Characterization of BKC-1 Class A Carbapenemase from Klebsiella pneumoniae Clinical Isolates in Brazil

ABSTRACTThreeKlebsiella pneumoniaeclinical isolates demonstrating carbapenem resistance were recovered from different patients hospitalized at two medical centers in São Paulo, Brazil. Resistance to all β-lactams, quinolones, and some aminoglycosides was observed for these isolates that were susceptible to polymyxin B. Carbapenem hydrolysis, which was inhibited by clavulanic acid, was observed for allK. pneumoniaeisolates that belonged to the same pulsed-field gel electrophoresis (PFGE) type and a novel sequence type (ST), ST1781 (clonal complex 442 [CC442]). A 10-kb nonconjugative incompatibility group Q (IncQ) plasmid, denominated p60136, was transferred toEscherichia colistrain TOP10 cells by electroporation. The full sequencing of p60136 showed that it was composed of a mobilization system, ISKpn23, the phosphotransferase aph3A-VI, and a 941-bp open reading frame (ORF) that codified a 313-amino acid protein. This ORF was namedblaBKC-1. BrazilianKlebsiellacarbapenemase-1 (BKC-1) showed a pI of 6.0 and possessed the highest identity (63%) with a β-lactamase ofSinorhizobium meliloti, an environmental bacterium. Hydrolysis studies demonstrated that purified BKC-1 not only hydrolyzed carbapenems but also penicillins, cephalosporins, and monobactams. However, the carbapenems were less efficiently hydrolyzed due to their very lowkcatvalues (0.0016 to 0.031 s−1). In fact, oxacillin was the best substrate for BKC-1 (kcat/Km, 53,522.6 mM−1s−1). Here, we report a new class A carbapenemase, confirming the diversity and rapid evolution of β-lactamases inK. pneumoniaeclinical isolates.

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Molecular and Epidemiological Characteristics of Carbapenemase-Producing Klebsiella pneumoniae Clinical Isolates in Japan

mSphere ◽

10.1128/msphere.00490-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Shinsuke Yonekawa ◽

Tomoki Mizuno ◽

Ryuichi Nakano ◽

Akiyo Nakano ◽

Yuki Suzuki ◽

...

Keyword(s):

Public Health ◽

Klebsiella Pneumoniae ◽

Virulence Genes ◽

Carbapenem Resistance ◽

Clinical Isolates ◽

Molecular Characteristics ◽

Content Type ◽

Health Threat ◽

Public Health Threat ◽

Epidemiological Characteristics

ABSTRACT Carbapenemase-producing Enterobacteriaceae represent a serious public health threat worldwide. Carbapenemase genes, harbored on a transferable plasmid, have been isolated globally with distinct geographical features. Klebsiella pneumoniae, included in Enterobacteriaceae, also produces carbapenemase and often shows hypervirulence. Overlapping carbapenem resistance and hypervirulence in K. pneumoniae have been reported, but such strains have not yet been found in Japan. Here, we screened 104 carbapenemase-producing K. pneumoniae isolates collected from 37 hospitals and outpatient clinics in Japan between September 2014 and July 2015. PCR and DNA sequencing demonstrated IMP-1 in 21 isolates and IMP-6 in 83 isolates, 77 of which coharbored CTX-M-2. Most of the isolates showed low MICs toward imipenem and meropenem but high MICs toward penicillin and cephalosporins. Conjugation experiments with an Escherichia coli J53 recipient showed that most of the plasmids in IMP-6 producers were transferable, whereas only one-half of the plasmids in IMP-1 producers were transferable. PCR-based replicon typing and multiplex PCR identified five isolates belonging to the CG258 non-tonB79 cluster and no isolate belonging to the CG258-tonB79 cluster or sequence type 307 (ST307). Four K1-ST23 isolates, 10 K2-ST65 isolates, and 7 K2-ST86 isolates were detected that harbored virulence genes. The resistance genes in 85 isolates were transferable, but the virulence genes were not transferred. These results demonstrate the acquisition of IMP-type carbapenemase genes and CTX-M-type genes among hypervirulence isolates in Japan, warranting further attention and countermeasures. In this study, we have determined the molecular characteristics and epidemiology of IMP-6 producers that coharbored various CTX-M genes in Japan. IMPORTANCE Carbapenems serve as a last resort for the clinical treatment of multidrug-resistant infections. Therefore, the rapid spread of carbapenemase-producing strains represents a serious public health threat, further limiting antibiotic choices. The current findings of hypervirulent carbapenemase-producing Klebsiella pneumoniae clinical isolates in Japan demonstrate the potential broad spread and transfer of these genes, necessitating close surveillance.

