The Swiss wastewater isolate Klebsiella pneumoniae 704SK6, encoding OXA-48 and CTX-M-15 ß-lactamases, was fully sequenced. The assembly resulted in an open chromosome of 5,208,104 bp in size (G+C content, 57.6%) and four closed plasmid sequences of 209,651, 197,670, 65,998, and 63,605 bp in size. Copyright © 2017 Marti et al.
Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited.To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains.The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS.All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2?mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype.The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 µg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype. Copyright © 2017 American Society for Microbiology.
The diversity of OXA-48-like carbapenemases is continually expanding. In this study, we describe the dissemination and characteristics of a novel carbapenem-hydrolyzing class D carbapenemase (CHDL) named OXA-436. In total, six OXA-436-producing Enterobacteriaceae isolates including Enterobacter asburiae (n=3), Citrobacter freundii (n=2) and Klebsiella pneumoniae (n=1) were identified in four patients in the period between September 2013 and April 2015. All three species of OXA-436-producing Enterobacteriaceae were found in one patient. The amino acid sequence of OXA-436 showed 90.4-92.8% identity to other acquired OXA-48-like variants. Expression of OXA-436 in Escherichia coli and kinetic analysis of purified OXA-436 revealed an activity profile similar to OXA-48 and OXA-181 with activity against penicillins including temocillin, limited or no activity against extended-spectrum cephalosporins and activity against carbapenems. The blaOXA-436 gene was located on a conjugative ~314 kb IncHI2/IncHI2A plasmid belonging to pMLST ST1, in a region surrounded by chromosomal genes previously identified adjacent to blaOXA-genes in Shewanella spp. In conclusion, OXA-436 is a novel CHDL with similar functional properties as OXA-48-like CHDLs. The described geographical spread among different Enterobacteriaceae and plasmid location of blaOXA-436 illustrates its potential for further dissemination. Copyright © 2017 American Society for Microbiology.
Genetic determinants of a clinical Klebsiella pneumoniae isolate (KP1814) coproducing IMP-4 and a rare ESBL gene SFO-1 was investigated. KP1814 belongs to a novel sequence type (ST) assigned to ST2270. WGS identified four circular DNA sequences in KP1814, including two multidrug-resistance (MDR) plasmids, one virulence plasmid, and one circular form. The MDR plasmid pKP1814-1 (299.9 Kb) is untypeable, and carries two large mosaic multiresistance regions (MRRs). bla SFO-1 and bla IMP-4 co-exists on MRR1, and bla SFO-1 is associated with an IS/Tn-independent genetic context. bla IMP-4 is carried by a novel In804-like integron (intlI-bla IMP-4-Kl.pn.I3-qacG2-aacA4-catB3?) associated with a novel Tn1696-like transposon (designed Tn6404) flanked by IS5075. The other MDR plasmid pKP1814-3 is a 95,701-bp IncFII plasmid, and is a hybrid of a Shigella flexneri plasmid pSF07201 and an E. coli plasmid pCA08. All resistance genes of pKP1814-3 were detected in a ~16-kb IS26-flanked composite transposon carried by a Tn5396 transposon. The circular form (18.3 Kb) was composed of two parts belonging to pKP1814-1 and pKP1814-3, respectively. The plasmid pKP1814-2, carrying multiple virulence factors, encodes IncFIBK and IncFIIK replicons with a size of 187,349?bp. The coexistence of MDR and virulence plasmids largely enhances the bacterial fitness in the host and environment.
