From 2008 to 2010 and from 2015 to 2018, 338 clinical isolates of K. pneumoniae were obtained from a tertiary hospital in Jeonbuk, Korea (Jeonbuk National University Hospital Culture Collection for Pathogens). The primary identification and antimicrobial susceptibility test were performed with a VITEK-2 system (bioMérieux, Marcy l’Etoile, France). The minimum inhibitory concentration (MIC) of antibiotics was determined by the macrodilution method using 2 ml of cation-adjusted Muller-Hinton broth in a standard glass test tube according to Clinical and Laboratory Standards Institute guidelines (8, 11). The MIC also was measured using the E-test carried out on a Muller-Hinton agar plate according to the manufacturer’s instructions (bioMérieux, Marcy l’Etoile, France; Liofilchem Inc., MA, USA). As a control, K. pneumoniae ATCC 13883 purchased from the American Type Culture Collection (ATCC, VA, USA) was used.

MLST of K. pneumoniae isolates was performed with seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB), according to a previously described protocol (12). Allele sequences and ST were assigned according to Institute Pasteur’s MLST scheme for K. pneumoniae.

Whole-bacterial DNA was prepared from bacterial cells lysed by heating at 95°C for 10 min. The colistin resistance- associated genes (pmrA, pmrB, pmrC, pmrD, pmrE, pmrK, phoP, phoQ, and mgrB) were amplified using specific primers (Table 1) (13, 14, 15). PCR amplification detection for plasmid-mediated mcr genes was performed using previously described primers (16, 17). The presence of extended-spectrum β-lactamase and carbapenem-hydrolyzing β-lactamase genes, such as bla_CTX-M, bla_SHV, bla_TEM, bla_KPCs, and bla_OXA-48, was screened as previously described (18, 19, 20). PCR analysis for the ompK35 and ompK36 genes, K. pneumoniae porin genes associated with carbapenem resistance, was carried using the following primers: ompK35 forward, 5′- GCCGCCCGCACTCCACGCTG-3′; ompK35 reverse, 5′-CAGAAAGTTATGGAGCCACG-3′; ompK36 forward, 5′-GTATCATATTCTTGTTGGATTA-3′; and ompK36 reverse, 5′-GAAAGTCATATCCATTAATCG-3′. PCR analysis was used with previously described primers to identify the genes encoding AmpC-type β-lactamases from the following six families (21): MOX (MOX-1, MOX-2, CMY-1, and CMY-8 to CMY-11), CIT (LAT-1 to LAT-4, CMY-2 to CMY-7, and BIL-1), DHA (DHA-1 and DHA-2), ACC, EBC (MIR-1T and ACT-1), and FOX (FOX-1 to FOX-5b). The amplified PCR products were confirmed by agarose gel electrophoresis, purified from the agarose gel, and sequenced. The nucleotide and deduced protein sequences were analyzed at the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov) using the Basic Local Alignment Search Tool (BLAST) program.

CRKP has been frequently identified as multidrug-resistant (MDR), including the potential development of resistance to colistin, a last-line antibiotic, and is associated with high rates of morbidity and mortality worldwide (22). To investigate the prevalence and the detailed mechanisms of MDR K. pneumoniae with carbapenem and colitis resistance, we assessed carbapenem and colistin resistance from the K. pneumoniae isolates collected by a Korean tertiary hospital, Jeonbuk National University Hospital Culture Collection for Pathogens. Out of 338 K. pneumoniae isolates, two (0.6%) were identified as both carbapenem and colistin resistance. Interestingly, a high rate of colistin resistance in CRKP was observed as previously reported (23, 24). Two (22%) of nine CRKP possessed colistin resistance (Table 2); the colistin MIC was ≥16 μg/ml using the broth culture macrodilution method, but control K. pneumoniae ATCC13883 was not resistant to colistin. The two colistin-resistant isolates, LTH36 and LTH51, were resistant to all tested β-lactam antibiotics, including carbapenems (ertapenem, imipenem, and meropenem) and broad spectrum β-lactams (ceftazidime, cefotaxime, ciprofloxacin, and cefpirome) (Table 2). Intriguingly, the MIC (≥32 μg/ml) for imipenem was higher in the two isolates than those of most CRKP within our health system (imipenem MIC range, 1 to 4 μg/ml) (25). In addition, the isolates were resistant to aztreonam and aminoglycoside antibiotics (amikacin and gentamicin) (Table 2). However, one isolate, LTH51, remained susceptible only to tigecycline, as previously reported in other colistin-resistant K. pneumoniae, although the other isolate, LTH36, was not resistant to tigecycline and trimethoprim/sulfamethoxazole (Table 2) (22, 26).