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Network Integrative Genomic and Transcriptomic Analysis of Carbapenem-ResistantKlebsiella pneumoniaeStrains Identifies Genes for Antibiotic Resistance and Virulence

mSystems ◽

10.1128/msystems.00202-19 ◽

2019 ◽

Vol 4 (4) ◽

Cited By ~ 5

Author(s):

Muyoung Lee ◽

Naina Adren Pinto ◽

Chan Yeong Kim ◽

Sunmo Yang ◽

Roshan D’Souza ◽

...

Keyword(s):

Antibiotic Resistance ◽

Klebsiella Pneumoniae ◽

Carbapenem Resistance ◽

Clinical Isolates ◽

Functional Modules ◽

Antibiotic Resistant ◽

Content Type ◽

Functional Association ◽

Carbapenem Resistant ◽

Tract Infections

ABSTRACTGlobal increases in the use of carbapenems have resulted in several strains of Gram-negative bacteria acquiring carbapenem resistance, thereby limiting treatment options.Klebsiella pneumoniaeis a common carbapenem-resistant pathogenic bacterium that is widely studied to identify novel antibiotic resistance mechanisms and drug targets. Antibiotic-resistant clinical isolates generally harbor many genetic alterations, and the identification of responsible mutations would provide insights into the molecular mechanisms of antibiotic resistance. We propose a method to prioritize mutated genes responsible for antibiotic resistance on the basis of expression changes in their local subnetworks, hypothesizing that mutated genes that show significant expression changes among the corresponding functionally associated genes are more likely to be involved in the carbapenem resistance. For network-based gene prioritization, we developed KlebNet (www.inetbio.org/klebnet), a genome-scale cofunctional network ofK. pneumoniaegenes. Using KlebNet, we reconstructed the functional modules for carbapenem resistance and virulence and identified the functional association between antibiotic resistance and virulence. Using complementation assays with the top candidate genes, we were able to validate a novel gene that negatively regulated carbapenem resistance and four novel genes that positively regulated virulence inGalleria mellonellalarvae. Therefore, our study demonstrated the feasibility of network-based identification of genes required for antibiotic resistance and virulence of human-pathogenic bacteria.IMPORTANCEKlebsiella pneumoniaeis a major bacterial pathogen that causes pneumonia and urinary tract infections in human.K. pneumoniaeinfections are treated with carbapenem, but carbapenem-resistantK. pneumoniaehas been spreading worldwide. We are able to identify antimicrobial-resistant genes among mutated genes of the antibiotic-resistant clinical isolates. However, they usually harbor many mutated genes, including those that cause weak or neutral functional effects. Therefore, we need to prioritize the mutated genes to identify the more likely candidates for the follow-up functional analysis. For this study, we present a functional network ofK. pneumoniaegenes and propose a network-based method of prioritizing the mutated genes of the resistant clinical isolates. We also reconstructed the network-based functional modules for carbapenem resistance and virulence and retrieved the functional association between antibiotic resistance and virulence. This study demonstrated the feasibility of network-based analysis of clinical genomics data for the study ofK. pneumoniaeinfection.

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Successive Emergence of Ceftazidime-Avibactam Resistance through Distinct Genomic Adaptations in bla KPC-2 -Harboring Klebsiella pneumoniae Sequence Type 307 Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02101-17 ◽

2017 ◽

Vol 62 (3) ◽

Cited By ~ 48

Author(s):

Marla J. Giddins ◽

Nenad Macesic ◽

Medini K. Annavajhala ◽

Stephania Stump ◽

Sabrina Khan ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Rapid Evolution ◽

