Exploring the Genome and Phenotype of Multi-Drug Resistant Klebsiella pneumoniae of Clinical Origin

Abstract

Klebsiella pneumoniae is an important nosocomial pathogen with an extraordinary resistant phenotype due to a combination of acquired resistant-elements and efflux mechanisms. In this study a detailed molecular characterization of 11 K. pneumoniae isolates of clinical origin was carried out. Eleven clinical isolates were tested for their susceptibilities, by disk diffusion and broth microdilution and interpreted according to CLSI guidelines. Efflux activity was determined by measuring the extrusion of ethidium bromide and biofilm formation was assessed following static growth in Müeller-Hinton and minimal media M9 broths at two temperatures and time points. Template DNA from all 11 isolates was extracted and sequenced. The study collection was found to be resistant to several (extended-spectrum beta-lactam) ESBL-type compounds along with several (fluoro)quinolones (FQ). Resistance to tetracycline accounted for 55% of the study collection (n = 6) and three of the 11 isolates were resistance to carbapenems. Genotyping identified bla_CTX-M-15 (82%), bla_SHV-12 (55%), and bla_TEM-1B (45%) ESBL encoding genes and FQ resistance was associated the presence of the oqxAB operon, identified in 10 of the 11 isolates and qnrB gene in one isolate. The polymorphisms detected in the quinolone resistance-determining regions (QRDRs) were associated with isolates of the clonal group CG15. Sequence types (ST) identified were representative of previously described clonal groups including CG258 (n = 7), CG15 (n = 3), and CG147 (n = 1). Plasmid replicon type databases were queried indicating the presence of IncFII and IncFIB replicon types in the majority of the isolates (91%), followed by IncFIA (45%), and IncR (45%). Two of the 11 isolates were found positive for yersiniabactin siderophore-encoding genes. No differences in the ability to efflux ethidium bromide were identified. Biofilm formation was stronger when the isolates were grown under stressed conditions at 37°C for a period up to 96 h. These data confirm the fact that well-recognized clonal groups of K. pneumoniae of importance to human health carries a diverse repertoire of antimicrobial resistance determinants, particularly related to critically important drugs in the ESBL and FQ classes. The capacity of most isolates to form strong biofilms, when stressed under laboratory-simulated conditions, supports the risk to human health associated with nosocomial infections deriving from indwelling medical devices.

Keywords: Klebsiella pneumonia; MDR phenotypes; antimicrobial resistance; biofilm formation; whole genome analysis.

Figure 1

Growth curve traces for all 11 Klebsiella pneumoniae isolates in Mueller-Hinton broth at 37°C with shaking at 200 rpm.

Figure 2

A dendrogram, constructed using the BioNumerics™ software (ver 7.5, Applied Maths), showing the antibiotic resistance profiles obtained following disk diffusion assays. The dendrogram shows two main clusters based on these resistance profiles. Breakpoints were interpreted according to CLSI guidelines 2013 with the following exceptions: Tigecycline, Moxifloxacin—the breakpoint used was that shown in the EUCAST guidelines 2016. Colistin and Polymyxin B—breakpoints were determined using the broth microdilution method and interpreted using EUCAST guidelines 2016. The following abbreviations apply to the listing compounds within drug classes: P, Penicillin; TIC, Ticarcillin; TIM, Ticarcillin-clavulanic acid; FEP, Cefepime; CTX, Cefotaxime; CAZ, Ceftazidime; ATM, Aztreonam; CN, Gentamicin; AK, Amikacin; DO, Doxycycline; MH, Minocycline; TE, Tetracycline; TGC, Tigecycline; CIP, Ciprofloxacin; LEV, Levofloxacin; MXF, Moxifloxacin; NA, Nalidixic Acid; NOR, Norfloxacin; DOR, Doripenem; ETP, Ertapenem; IPM, Imipenem; MEM, Meropenem; C, Chloramphenicol; CT, Colistin sulfate; PB, Polymyxin B; SXT, Trimethoprim-Sulfametoxazole.

Figure 3

Determination of the ethidium bromide efflux by a semi-automated fluorometric method. Klebsiella pneumoniae isolates were saturated with 50 μM ethidium bromide and efflux measured by fluorometry at 37°C for 50 min with an excitation and emission wavelengths of 518- and 606-nm, in the presence or absence of glucose and CCCP. Black arrows indicate when glucose (50 mM) was added after 3 min of initial reading.

Figure 4

Genomic analysis of Klebsiella pneumoniae clinical isolates. Antimicrobial resistance genes were grouped by class and the presence of at least one gene was considered as a positive result (black). The number of plasmids was identified from the gels (large plasmids >25 kb, small plasmids 2–25 kb).