Characterization of Extended-Spectrum Beta-Lactamase,Carbapenemase,and Plasmid Quinolone Determinants in Klebsiella pneumoniae Isolates Carrying Distinct Types of 16S rRNA Methylase Genes,and Their Association with Mobile Genetic Elements

Bacterial strains and antimicrobial susceptibilities

A total of 84 nonduplicate MDR-KP clinical isolates with MIC ≥2 mg/L to amikicin were collected between January and October 2012 in a university hospital located in the southeastern region of China. The identification of K. pneumoniae was performed with a Vitek 2 automated system (Biomerieux). Antimicrobial susceptibility testing was performed using the automated broth microdilution system (Vitek 2), according to the Clinical and Laboratory Standards Institute. ⁵ Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as quality control strains in susceptibility tests with results within the expected ranges. E. coli J53 was used in the conjugation experiments. The presence of metallo-β-lactamase (MBL) production was determined in the K. pneumoniae with the three screening methods (Imipenem-EDTA combined disc test, IMP-EDTA double-disc synergy test, and MBL E-test).^16,36 All isolates fulfilled the CLSI criterion for performing carbapenemase detection by the modified Hodge test (MHT) as previously described. ¹⁶

DNA preparation, polymerase chain reaction amplification, and sequencing

Genomic DNA was extracted by boiling bacterial colonies for 10 min in sterile distilled water. After centrifugation, the supernatant was used as a DNA template. Single polymerase chain reaction (PCR) was used to analyze genes 16S rRNA methylase (armA, rmtB, npmA, rmtC, and rmtD), plasmid-mediated quinolone resistance [PMQR; qnrA, qnrB, qnrS, and acc(6′)-Ib-cr], ESBL (CTX-M, SHV, and TEM), and carbapenemase genes (KPC, IMP, VIM, NDM, and OXA-48) with specific primers for each one, as previously reported.^14,20,35 The strains were also screened for the presence of other β-lactamase genes (bla_OXA-1 and bla_OXA-10) as well as various mobile elements, such as ISEcp1, ISCR1, IS26, ISAba125, and sul-1 integron. ¹³ PCR products were purified by using a Qiaquick PCR purification kit (Qiagen). Bidirectional DNA sequencing was performed using the Big-Dye Terminator kit and analyzed by ABI 7300 sequencer (Applied Biosystems). The nucleotides were compared with reference sequences deposited in GenBank databases.

Screening of physical linkages between genetic elements and resistance genes

Physical linkages between insertion sequences or integron and resistance genes were determined using a combination of primers specific for various genetic elements (ISEcp1, ISCR1, IS26, ISAba125, and sul-1 integron) and consensus primers for all the resistance genes. A long-range polymerase (LongAmp Taq DNA Polymerase; New England Biolabs) was used in all reactions for physical linkages. A slow ramping rate of between 0.2°C/sec and 0.3°C/sec was set for the annealing step. The extension time was set at 72°C for 2 min and a final extension of 72°C for 15 min was carried out after 40 cycles of denaturation, annealing, and extension.

Conjugation, transformation, southern hybridization, and plasmid replicon typing

E. coli J53 was used as the recipient in the conjugation studies, and mating was carried out on blood agar medium without selection. After 18 hr, the mixed cultures were taken from the plates, suspended in saline, and plated onto MacConkey medium containing sodium azide (150 μg/ml) and amikacin (30 μg/ml). Confirmation that conjugation had taken place in E. coli J53 was determined by the Vitek 2 automated system and testing for the presence of the armA and rmtB genes by PCR analysis. Plasmid DNAs of donor isolates and E. coli transconjugants were extracted using the Kieser technique. ¹² Plasmid DNA was purified by the Qiagen Plasmid Midikit (Qiagen, Inc.). Purified plasmids were used to transform MAX Efficiency DH5α E. coli chemically competent cells (Invitrogen). DH5α was chosen as the recipient because it is capable of being transformed efficiently with large plasmids.

