Molecular epidemiology and genetic characterization of multi-drug resistant Klebsiella pneumoniae

Abstract

Multidrug resistant Klebsiella pneumoniae infections are a growing worldwide issue. Especially ESBL- producer and carbapenem resistant K. pneumoniae CRKP have been recognized as a hazard over the past decade. These MDR K. pneumoniae can cause serious infections, prolong hospital stay resulting in increased cost of the treatment and increased mortality. It is need of the hour to evaluate the presence of MDR K. pneumoniae in healthcare settings and understand the molecular epidemiology of these MDR K. pneumoniae to form strategies to decrease the overall burden of infections caused by these opportunistic bacteria. Current study was carried out to characterize the MDR K. pneumoniae encountered at a tertiary care hospital in Pakistan. For this purpose, 252 sequentially encountered K. pneumoniae received at the Microbiology laboratory at a tertiary care hospital in Islamabad were collected over a period of six months. The basic patient demographics such as gender, age, specimen and ward were recorded. Susceptibility profiles were determined using disk diffusion method against routinely used antibiotics. PCR amplifications were performed for ESBL genes, blaTEM, blaSHV and blaCTX-M. PCR amplifications were performed for carbapenemase genes, blaIMP, blaVIM, blaSPM, blaNDM, blaKPC, blaBIC, blaAIM, blaDIM, blaGIM and blaSIM. PCR amplifications were performed for PMQR genes, qnrA, qnrB, qnrS, qnrC, qnrD, qepA, oqxAB and aac(6′)-Ib-cr. Sequencing method was used to identify variants of ESBL and carbapenemase genes. PCR amplification and sequencing of porin genes with promotor regions of OmpK35 and OmpK36 of CRKP without any carbapenemase production was performed to identify variations. ERIC-PCR was performed and dendrograms were made using Gelcompar II, for the purpose of investigating the clonal relatedness among MDR isolates. Multilocus sequence typing was performed for seven loci for the determination of sequence types of a subgroup of isolates Page xvi encoding genes for carbapenemases. Plasmid replicon typing was performed for the carbapenemase producer K. pneumoniae. Plasmid stability testing was performed by serial passage without antibiotic selection for up to 100 generations to observe loss of carbapenemase encoding plasmid in MDR isolates. PCR amplification was used to confirm loss of plasmid. Cost of the plasmid was calculated in isolates with a loss of plasmid in stability testing. A subset of carbapenemase encoding isolates was selected for the whole genome sequencing using Illumina Miseq. The genetic environment of resistance markers was studies, identification of virulence markers, plasmid analysis and porin analysis using CLC genomic workbench. PacBio sequencing was performed for one isolate to study the genetic makeup of the strain. Susceptibility testing of Klebsiella pneumoniae showed a high prevalence of antibiotic resistance i.e. 78% MDR and 1% XDR. Highest resistance was observed against cephalosporins while colistin and tigecycline were found to be the most effective antibiotics in-vitro. Of all MDR isolates, 91% were ESBL positive and 55% of MDR K. pneumoniae encoded for carbapenemases. The most prevalent ESBL was found to be CTX-M-1 and the most prevalent carbapenemase was found to be NDM-1. Other ESBL identified included SHV- and TEM (IRT). Other carbapenemases included OXA-48-like and VIM-type. Variants included CTX-M-15, CTX-M-14, SHV-12, SHV-33, SHV-132, SHV-76, IRT (Inhibitor resistant TEM), NDM-1, OXA-48, OXA-181, VIM-1 and VIM-34. A high prevalence of PMQR i.e. 84% of MDR K. pneumoniae carried at least one PMQR gene while one isolate encoded for qnrD gene. The porin analysis of 15 CRKP not encoding carbapenemase showed that eleven of these either have a premature stop codon in one of the porins (n=7) or one of the porin genes couldn’t be amplified (due to loss of porin/ large sequence insertion). These disruptions may be the reason behind the carbapenem resistance. Previously described variants ompK36_v4 and ompK36_v5 were also found in two isolates. MDR isolates included five major clonal complexes comprising of 15-27 isolates, made up almost half (47%) of MDR isolates. The MLST of 33 K. pneumoniae gave 9 sequence types with 19 isolates belonging to CC11/CC258. ST29 and ST147 were found to be the major sequence types. Page xvii BlaNDM-1 was encoded by sequence types, ST29, ST147, ST437, ST340 and ST628. BlaOXA-48 was encoded by ST893 and ST43 while blaOXA-181 by ST147. Carbapenemase VIM- was encoded by ST147, ST37 and ST1787. Plasmid replicon typing of carbapenemase producers showed the genes encoded by a variety of Inc groups previously associated with these genes. The plasmid stability analysis showed that blaNDM-1 was stable in all isolates over 100 generations. On the other hand blaOXA-48 in one isolate and blaVIM-1 in two isolates was found to be unstable. Cost was observed for blaOXA-48 and one of the blaVIM-1 isolate. The cost for blaVIM-1 K. pneumoniae was statistically significant. All isolates selected for WGS carried a number of resistance markers with porin variations and encoded for a number of virulence markers predicting the pathogenic nature of these isolates. ICEs were found to be responsible for the transfer of resistance markers between isolates. A novel mutation Q336H was identified in PmrB gene of colistin resistant isolates. A novel variant of OXA-1 with Arg155Ile mutation was also identified. A variant of blaOXA-48 harboring plasmid pKPoxa-48N1 was identified. We were able to explore further the blaNDM-1 harboring plasmid pPN66-Ecl-NDM-1 identified previously in E. coli from Pakistan and a number of resistance markers including aacA4, sul1 and rmtC were identified upstream of blaNDM-1. A complete analysis of DA48996, a ST147 MDR K. pneumoniae was performed in comparison with a previously described PDR isolate MS6671 from UAE. The analysis showed that both isolates were very closely related with 46 individual differences between genomes and 14 structural variations. Important mutations in efflux pumps, MarA S50G and RcSC L60M were identified while two mutations were missing in DA48896, AcrR R18L and MarA V26D. This may have been the basis of difference in the resistance state of both isolates. DA48896 carried variants of 3 of the 5 plasmids (missing MS6671 plasmid A (LN824134) and C (LN824136)) of K. pneumoniae MS6671 but in addition also three plasmids not found in MS6671 A variant of plasmid pMS6671_E was identified as pDA48896_1 with >70 kbp insertion carrying multiple resistance markers. The variations between the two isolates could explain the transformation of MDR isolate to PDR isolate over time. Hence DA48896 represents an intermediate stage towards pan-drug resistance.