Carbapenem Resistance ◽

Sequence Type ◽

Content Type ◽

Carbapenem Resistant ◽

Oxford Nanopore ◽

Long Read ◽

Stepwise Evolution ◽

Emergence Of Resistance

ABSTRACT Ceftazidime-avibactam (CAZ-AVI) is a promising novel treatment for infections caused by carbapenem-resistant Enterobacteriaceae (CRE). Despite improved treatment outcomes compared to those achieved with aminoglycoside- and colistin-based regimens, the rapid evolution of CAZ-AVI resistance during treatment has previously been reported in Klebsiella pneumoniae sequence type 258 (ST258) bla KPC-3 -harboring isolates. Here, we report the stepwise evolution and isolation of two phenotypically distinct CAZ-AVI-resistant Klebsiella pneumoniae isolates from a patient with pancreatitis. All susceptible ( n = 3) and resistant ( n = 5) isolates were of the ST307 clonal background, a rapidly emerging clone. Taking advantage of short-read Illumina and long-read Oxford Nanopore sequencing and full-length assembly of the core chromosome and plasmids, we demonstrate that CAZ-AVI resistance first occurred through a 532G → T bla KPC-2 point mutation in bla KPC-2 (D179Y protein substitution) following only 12 days of CAZ-AVI exposure. While subsequent isolates exhibited substantially decreased meropenem (MEM) MICs (≤2 μg/ml), later cultures demonstrated a second CAZ-AVI resistance phenotype with a lower CAZ-AVI MIC (12 μg/ml) but also MEM resistance (MIC > 128 μg/ml). These CAZ-AVI- and MEM-resistant isolates showed evidence of multiple genomic adaptations, mainly through insertions and deletions. This included amplification and transposition of wild-type bla KPC-2 into a novel plasmid, an IS 1 insertion upstream of ompK36 , and disruption of the rfb gene locus in these isolates. Our findings illustrate the potential of CAZ-AVI resistance to emerge in non- K. pneumoniae ST258 clonal backgrounds and alternative bla KPC variants. These results raise concerns about the strong selective pressures incurred by novel carbapenemase inhibitors, such as avibactam, on isolates previously considered invulnerable to CAZ-AVI resistance. There is an urgent need to further characterize non-KPC-mediated modes of carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance during treatment.

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Genome Sequences of Five Clinical Isolates of Klebsiella pneumoniae

Genome Announcements ◽

10.1128/genomea.00040-16 ◽

2016 ◽

Vol 4 (2) ◽

Cited By ~ 3

Author(s):

L. Letti Lopez ◽

Brigida Rusconi ◽

Heidi Gildersleeve ◽

Chao Qi ◽

Milena McLaughlin ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Broad Spectrum ◽

Carbapenem Resistance ◽

Clinical Isolates ◽

Nosocomial Pathogen ◽

Genome Sequences ◽

Class A ◽

Carbapenem Resistant ◽

Broad Spectrum Antibiotics

Klebsiella pneumoniae is a nosocomial pathogen of emerging importance and displays resistance to broad-spectrum antibiotics, such as carbapenems. Here, we report the genome sequences of five clinical K. pneumoniae isolates , four of which are carbapenem resistant. Carbapenem resistance is conferred by hydrolyzing class A β-lactamases found adjacent to transposases.

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Contribution of β-Lactamases and Porin Proteins OmpK35 and OmpK36 to Carbapenem Resistance in Clinical Isolates of KPC-2-Producing Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02045-12 ◽

2013 ◽

Vol 58 (2) ◽

pp. 1214-1217 ◽

Cited By ~ 41

Author(s):

Ying Zhang ◽

Xiaofei Jiang ◽

Yanyan Wang ◽

Gang Li ◽

Yueru Tian ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Carbapenem Resistance ◽

Clinical Isolates ◽

Content Type ◽

Carbapenem Resistant ◽

Sds Page ◽

Porin Proteins ◽

A Minor ◽

Sequence Types ◽

M 14

ABSTRACTFifty-seven carbapenem-resistantKlebsiella pneumoniaeisolates belonging to ST11 (50 isolates), ST423 (5 isolates), and two other sequence types were studied. All were positive forblaKPC-2,blaTEM-1, andblaCTX-M-14. SDS-PAGE analysis of six representative isolates demonstrated varied porin expression. Nevertheless, whenblaKPC-2was deleted, carbapenem resistance was markedly reduced. Additionally, SHV-12, DHA-1, and/or VIM-1 appeared to contribute to accessory carbapenemase activity. In contrast, OmpK35 and/or OmpK36 deficiency seemed to serve only as a minor cooperative factor.

