While all isolates revealed an over-expressed pmrK, only 55% of isolates exhibited pmrC upregulation implying that pmrC-mediated LPS modification was inferior to that of pmrK with regard to colistin resistance. However, one isolate (KP02) revealed partially higher expression level for pmrC (3.2-fold) compared to pmrK gene (2.5-fold). Jayol et al. (2014) reported that Thr157Pro substitution in PmrB was responsible for the overexpression of pmrCAB and pmrHFIJKLM operons with the pmrC being more overexpressed (170-fold) compared to pmrK (40-fold). We also observed that strains with the same mutational profile and colistin MIC values revealed distinct expression level/profile of pmrK or pmrC genes. This can be explained by the fact that determinants other than PmrB/MgrB (such as CrrAB TCRS) might contribute to regulation of operons involved in the LPS modification. Also, different MIC values between the isolates with the same genetic alterations in the studied loci can be attributed to alterations in other unknown loci or involvement of other resistance mechanisms such as increased amount of capsular polysaccharide or efflux pumps (Olaitan et al., 2014b).

In summary, we found that MgrB alterations play major role in colistin resistance in K. pneumoniae isolates studied in the current work. We also found that, pmrK operon and therefore LPS modification with L-Ara4N plays prominent role in resistance conferring followed by pmrC operon and PEtN modification. This is the first report about molecular mechanisms of colistin resistance in K. pneumoniae isolates from Iran. Evaluating the role of CrrAB system in mediating colistin resistance will be the next step of this ongoing work to provide a comprehensive understanding of mechanisms involved in colistin non-susceptibility in K. pneumoniae isolates. In addition, multilocus sequence typing should be performed to determine whether these Iranian col-R KP isolates belong to predominant sequence types in Iran or to those, which are common in the world.

MH is the corresponding author who designed the experiments, analyzed the experiment data, and wrote the manuscript. AJ performed the experiments, analyzed the experiment data, and drafted the manuscript. JM and ZJ contributed to performance of complementation assays and PFGE analysis respectively. MF designed the experiments and wrote the manuscript. EB participated in the design and performance of RNA extraction and RT-qPCR and editing the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.