Since the isolates exhibited resistant phenotypes to a variety of β-lactam antibiotics, including carbapenems, we investigated the presence of extend-spectrum-β-lactamase (ESBL) and carbapenem-hydrolyzing β-lactamase genes using PCR analysis and nucleotide sequencing. For ESBL genes, bla_CTX-M, bla_SHV, and bla_TEM genes were analyzed. The LTH36 isolate carried the bla_SHV-31 and bla_TEM-116 genes, and the LTH51 isolate revealed the presence of the bla_CTX-M-3, bla_SHV-11, and bla_TEM-1 genes. For detection of genes associated with carbapenem resistance, we evaluated the most prevalent carbapenem-hydrolyzing β-lactamase genes, bla_KPCs and bla_OXA-48. However, the bla_KPC and bla_OXA-48 genes were not identified in the two isolates. Since carbapenem resistance can occur with various types of carbapenemases, we screened for other carbapenem- hydrolyzing β-lactamase genes with previously described primers to amplify the following genes (18): bla_OXA-48-like including OXA-199 and OXA-370, bla_VIM-type, bla_NDM-1, bla_IMP variants except IMP-3, 16, 27, 31, 34, and 35, bla_OXA-23-like, bla_{SME-1 to 5}, bla_IMI-type, bla_IMP-27, bla_{NDM-2 to 10 and 12}, bla_{IMP-3 and 34}, bla_{GES-1 to 22 and 24}, bla_GIM-1, bla_{OXA-2-, 10-, 51-, and 58-like}, bla_DIM-1, and bla_CMY-2. Unexpectedly, the tested genes involved in carbapenem resistance were not detected in the two K. pneumoniae isolates, LTH36 and LTH51.

To investigate diversity, distribution, and relationship of our bla_KPC- and bla_OXA-48-negative CRKP isolates with global isolates, we performed MLST. Interestingly, the STs of our colistin-resistant CRKP isolates LTH36 and LTH51 were ST11 and ST258, respectively, which are the most global STs of KPC-producing CRKP (3, 5, 27). Although the CRKP isolates with ST11 and ST258 were previously isolated in Korea, they carried KPC and showed a different susceptibility to colistin: the ST11 exhibited a colistin-resistant phenotype, but the ST258 did not (10, 28, 29). Taken together, we first isolated K. pneumoniae ST11 and ST258 in Korea possessing both carbapenem and colistin resistance despite the absence of previously known carbapenem-hydrolyzing β-lactamase genes.

Because our two isolates were resistant to carbapenems without producing the most prevalent carbapenemases, we investigated an alternative mechanism for carbapenem resistance, which could result from mutation(s) in outer membrane major porins, OmpK35 and OmpK36, in association with AmpC-type β-lactamases. The deficiency of porin as a selective barrier would result in carbapenem resistance through reducing the permeability of porin; in addition, AmpC β-lactamases without carbapenemase activity could be involved in trapping carbapenems, which prevent the antibiotics from accessing their targets (6).