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Comparative Genomics of Klebsiella pneumoniae Strains with Different Antibiotic Resistance Profiles

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00052-11 ◽

2011 ◽

Vol 55 (9) ◽

pp. 4267-4276 ◽

Cited By ~ 48

Author(s):

Vinod Kumar ◽

Peng Sun ◽

Jessica Vamathevan ◽

Yong Li ◽

Karen Ingraham ◽

...

Keyword(s):

Antibiotic Resistance ◽

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Susceptible Strain ◽

Clinical Isolates ◽

Whole Genome ◽

Content Type ◽

Extended Spectrum

ABSTRACTThere is a global emergence of multidrug-resistant (MDR) strains ofKlebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. While the epidemiology ofK. pneumoniaestrains and occurrences of specific antibiotic resistance genes, such as plasmid-borne extended-spectrum β-lactamases (ESBLs), have been extensively studied, only four complete genomes ofK. pneumoniaeare available. To better understand the multidrug resistance factors inK. pneumoniae, we determined by pyrosequencing the nearly complete genome DNA sequences of two strains with disparate antibiotic resistance profiles, broadly drug-susceptible strain JH1 and strain 1162281, which is resistant to multiple clinically used antibiotics, including extended-spectrum β-lactams, fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles. Comparative genomic analysis of JH1, 1162281, and other publishedK. pneumoniaegenomes revealed a core set of 3,631 conserved orthologous proteins, which were used for reconstruction of whole-genome phylogenetic trees. The close evolutionary relationship between JH1 and 1162281 relative to otherK. pneumoniaestrains suggests that a large component of the genetic and phenotypic diversity of clinical isolates is due to horizontal gene transfer. Using curated lists of over 400 antibiotic resistance genes, we identified all of the elements that differentiated the antibiotic profile of MDR strain 1162281 from that of susceptible strain JH1, such as the presence of additional efflux pumps, ESBLs, and multiple mechanisms of fluoroquinolone resistance. Our study adds new and significant DNA sequence data onK. pneumoniaestrains and demonstrates the value of whole-genome sequencing in characterizing multidrug resistance in clinical isolates.

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Dynamics of blaKPC-2 Dissemination from Non-CG258 Klebsiella pneumoniae to Other Enterobacterales via IncN Plasmids in an Area of High Endemicity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01743-20 ◽

2020 ◽

Vol 64 (12) ◽

Cited By ~ 1

Author(s):

Ana M. Rada ◽

Elsa De La Cadena ◽

Carlos Agudelo ◽

Cesar Capataz ◽

Nataly Orozco ◽

...

Keyword(s):

Public Health ◽

Klebsiella Pneumoniae ◽

Neonatal Intensive Care ◽

Carbapenem Resistance ◽

Health Interventions ◽

Global Public Health ◽

Public Health Interventions ◽

Content Type ◽

Carbapenem Resistant ◽

Long Read

ABSTRACT Carbapenem-resistant Enterobacterales (CRE) pose a significant threat to global public health. The most important mechanism for carbapenem resistance is the production of carbapenemases. Klebsiella pneumoniae carbapenemase (KPC) represents one of the main carbapenemases worldwide. Complex mechanisms of blaKPC dissemination have been reported in Colombia, a country with a high endemicity of carbapenem resistance. Here, we characterized the dynamics of dissemination of blaKPC gene among CRE infecting and colonizing patients in three hospitals localized in a highly endemic area of Colombia (2013 and 2015). We identified the genomic characteristics of KPC-producing Enterobacterales recovered from patients infected/colonized and reconstructed the dynamics of dissemination of blaKPC-2 using both short and long read sequencing. We found that spread of blaKPC-2 among Enterobacterales in the participating hospitals was due to intra- and interspecies horizontal gene transfer (HGT) mediated by promiscuous plasmids associated with transposable elements that was originated from a multispecies outbreak of KPC-producing Enterobacterales in a neonatal intensive care unit. The plasmids were detected in isolates recovered in other units within the same hospital and nearby hospitals. The gene “epidemic” was driven by IncN-pST15-type plasmids carrying a novel Tn4401b structure and non-Tn4401 elements (NTEKPC) in Klebsiella spp., Escherichia coli, Enterobacter spp., and Citrobacter spp. Of note, mcr-9 was found to coexist with blaKPC-2 in species of the Enterobacter cloacae complex. Our findings suggest that the main mechanism for dissemination of blaKPC-2 is HGT mediated by highly transferable plasmids among species of Enterobacterales in infected/colonized patients, presenting a major challenge for public health interventions in developing countries such as Colombia.

