Whole-Genome Sequences of Two Carbapenem-Resistant Klebsiella quasipneumoniae Strains Isolated from a Tertiary Hospital in Johor, Malaysia.
ABSTRACT: We report the whole-genome sequences of two carbapenem-resistant clinical isolates of Klebsiella quasipneumoniae subsp. similipneumoniae obtained from two different patients. Both strains contained three different extended-spectrum β-lactamase genes and showed strikingly high pairwise average nucleotide identity of 99.99% despite being isolated 3 years apart from the same hospital.
Project description:Klebsiella quasipneumoniae is an emerging pathogen in human medicine. We report draft genome sequences of NDM-1- and KPC-2-producing K. quasipneumoniae strains from inpatients in Brazil. K. quasipneumoniae subsp. quasipneumoniae and K. quasipneumoniae subsp. similipneumoniae harbored broad resistomes. These data could contribute to a better understanding of acquired resistance in K. quasipneumoniae.
Project description:The aim of this study was to unravel the genetic determinants responsible for multidrug (including carbapenems) resistance and virulence in a clinical isolate of Klebsiella quasipneumoniae subsp. similipneumoniae by whole-genome sequencing and comparative analyses. Eighty-three clinical isolates initially identified as carbapenem-resistant K. pneumoniae were collected from nosocomial infections in southeast Brazil. After RAPD screening, the KPC-142 isolate, showing the most divergent DNA pattern, was selected for complete genome sequencing in an Illumina HiSeq 2500 instrument. Reads were assembled into scaffolds, gaps between scaffolds were resolved by in silico gap filling and extensive bioinformatics analyses were performed, using multiple comparative analysis tools and databases. Genome sequencing allowed to correct the classification of the KPC-142 isolate as K. quasipneumoniae subsp. similipneumoniae. To the best of our knowledge this is the first complete genome reported to date of a clinical isolate of this subspecies harboring both class A beta-lactamases KPC-2 and OKP-B-6 from South America. KPC-142 has one 5.2 Mbp chromosome (57.8% G+C) and two plasmids: 190 Kbp pKQPS142a (50.7% G+C) and 11 Kbp pKQPS142b (57.3% G+C). The 3 Kbp region in pKQPS142b containing the blaKPC-2 was found highly similar to that of pKp13d of K. pneumoniae Kp13 isolated in Southern Brazil in 2009, suggesting the horizontal transfer of this resistance gene between different species of Klebsiella. KPC-142 additionally harbors an integrative conjugative element ICEPm1 that could be involved in the mobilization of pKQPS142b and determinants of resistance to other classes of antimicrobials, including aminoglycoside and silver. We present the completely assembled genome sequence of a clinical isolate of K. quasipneumoniae subsp. similipneumoniae, a KPC-2 and OKP-B-6 beta-lactamases producer and discuss the most relevant genomic features of this important resistant pathogen in comparison to several strains belonging to K. quasipneumoniae subsp. similipneumoniae (phylogroup II-B), K. quasipneumoniae subsp. quasipneumoniae (phylogroup II-A), K. pneumoniae (phylogroup I), and K. variicola (phylogroup III). Our study contributes to the description of the characteristics of a novel K. quasipneumoniae subsp. similipneumoniae strain circulating in South America that currently represent a serious potential risk for nosocomial settings.
Project description:Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603, formerly known as K. pneumoniae K6, is known for producing extended-spectrum ?-lactamase (ESBL) enzymes that can hydrolyze oxyimino-?-lactams, resulting in resistance to these drugs. We herein report the complete genome of strain ATCC 700603 and show that the ESBL genes are plasmid-encoded.
Project description:Klebsiella pneumoniae is a Gram-negative organism and a major public health threat. In this study, we used whole-genome sequences to characterize 32 carbapenem-resistant K. pneumoniae (CRKP) and two carbapenem-resistant K. quasipneumoniae (CRKQ). Antimicrobial resistance was assessed using disk diffusion and E-test, while virulence was assessed in silico. The capsule type was determined by sequencing the wzi gene. The plasmid diversity was assessed by PCR-based replicon typing to detect the plasmid incompatibility (Inc) groups. The genetic relatedness was determined by multilocus sequence typing, pan-genome, and recombination analysis. All of the isolates were resistant to ertapenem together with imipenem and/or meropenem. Phenotypic resistance was due to blaOXA-48, blaNDM-1, blaNDM-7, or the coupling of ESBLs and outer membrane porin modifications. This is the first comprehensive study reporting on the WGS of CRKP and the first detection of CRKQ in the region. The presence and dissemination of CRKP and CRKQ, with some additionally having characteristics of hypervirulent clones such as the hypermucoviscous phenotype and the capsular type K2, are particularly concerning. Additionally, mining the completely sequenced K. pneumoniae genomes revealed the key roles of mobile genetic elements in the spread of antibiotic resistance and in understanding the epidemiology of these clinically significant pathogens.