Southern hybridization was performed by standard methods with armA-specific and rmtB-specific digoxigenin (DIG)–labeled probes formed by the PCR DIG detection system (Roche Diagnostics GmbH) as described previously. ³⁷ Plasmid profiles and size estimation were determined by electrophoresis using a 0.7% agarose gel. PCR-based replicon typing of the main plasmid incompatibility groups reported for K. pneumoniae was performed as described. ³ Specifically, 18 replicon types (FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA) were screened by the methods of Carattoli et al., ³ and additional three types (ColE, R, and U) were investigated by the method of Pallecchi et al. ²³ The colE PCR was devised to amplify all the colE-like plasmids. Amplicons were sequenced for confirmation.

Enterobacterial repetitive intergenic consensus polymerase chain reaction and multilocus sequence typing

Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) was performed and isolates were typed according to their band patterns as described previously. ¹⁸ BioNumerics software (version 4.0; Applied Maths) was used to analyze the DNA patterns and determine their similarity. Multilocus sequence typing (MLST) of K. pneumoniae was performed as described by Liu et al. ¹⁹ Experimentally determined DNA sequences were uploaded into the MLST database (www.pasteur.fr/recherche/genopole/PF8/mlst/K.pneumoniae.html), and allelic numbers as well as sequence types (STs) were obtained.

Antimicrobial drug susceptibility

Among the 84 MDR-KP isolates, 19 highly amikacin-resistant isolates (MICs >32 mg/L) were collected and the aminoglycoside resistance was due to the production of the 16S rRNA methylase (armA or rmtB). The majority of the isolates were recovered from blood samples (9/47.4%), followed by surgical wounds (5/26.3%), urine (3/15.8%), and catheter tips (2/10.5%). Antimicrobial resistance rates against 19 MDR-KP isolates are shown in Table 1. Most of the isolates showed high-level resistance for aminoglycosides (amikacin, gentamicin, and tobramycin) and quinolone (ciprofloxacin and levofloxacin); for the aminolycosides, MIC₅₀ was >64 mg/L and for the quinolone, MIC₅₀ was >16 mg/L. Thirteen out of the 19 isolates showed resistance to imipenem (MIC₅₀ of 32 mg/L) and all of the isolates were susceptible to tigecycline (MIC₅₀ of 0.38 mg/L, respectively). Seven isolates showed a positive result in the MHT. In addition, three isolates were phenotypically positive for MBL production by the three screening methods used. Overall, all of the isolates showed resistance to most of the tested antimicrobial agents excluding tigecycline (Table 1).

Resistance genes

PCR and sequencing performed as previously described revealed that all 19 isolates possessed armA or rmtB gene. PCR on the total DNA of all armA- or rmtB-positive isolates showed different β-lactamase gene combinations (Table 2). In addition to bla_CTX-Ms, isolates also harbored several acquired and plasmid-encoded genes: bla_SHV-12, 31.6%; bla_TEM-1, 100%; bla_OXA-1, 100%; bla_KPC-2, 26.3%; bla_NDM-1, 5.3%; bla_IMP-4, 10.5%; and bla_CMY-2, 42.1% (Table 2).

Interestingly, genes for qnr or aac(6′)-Ib-cr were detected in 100% of the 19 isolates (Table 2). Cooccurrence of PMQR determinants was often observed, being of different types [qnrA1, qnrB4, qnrS1, and aac(6′)-Ib-cr]. Among the PMQR genes, the presence of qnrA1 was detected in 16 isolates, including 15 coexisting with acc(6′)-Ib-cr gene, 9 coharboring with qnrS1 gene, and 7 cocarrying with qnrB4 gene. Various combinations of bla_CTX-Ms, bla_CMY-2, bla_KPC-2, qnr, aac(6′)-Ib-cr, and armA genes were found in majority of the isolates.

Mobile elements ISEcp1, IS26, ISCR1, ISAba125, and sul-1 integron were detected in 19/19 (100%), 16/19 (84.2%), 18/19 (94.7%), 9/19 (47.4%), and 18/19 (94.7%) isolates, respectively. The isolates were also analyzed in reference to various combinations of observed genetic elements, and ISEcp1 plus ISCR1 plus sul-1 integron was found to be the most common combination, followed by ISEcp1 plus IS26 plus ISCR1 plus sul-1 integron. The frequencies of occurrence of mobile element combinations were different in the different resistance genes (Table 3).