Carbapenem resistant Klebsiella pneumoniae (CR-KP) infections are an ever-increasing global issue, especially in the Indian subcontinent. Here we report genetic insight into a blaOXA-181 harbouring Klebsiella pneumoniae, belonging to the pandemic lineage ST147, that represents an intermediate stage towards pan-drug resistance. The CR-KP isolate DA48896 was isolated from a patient from Pakistan and was susceptible only to tigecycline and colistin. It harboured blaOXA-181 and was assigned to sequence type ST147. Analysis from whole genome sequencing revealed a very high sequence similarity to the previously sequenced pan-resistant K. pneumoniae isolate MS6671 from the United Arab Emirates. The two isolates are very closely related with only 46 chromosomal nucleotide differences, 14 indels and differences in plasmid content. Both carry a substantial number of plasmid-borne and chromosomally encoded resistance determinants. Interestingly, the two differences in susceptibility between the isolates could be attributed to DA48896 lacking an insertion of blaOXA-181 into the mgrB gene that results in colistin resistance in MS6671 and SNPs affecting AcrAB efflux pump expression likely to result in tigecycline resistance. These differences between the otherwise very similar isolates indicate that strong selection has occurred for resistance towards these last-resort drugs and illustrates the trajectory of resistance evolution of OXA-181-producing versions of the ST147 international risk clone.
Objective. Due to the fact that K. variicola, K. quasipneumoniae and K. pneumoniae are closely related bacterial species, misclassification can occur due to mistakes either in normal biochemical tests or during submission to public databases. The objective of this work was to identify K. variicola and K. quasipneumoniae genomes misclassified in GenBank database. Materials and methods. Both rpoB phylogenies and average nucleotide identity (ANI) were used to identify a significant number of misclassified Klebsiella spp. genomes. Results. Here we report an update of K. variicola and K. quasipneumoniae genomes correctly classified and a list of isolated genomes obtained from humans, plants, animals and insects, described originally as K. pneumoniae or K. variicola, but known now to be misclassified. Conclusions. This work contributes to recognize the extensive presence of K. variicola and K. quasipneumoniae isolates in diverse sites and samples.
Complete nucleotide sequences were determined for two plasmids bearing rmtD group 16S rRNA methyltransferase genes. pKp64/11 was 78 kb in size, belonged to the IncL/M group, and harbored blaTEM-1b, sul1, qacE?1, dfrA22, and rmtD1 across two multidrug resistance regions (MRRs). pKp368/10 was 170 kb in size, belonged to the IncA/C group, and harbored acrB, sul1, qacE?1, ant(3?)-Ia, aac(6′)-Ib, cat, rmtD2, and blaCTX-M-8 across three MRRs. The rmtD-containing regions shared a conserved motif, suggesting a common origin for the two rmtD alleles. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Klebsiella pneumoniae J1 is a Gram-negative strain, which belongs to a protein-based microbial flocculant-producing bacterium. However, little genetic information is known about this species. Here we carried out a whole-genome sequence analysis of this strain and report the complete genome sequence of this organism and its genetic basis for carbohydrate metabolism, capsule biosynthesis and transport system. Copyright © 2016. Published by Elsevier B.V.
The prevalence of Klebsiella pneumoniae coproducing carbapenemase metallo-ß-lactamase 1 (NDM-1) and OXA-48 has been increasing globally since 2013. The complete genome of KP617 was sequenced and assembled into a circular chromosome and two plasmids. This sequence provides the genetic background for understanding the evolution of carbapenemase genes in K. pneumoniae KP617.