Project description:Background:Nosocomial infections caused by multi-drug resistant Enterobacteriaceae are a global public health threat that ought to be promptly identified, reported, and addressed accurately. Many carbapenem-resistant Enterobacteriaceae-associated genes have been identified in Saudi Arabia but not the endemic Klebsiella pneumoniae carbapenemases (KPCs), which are encoded by bla KPC-type genes. KPCs are known for their exceptional spreading potential. Methods:We collected n?=?286 multi-drug resistant (MDR) Klebsiella spp. isolates as part of screening for resistant patterns from a tertiary hospital in Saudi Arabia between 2014 and 2018. Antimicrobial susceptibility testing was carried out using both VITEK II and the broth microdilution of all collected isolates. Detection of resistance-conferring genes was carried out using Illumina whole-genome shotgun sequencing and PacBio SMRT sequencing protocols. Results:A Carbapenem-resistant Enterobacteriaceae (CRE) Klebsiella quasipneumoniae subsp. similipneumoniae strain was identified as a novel ST-3510 carrying a bla KPC-2 carbapenemase encoding gene. The isolate, designated as NGKPC-421, was obtained from shotgun Whole Genome Sequencing (WGS) surveillance of 286 MDR Klebsiella spp. clinical isolates. The NGKPC-421 isolate was collected from a septic patient in late 2017 and was initially misidentified as K. pneumoniae. The sequencing and assembly of the NGKPC-421 genome resulted in the identification of a putative ~?39.4?kb IncX6 plasmid harboring a bla KPC-2 gene, flanked by transposable elements (ISKpn6-bla KPC-2-ISKpn27). Conclusion:This is the first identification of a KPC-2-producing CRE in the Gulf region. The impact on this finding is of major concern to the public health in Saudi Arabia, considering that it is the religious epicenter with a continuous mass influx of pilgrims from across the world. Our study strongly highlights the importance of implementing rapid sequencing-based technologies in clinical microbiology for precise taxonomic classification and monitoring of antimicrobial resistance patterns.
Project description:A clinical isolate of extended-spectrum-β-lactamase-producing Klebsiella quasipneumoniae subsp. similipneumoniae 06-219 with hypermucoviscosity phenotypes obtained from a urine culture of an adult patient was used for whole-genome sequencing. Here, we report the draft genome sequences of this strain, consisting of 53 contigs with an ~5.6-Mb genome size and an average G+C content of 57.36%. The annotation revealed 6,622 coding DNA sequences and 77 tRNA genes.
Project description:Klebsiella pneumoniae (phylogroup Kp1), one of the most problematic pathogens associated with antibiotic resistance worldwide, is phylogenetically closely related to K. quasipneumoniae [subsp. quasipneumoniae (Kp2) and subsp. similipneumoniae (Kp4)], K. variicola (Kp3) and two unnamed phylogroups (Kp5 and Kp6). Together, Kp1 to Kp6 make-up the K. pneumoniae complex. Currently, the phylogroups can be reliably identified only based on gene (or genome) sequencing. Misidentification using standard laboratory methods is common and consequently, the clinical significance of K. pneumoniae complex members is imprecisely defined. Here, we evaluated and validated the potential of MALDI-TOF mass spectrometry (MS) to discriminate K. pneumoniae complex members. We detected mass spectrometry biomarkers associated with the phylogroups, with a sensitivity and specificity ranging between 80-100% and 97-100%, respectively. Strains within phylogroups Kp1, Kp2, Kp4, and Kp5 each shared two specific peaks not observed in other phylogroups. Kp3 strains shared a peak that was only observed otherwise in Kp5. Finally, Kp6 had a diagnostic peak shared only with Kp1. Kp3 and Kp6 could therefore be identified by exclusion criteria (lacking Kp5 and Kp1-specific peaks, respectively). Further, ranked Pearson correlation clustering of spectra grouped strains according to their phylogroup. The model was tested and successfully validated using different culture media. These results demonstrate the potential of MALDI-TOF MS for precise identification of K. pneumoniae complex members. Incorporation of spectra of all K. pneumoniae complex members into reference MALDI-TOF spectra databases, in which they are currently lacking, is desirable. MALDI-TOF MS may thereby enable a better understanding of the epidemiology, ecology, and pathogenesis of members of the K. pneumoniae complex.