To test this possibility, we analyzed the sequences of porin genes, ompK35 and ompK36, and the presence of bla_AmpC genes from six families: ACC, CIT, DHA, EBC, FOX, and MOX. In the sequence analysis of ompK35 and ompK36 compared with those of a control K. pneumoniae ATCC13883 (ompK35 and ompk36, GenBank accession no. CP040993.1), our LTH36 and LTH51 isolates revealed the lack of one or both of the two major porins. The LTH36 isolate revealed 1-bp deletion at position 54 and 1-bp insertion at position 419 of the ompK35 and ompk36 genes, respectively, which created amino acid changes and premature terminations after Gly-18 and Ser-139 in OmpK35 and OpmK36, respectively. In the LTH51 isolate, ompk36 sequencing reveled an ochre nonsense mutation after the start codon that could lead to the loss of OpmK36 porin, although there was wild type of the ompK35 gene. Furthermore, PCR and sequencing analysis of genes encoding AmpC β-lactamases showed that all of the nCP-CRKP isolates with colistin resistance harbored the DHA-1 ampC β-lactamase gene. These results suggest that the carbapenem resistance of the nCP-CRKP isolates LTH36 and LTH51 could be associated with loss of the major porins OmpK35 and/or OpmK36 and involved in DHA-1 AmpC β-lactamase production.

To investigate the molecular mechanisms related to colistin resistance in the nCP-CRKP isolates with colistin resistance, we evaluated genetic mutations in the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems, the PmrD connector of the two-component systems, the MgrB negative regulator of the PhoP/PhoQ, and LPS-modifying enzymes including PmrC, PmrE, and PmrK. Sequence analysis revealed amino acid variations in PmrB, PmrC, PmrE, PmrK, and MgrB compared with the colistin-susceptible K. pneumoniae reference strain, MGH78578 (Table 3). The same amino acid changes that were previously reported as contributing to colistin resistance were observed in PmrB (T246A and R256G) and PmrC (C27F, I138V, and Q319R) from the two isolates (8, 30, 31). Furthermore, we identified novel amino acid substitutions in PmrE (V17I, A33S, H68Q, T105A, A165E, D172N, A274G, N354D/A, E371D, and T373A) and PmrK (M114L, V117I, R372K, and G462R) from the LTH36 or LTH51 isolates, although more studies are required to prove a connection between the mutations and colistin resistance. Interestingly, although the two K. pneumoniae isolates belonged to different STs, there were the amino acid changes at the same position in PmrB, PmrC, PmrE, and PmrK: PmrB, T246A, and R256G; PmrC, C27F, I138V, and Q319R; PmrE, N354D/A and T373A; PmrK, M114L, V117I, and R372K. In the mgrB gene analysis of the two isolates, insertion sequences (ISs) were found in all isolates and caused insertional inactivation of the MgrB protein by missense and frameshift mutations, which resulted in amino acid residue and terminational changes after Leu-17 and Cys-39 of MgrB from the LTH36 and LTH51 isolates, respectively. However, PmrA, PmrD, PhoP, and PhoQ of the isolates were identical to the wild-type proteins of the reference strain K. pneumoniae MGH78578, and plasmid-mediated colistin resistance genes, mcr-1 to mcr-9, were not detected in the isolates. These results suggest that the amino acid changes in PmrB, PmrC, PmrE, PmrK, and MgrB of the colistin-resistant CRKP isolates might be associated with colistin resistance.

In conclusion, we detected a high rate (22%, 2 of 9) of colistin resistance among CRKP although two (0.6%) K. pneumoniae isolates with carbapenem and colistin resistance were found in 338 clinical isolates collected by a Korean tertiary hospital. The two isolates belonged to ST11 and ST258, the most prevalent STs of KPC-producing CRKP, but these carbapenem resistances were related to truncating mutations in the major porin ompK35 and/or ompK36 genes associated with DHA-1 AmpC β-lactamase. In colistin resistance, these isolates carried novel and previously reported mutations in the pmrB, pmrC, pmrE, pmrK, and mgrB genes that could confer colistin resistance (Table 3). Taken together, we identified and characterized the detail molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae ST11 and ST258 isolate that have not been reported in Korea. These results suggest that colistin-resistant nCP-CRKP with novel mutations emerged in Korea and could spread throughout the country. In addition, this study showed the importance of judicious use of antibiotics with proper susceptibility analysis as well as continuous surveillance and strict infection control measures on colistin-resistant CRKP to prevent the resistant bacteria from becoming prevalent in the hospital setting.