Panel strain of Klebsiella pneumoniae for beta-lactam antibiotic evaluation: their phenotypic and genotypic characterization

Bacterial isolates

K. pneumoniae isolates were collected during 2009 to 2013 from a tertiary care university affiliated hospital in Seoul, Korea. Bacteria identification was performed using VITEK 32 GN system (BioMérieux, Marcy l’Etoile, France), and was confirmed using the direct colony method with MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). These isolates were screened for specific phenotypes such as high level acquired penicillinase, over produced AmpC, ESBL, and carbapenemase based on Bonnet R, et al. in “Antibiogram” (LeClercq, Courvalin & Rice, 2010) and “From antibiogram to prescription” book (François et al., 2004) from the hospital database.

Susceptibility tests and MIC determinations

Around 3,000 K. pneumoniae isolates were shortlisted for antibiotic susceptibility test. This was performed by the disc diffusion method using piperacillin, ampicillin, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, meropenem, ciprofloxacin, ceftazidime-clavulanate, ampicillin-sulbactam, aztreonam on Mueller-Hinton agar. Further confirmatory tests were performed for the above antibiotics by agar-dilution technique. All the results were interpreted according to the Clinical and laboratory standards Institute (CLSI) guidelines (2015).

ESBL isolates were sorted out using double disk synergy test using cefepime and clavulanate and Hodge test using cefoxitin. Imipenem and EDTA double disk synergy along with Hodge test were used to select the carbapenemase-producing isolates. High level AmpC were selected using ertapenem and aminophenyl boronic acid (APBA) double disk synergy test.

Resistance gene confirmation

Phenotypically confirmed isolates were cultured overnight and suspended in distilled water, heated at 95 °C for 10 min. The suspension was centrifuged for 1 min at 6,000 rpm and the supernatant was used as a DNA template. Primers were designed for resistance genes (Table S1) and ordered from Macrogen (Seoul, Korea) and PCR was performed using accupower PCR premix (Bioneer, Korea).

DNA isolation

Random strains were picked from each resistance phenotypes and cultured overnight. Both genomic and plasmid DNA were isolated using Wizard genomic DNA purification kit (Promega, Madison, WI, USA) with little modification to the manufacturer’s protocol, and Qiaprep spin miniprep kit (Qiagen, Hilden, Germany), respectively. DNA concentration was estimated through Qubit dsDNA BR assay kit (Molecular Probes, Eugene, OR, USA).

Ion Torrent PGM sequencing

Whole genome library was performed using Ionplus fragment library kit (Life Technologies, Carlsbad, CA, USA). Emulsion PCR was carried out using the IonOnetouch 200 Template kit v2 DL (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Sequencing of the libraries was carried out on a 318 chip using the Ion Torrent PGM system and Ion Sequencing 200kit (Life Technologies, Carlsbad, CA, USA).

Sequence assembly, annotation, multilocus sequence typing (MLST) and resistome analysis

Reads from Ion Torrent PGM system were assembled using MIRA plug-in available in Torrent suite software. Annotations were performed using the RAST annotation pipeline with manual scrutiny. Genomic analysis was performed using Geneious pro 8.0 (http://www.geneious.com; Kearse et al., 2012). Resistance genes were screened using Resfinder (Zankari et al., 2012; https://cge.cbs.dtu.dk/services/ResFinder/) and they were further verified using NCBI BLAST. All the references used to annotate the resistance genes are listed in Table S2. Bacterial typing was performed using online tool MLST 1.8 (Zankari et al., 2013; https://cge.cbs.dtu.dk/services/MLST/).

OMP detection

Bacterial cells were grown in high-osmolarity MHB to the logarithmic phase and lysed by sonication at 18–20% amplitude for 2 × 30s cycles, each comprised 6 × 5s sonication steps separated by 1s of no sonication, and 30s of no sonication between the two cycles. Unbroken cells were separated using centrifugation at 3,000 g for 5 min and Outer membrane proteins (OMP) were extracted with Sodium lauroyl sarcosinate and recovered by ultracentrifugation, as described previously (Hernandez-Alles et al., 1999). The OMP profiles were determined using SDS-PAGE using Mini-Protean TGX gels followed by coomassie blue staining (Bio-Rad, Hercules, CA, USA). Additionally, OMP’s were detected using Matrix-Assisted Laser Desorption-Time of Flight Mass Spectrometry on Tinkerbell LT (ASTA, Suwon, Korea) as described in Cai et al. (2012). All the experiments were repeated thrice to check the reproducibility of the results.