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Elucidating the Regulon of Multidrug Resistance Regulator RarA in Klebsiella pneumoniae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01998-12 ◽

2013 ◽

Vol 57 (4) ◽

pp. 1603-1609 ◽

Cited By ~ 31

Author(s):

Shyamasree De Majumdar ◽

Mark Veleba ◽

Sarah Finn ◽

Séamus Fanning ◽

Thamarai Schneiders

Keyword(s):

Klebsiella Pneumoniae ◽

Cell Envelope ◽

Polymyxin B ◽

Multidrug Resistant ◽

Protein Synthesis Inhibitors ◽

Content Type ◽

Phenotypic Microarray ◽

Genes Encoding ◽

Clusters Of Orthologous Groups ◽

Microarray Analyses

ABSTRACTRarA is an AraC-type regulator inKlebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both theacrABandoqxABefflux genes. IncreasedrarAexpression has also been shown to be integral in the development of tigecycline resistance in the absence oframAinK. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8ΔrarA/pACrarA-2 (rarA-expressing construct) and Ecl8ΔrarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences ofK. pneumoniaeMGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show thatrarAoverexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141,sdaCB, andleuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g.,nuoA,narJ, andproWX) were found to be downregulated. Biolog phenotype analyses demonstrated thatrarAoverexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype ofK. pneumoniae.

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Comparative Effectiveness of Aminoglycosides, Polymyxin B, and Tigecycline for Clearance of Carbapenem-Resistant Klebsiella pneumoniae from Urine

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00387-11 ◽

2011 ◽

Vol 55 (12) ◽

pp. 5893-5899 ◽

Cited By ~ 79

Author(s):

Michael J. Satlin ◽

Christine J. Kubin ◽

Jill S. Blumenthal ◽

Andrew B. Cohen ◽

E. Yoko Furuya ◽

...

Keyword(s):

New York ◽

Klebsiella Pneumoniae ◽

Urine Culture ◽

Active Agent ◽

Polymyxin B ◽

Content Type ◽

Carbapenem Resistant ◽

Tract Infections

ABSTRACTCarbapenem-resistantKlebsiella pneumoniae(CRKP) is an increasingly common cause of health care-associated urinary tract infections. Antimicrobials within vitroactivity against CRKP are typically limited to polymyxins, tigecycline, and often, aminoglycosides. We conducted a retrospective cohort study of cases of CRKP bacteriuria at New York-Presbyterian Hospital from January 2005 through June 2010 to compare microbiologic clearance rates based on the use of polymyxin B, tigecycline, or an aminoglycoside. We constructed three active antimicrobial cohorts based on the active agent used and an untreated cohort of cases that did not receive antimicrobial therapy with Gram-negative activity. Microbiologic clearance was defined as having a follow-up urine culture that did not yield CRKP. Cases without an appropriate follow-up culture or that received multiple active agents or less than 3 days of the active agent were excluded. Eighty-seven cases were included in the active antimicrobial cohorts, and 69 were included in the untreated cohort. The microbiologic clearance rate was 88% in the aminoglycoside cohort (n= 41), compared to 64% in the polymyxin B (P= 0.02;n= 25), 43% in the tigecycline (P< 0.001;n= 21), and 36% in the untreated (P< 0.001;n= 69) cohorts. Using multivariate analysis, the odds of clearance were lower for the polymyxin B (odds ratio [OR], 0.10;P= 0.003), tigecycline (OR, 0.08;P= 0.001), and untreated (OR, 0.14;P= 0.003) cohorts than for the aminoglycoside cohort. Treatment with an aminoglycoside, when activein vitro, was associated with a significantly higher rate of microbiologic clearance of CRKP bacteriuria than treatment with either polymyxin B or tigecycline.

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