The objective of this study was to conduct a comparative analysis with reported IncR plasmids of a Klebsiella pneumoniae IncR plasmid carrying an MDR region.MDR K. pneumoniae isolates were serially identified from two inpatients at a hospital in the USA in 2014. MDR plasmid pYDC676 was fully sequenced, annotated and compared with related plasmids. Antimicrobial susceptibility testing, PFGE and MLST were also conducted.The K. pneumoniae isolates were identical by PFGE, belonged to ST37 and harboured an identical ~50 kb IncR plasmid (pYDC676). pYDC676 possessed the backbone and multi-IS loci closely related to IncR plasmids reported from aquatic bacteria, as well as animal and human K. pneumoniae strains, and carried an MDR region consisting of armA, blaDHA-1 and qnrB4, a combination that has been reported in IncR plasmids from K. pneumoniae ST11 strains in Europe and Asia. A plasmid with the identical IncR backbone and a similar MDR region containing blaDHA-1 and qnrB4 has also been reported in ST37 strains from Europe, suggesting potential dissemination of this lineage of IncR plasmids in K. pneumoniae ST37.K. pneumoniae ST37 strains with an MDR IncR plasmid carrying armA, blaDHA-1 and qnrB4 were identified in a hospital in the USA, where these resistance genes remain rare. The IncR backbone may play a role in the global dissemination of these resistance genes.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Klebsiella pneumoniae U25 is a multidrug resistant strain isolated from a tertiary care hospital in Chennai, India. Here, we report the complete annotated genome sequence of strain U25 obtained using PacBio RSII. This is the first report of the whole genome of K. pneumoniaespecies from Chennai. It consists of a single circular chromosome of size 5,491,870-bp and two plasmids of size 211,813 and 172,619-bp. The genes associated with multidrug resistance were identified. The chromosome of U25 was found to have eight antibiotic resistant genes [blaOXA-1,blaSHV-28, aac(6′)1b-cr,catB3, oqxAB, dfrA1]. The plasmid pMGRU25-001 was found to have only one resistant gene (catA1) while plasmid pMGRU25-002 had 20 resistant genes [strAB, aadA1,aac(6′)-Ib, aac(3)-IId,sul1,2, blaTEM-1A,1B,blaOXA-9, blaCTX-M-15,blaSHV-11, cmlA1, erm(B),mph(A)]. A mutation in the porin OmpK36 was identified which is likely to be associated with the intermediate resistance to carbapenems in the absence of carbapenemase genes. U25 is one of the few K. pneumoniaestrains to harbour clustered regularly interspaced short palindromic repeats (CRISPR) systems. Two CRISPR arrays corresponding to Cas3 family helicase were identified in the genome. When compared to K. pneumoniaeNTUHK2044, a transposase gene InsH of IS5-13 was found inserted.
Carbapenem resistance in Klebsiella pneumoniae, frequently conferred by the blaKPC gene, is a major public health threat. We sequenced a blaKPC-containing strain of K. pneumoniae belonging to the emergent lineage ST941, in order to better understand the evolution of blaKPC within this species. Copyright © 2016 Sheppard et al.
blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1 and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A and ISCR1 may have been involved in acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones but different regions carrying blaNDM are found in different locations. Tn3-derived Inverted-repeat Transposable Elements (TIME) appear to have been involved in acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterisation of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements and plasmids and provide insights into the possible routes for transmission of blaNDM genes amongst species of the Enterobacteriaceae family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
The complete 284,628bp sequence of pH11, an IncHI2 plasmid, was determined through single-molecule, real-time (SMRT) sequencing. Harbored by a clinical Klebsiella pneumoniae strain H11, and isolated in Beijing, this plasmid contains multiple antibiotic resistance genes, including catA2, aac(6′)-Ib, strB, strA, dfrA19, blaTEM-1, blaSHV-12, sul1, qacE delta 1, ereA, arr2, and aac3. The aac(6′)-Ib is carried by a class I integron. Plasmid pH11 also carries several genes associated with resistance to heavy metals, such as tellurium, mercury, cobalt, zinc, nickel, copper, lead and cadmium. This plasmid exhibits numerous characteristics, including HipBA and RelBE toxin-antitoxin systems, two major transfer (Tra) regions closely related to those of Salmonella enterica serovar plasmid pRH-R27, a type II restriction modification system (EcoRII R-M system), several methyltransferases and methylases and genes encoding Hha and StpA. These characteristics suggest that pH11 may adapt to various hosts and environments. Multiple insertion sequence elements, transposases, recombinases, resolvases and integrases are scattered throughout pH11. The presence of these genes may indicate that horizontal gene transfer occurs frequently in pH11 and thus may facilitate the dissemination of antimicrobial resistance determinants. Our data suggest that pH11 is a chimera gradually assembled through the integration of different horizontally acquired DNA segments via transposition or homologous recombination. Copyright © 2016 Elsevier Inc. All rights reserved.
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