High level acquired Penicillinase

K. pneumoniae YMC2011/8/B10311 was susceptible to piperacillin-tazobactam, ceftazidime, cefepime, imipenem, meropenem, ciprofloxacin, ceftazidime-clavulanate and cefoxitin but resistant to piperacillin, ampicillin, and ampicillin-sulbactam. WGS analysis shows the presence of bla_SHV-11 and bla_TEM-1 genes. Resistance to the piperacillin, ampicillin and ampicillin-sulbactam are due to hyperproduction of penicillinase TEM-1and SHV-11 beta- lactamase. OmpK35 gene was present while OmpK36 gene expression was truncated or terminated due to the mutations present as observed in the WGS. It was also confirmed using SDS-PAGE, which revealed OmpK35 porin alone.TEM-1 beta-lactamase offers resistance to α- amino and - carboxy - penicillins in E. coli and Enterobacteriaceae. Generally, in high level acquired penicillinase strains, there was increased production of TEM-1, which can be inhibited by piperacillin-tazobactam efficiently than the ampicillin-sulbactam combination (Livermore & Seetulsingh, 1991), as confirmed above.

Extended spectrum beta-lactamase

ESBL producing strains were phenotypically confirmed using the double disk and Hodge test. Most of these strains belonged to ST11, which are predominant in Korea since 2010 (Ko et al., 2010).

High level AmpC beta-lactamase

K. pneumoniae YMC 2010/8/B2027 was found to be susceptible to cefepime, imipenem and meropenem and intermediate to ciprofloxacin but resistant to piperacillin, piperacillin-tazobactam, ceftazidime, ceftazidime-clavulanate, cefoxitin, ampicillin and ampicillin-sulbactam. The resistance phenotype can be because of the presence of AmpC genes i.e.bla_DHA-1and bla_CMY-2. It also carries the broad spectrum beta-lactamase bla_SHV-11 andpenicillinase gene bla_TEM-1.Ciprofloxacin resistance has been conferred by the presence of aac(6′)Ib-cr, strA and strB.

Carbapenemases

K. pneumoniae YMC 2012/8/C631 was found to be susceptible to piperacillin-tazobactam and ciprofloxacin but resistant to piperacillin, ceftazidime, imipenem, meropenem, ceftazidime-clavulanate, cefoxitin, ampicillin and ampicillin-sulbactam. The cefoxitin resistance was due to the presence of bla_DHA-1. Even though IMP-1 enzyme is known to hydrolyze piperacillin and tazobactam, this strain appeared susceptible. Similar cases have been reported earlier (Chen et al., 2009; Koh, Wang & Sng, 2004; Mushtaq, Ge & Livermore, 2004; Picao et al., 2013; Santella et al., 2010; Scheffer et al., 2010), which may be due to inherent susceptibility to the particular antibiotics. Other resistances are conferred due to the presence of 3 copies of bla_TEM-1 and bla_SHV-11. While SDS-PAGE revealed the presence of OmpK35, in silico analysis of OmpK35 gene showed the insertion of IS102, thus affecting antibiotic passage through the membrane.

In addition, as opposed to the findings by Zankari et al. (2012) we found few discrepancy in the identification of antimicrobial resistance genes by using Resfinder using the whole genome sequencing, such as few genes were identified as bla_LEN-11 instead of bla_SHV-11/bla_SHV-12 in the above panel strains. Hence the above panel strains needed to be further evaluated for accurate identification. This software consists of database of resistance genes, which helps us to easily identify the resistance mechanism. However, manual scrutiny of the Resfinder results is essential to identify the true antibiotic resistance genes present in the pathogen. Figure S4 indicates the encoded amino acid alignment of the SHV type genes found in the panel strains. bla_SHV-11 is a broad spectrum β-lactamase gene, encodes 286 amino acid, in which the mutations Gly₂₃₄-Serine₂₃₄ and Glu₂₃₅-Lys₂₃₅ results in SHV-12 β-lactamase, Gln₃₁-Leu₃₁ results in SHV-187 β-lactamase and Thr₅₄-Ala₅₄ leads to SHV-158 β-lactamase.

The advent of NGS in clinical laboratory field has helped us to gain better insights of resistant mechanisms in detail compared to the traditional phenotypic detection. The analysis of WGS will also help us to understand the collective molecular network of pathogen offering the specific MIC with the relevant antibiotics (Tsai et al., 2013). In addition, it also plays an important role in clarifying the discrepancy observed due to false negative results generated from existing diagnostic assays (Koser, Ellington & Peacock, 2014). These assays mostly target the single resistance mechanism or phenotype, which is not sufficient to understand the complete underlying mechanism. Using WGS to predict the antibiotic resistance has demonstrated sensitivity and specificity of 96% and 97% respectively, compared to the phenotypic detection assays (Goldberg et al., 2015).