Analysis and transferability of armA-carrying plasmids

Nine of the 18 replicons tested—including I1, L/M, FrepB, FIA, FIB, FIC, A/C, colE, and N—were involved in the donors. In case of their incompatibility groups, IncL/M (9/19), IncA/C (7/19), and IncFIA (7/19) were the dominant ones. High-level aminoglycoside resistance could be transferred to E. coli J53 by conjugation with frequencies between 10⁻⁵ and 10⁻⁷ only from seven armA-positive donors through repeat attempts including using filter mating (data not shown). All the seven donor strains belonged to ST15. The transconjugants were highly resistant to amikacin, gentamicin, and tobramycin. The results of PCR and sequence analysis revealed that qnrS1, acc(6′)-Ib-cr, bla_TEM-1, bla_CMY-2, bla_NDM-1, and bla_CTX-M-15 were cotransferred with armA on plasmids to the recipients (Table 2). To analyze plasmids carrying the armA gene, plasmid DNA was prepared and transfer to E. coli DH5α by electroporation was attempted. armA-positive plasmids were successfully transferred by transformation from all the parental strains to the recipient E. coli DH5α strain. The transformants harbored a single plasmid with a size of ∼65 kb (data not shown). The plasmids of armA-positive isolates and their transconjugants were hybridized with the armA-specific probe (Fig. 1). The results of PCR typing revealed that the armA-carrying plasmids in this study all belonged to the IncA/C incompatibility group.

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FIG. 1.

The plasmid profile of the donors (A) and transconjugants (B); southern blot analysis of plasmid DNA of transconjugants (C). Lanes 1 to 7 show the plasmid profiles of strains Kpn14, Kpn20, Kpn29, Kpn33, Kpn34, Kpn35, and Kpn44, respectively. Lanes a and g show the plasmid profiles of transconjugant E. coli strains; lanes I and VII show the molecular probes specific for armA. Lane M, Escherichia coli 39R861. Chr, chromosomal DNA fragments.

Physical linkages between resistance genes and genetic elements

Table 3 illustrates selected examples of physical linkages between resistance genes and different genetic elements. Thirteen (68.4%) of the 19 armA were borne on strains containing the ISCR1 while 26.3% were borne on strains linked to the ISEcp1. However, only the ISCR1 element and sulI integrons were found upstream of rmtB. Close to 80% of isolates carrying bla_TEM-1 and bla_SHV-12 were physically linked to the IS26 as reported in Belgium and Germany (Table 3). In all cases, the ISEcp1 was the most common IS element associated with bla_CTX-M-14, bla_CTX-M-15, bla_OXA-1, and bla_CMY-2. Majority (81.3%) of qnr genes were physically linked to either integron-associated ISCR1 or the ISEcp1. In all cases tested, the qnrA1 gene was found immediately downstream of ISCR1 as part of a complex class 1 integron that included the 3′CS duplication. All isolates carrying aac(6′)-lb-cr or the qnr gene contained multiple genetic elements and were all MDR. ISAba125 was located upstream of the bla_NDM-1 gene and bla_CMY-2 gene.

MLST and ERIC-PCR

The 19 AmrA- or RmtB/CTX-M-producing isolates were evaluated by using the PubMLST (http://pubmlst.org/). By combining the seven gene loci, seven different STs (ST11, ST15, ST23, ST37, ST39, ST147, and ST395) were assigned for the isolates and the most dominant clone was ST15, with seven isolates (36.8%)—four belonged to ST395. The analysis of STs by eBURST (http://eburst.mlst.net/) showed that ST 11 and ST 395 are single-locus variants of ST 258. Of these isolates, seven belonged to CC14 and six belonged to CC11. These results mostly corresponded with the results of ERIC profile analysis, which revealed two clusters showing >60% similarity (clusters I and II). Cluster I comprised seven isolates belonging to CC14 and cluster II comprised six isolates belonging to CC11 (data not shown).