Project description:During March 2017, a neonatal patient with severe diarrhoea subsequently developed septicaemia and died, with Klebsiella isolated as the causative microorganism. In keeping with infection control protocols, the coincident illness of an attending staff member and three other neonates with Klebsiella infection triggered an outbreak response, leading to microbiological assessment of isolates collected from the staff member and all 21 co-housed neonates. Multilocus sequence typing and genomic sequencing identified that the isolates from the 21 neonates were of a new Klebsiella sequence type, ST2727, and taxonomically belonged to K. quasipneumoniae subsp. similipneumoniae (formerly referred to as KpIIB). Genomic characterization showed that the isolated ST2727 strains had diverged from other K. quasipneumoniae subsp. similipneumoniae strains at least 90 years ago, whereas the neonatal samples were highly similar with a genomic divergence of 3.6?months. There was no relationship to the Klebsiella isolate from the staff member. This demonstrates that no transmission occurred from staff to patient or between patients. Rather, the data suggest that ST2727 colonized each neonate from a common hospital source. Sequence-based analysis of the genomes revealed several genes for antimicrobial resistance and some virulence features, but suggest that ST2727 is neither extremely-drug resistant nor hypervirulent. Our results highlight the clinical significance and genomic properties of ST2727 and urge genome-based measures be implemented for diagnostics and surveillance within hospital environments. Additionally, the present study demonstrates the need to scale the power of genomic analysis in retrospective studies where relatively few samples are available.
Project description:We report a case of pyogenic liver abscess caused by community-acquired Klebsiella quasipneumoniae subsp. quasipneumoniae. The infecting isolate had 2 prominent features of hypervirulent K. pneumoniae strains: the capsular polysaccharide synthesis region for K1 serotype and the integrative and conjugative element ICEKp1, which encodes the virulence factors yersiniabactin, salmochelin, and RmpA.
Project description:The continuous emergence of carbapenemase-producing Enterobacteriaceae (CPE) presents a great public health challenge. Mitigation of CPE spread in the environment is crucial, particularly from a One Health perspective. Here we describe the isolation of CPE strain SNI47 from influent water of a sewage treatment plant in Japan. SNI47 was identified as Klebsiella quasipneumoniae subsp. quasipneumoniae by phylogenetic analysis and was resistant to ?-lactams, including carbapenems. Of four plasmids detected from SNI47, the 185,311-bp IncA/C2 plasmid (pTMSNI47-1), which carried 10 drug resistance genes, including genes for four ?-lactamases (bla CTX-M-2, bla DHA-1, bla KHM-1, and bla OXA-10), was transferred to Escherichia coli J53 via conjugation. The MICs of all tested ?-lactams for the transconjugant were higher than for the recipient. We constructed recombinant plasmids, into which each ?-lactamase gene was inserted, and used them to transform E. coli DH5? cells, demonstrating that KHM-1 enhanced carbapenem resistance. In addition, these ?-lactamases were responsible for a wide-spectrum ?-lactam resistance acquisition with mutual compensation. KHM-1, recognized as a rare type of metallo-?-lactamase, was detected in a transferable plasmid, from a sewage treatment plant, involved in horizontal gene transfer. The detection of such plasmids raises a health risk alarm for CPE dissemination.IMPORTANCE In our investigation of urban wastewater in Japan, carbapenem-resistant Klebsiella quasipneumoniae subsp. quasipneumoniae was isolated that carried the pTMSNI47-1 plasmid, which carries four ?-lactamase genes and has transferability among Enterobacteriaceae pTMSNI47-1 was found to encode a rarely reported carbapenemase, KHM-1. Cooperative effects of ?-lactamases encoded by pTMSNI47-1 appeared to have broad-spectrum resistance to ?-lactams. The detection of the KHM-1 gene in urban wastewater suggests that such a rare antimicrobial resistance (AMR) gene can be pooled in the environment, potentially emerging as an AMR determinant in a pathogen. When the number of ?-lactamase resistance genes is increased in one plasmid, the transfer of this plasmid can confer broad-spectrum resistance to ?-lactams, even if the individual gene confers narrow-spectrum resistance. The present study adds important information about the potential risk of sewage treatment plants as reservoirs and environmental suppliers of AMR genes, contributing to the public health from a One Health perspective.