scholarly journals In VivoEvolution to Colistin Resistance by PmrB Sensor Kinase Mutation in KPC-Producing Klebsiella pneumoniae Is Associated with Low-Dosage Colistin Treatment

2014 ◽  
Vol 58 (8) ◽  
pp. 4399-4403 ◽  
Author(s):  
Antonio Cannatelli ◽  
Vincenzo Di Pilato ◽  
Tommaso Giani ◽  
Fabio Arena ◽  
Simone Ambretti ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Analysis ◽  
Amino Acid Position ◽  
Comparative Genomic ◽  
Sensor Kinase ◽  
Wild Type ◽  
Colistin Resistance ◽  
Content Type ◽  
Before And After ◽  
Low Dosage

ABSTRACTColistin is a key drug for the treatment of infections caused by extensively drug-resistant strains ofEnterobacteriaceaeproducing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially amongKlebsiella pneumoniaestrains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription ofpmrAand ofpmrK, which is part of thepmrHFIJKLMoperon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-typepmrBin KPB-2 restored colistin susceptibility and reduced the transcription ofpmrAandpmrKto basal levels, while expression of PmrBL82Rin KPB-1 did not alter colistin susceptibility or upregulatepmrAandpmrKexpression, confirming the dominance of wild-type PmrB versus the PmrBL82Rmutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

scholarly journals In VivoEmergence of Colistin Resistance in Klebsiella pneumoniae Producing KPC-Type Carbapenemases Mediated by Insertional Inactivation of the PhoQ/PhoPmgrBRegulator

2013 ◽  
Vol 57 (11) ◽  
pp. 5521-5526 ◽  
Author(s):  
Antonio Cannatelli ◽  
Marco Maria D'Andrea ◽  
Tommaso Giani ◽  
Vincenzo Di Pilato ◽  
Fabio Arena ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Analysis ◽  
Negative Regulator ◽  
Resistant Isolate ◽  
Comparative Genomic ◽  
Colistin Resistance ◽  
Content Type ◽  
Insertional Inactivation ◽  
Resistant Mutants

ABSTRACTColistin is one of the few agents that retain activity against extensively drug-resistant strains ofKlebsiella pneumoniaeproducing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of themgrBgene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role ofmgrBinactivation in acquired colistin resistance was confirmed by complementation experiments with wild-typemgrB, which restored colistin susceptibility in KKBO-4, and by construction of anmgrBdeletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. InsertionalmgrBinactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of themgrBgene, upregulated transcription ofphoP,phoQ, andpmrK(which is part of thepmrHFIJKLMoperon) was detected. These findings confirmed the MgrB regulatory role inK. pneumoniaeand were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of thepmrHFIJKLMoperon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.

scholarly journals Proteomic Investigation of the Signal Transduction Pathways Controlling Colistin Resistance in Klebsiella pneumoniae

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Ching Hei Phoebe Cheung ◽  
Punyawee Dulyayangkul ◽  
Kate J. Heesom ◽  
Matthew B. Avison
Keyword(s):  
Klebsiella Pneumoniae ◽  
Protein Production ◽  
Wild Type ◽  
Colistin Resistance ◽  
Reciprocal Effect ◽  
Content Type ◽  
Direct Activation ◽  
Proteomic Investigation ◽  
Two Component Systems ◽  
Operon Expression

ABSTRACT Colistin resistance in Klebsiella pneumoniae is predominantly caused by mutations that increase expression of the arn (also known as pbg or pmrF) operon. Expression is activated by the PhoPQ and PmrAB two-component systems. Constitutive PhoPQ activation occurs directly by mutation or following loss of MgrB. PhoPQ may also cross-activate PmrAB via the linker protein PmrD. Using proteomics, we show that MgrB loss causes a wider proteomic effect than direct PhoPQ activation, suggesting additional targets for MgrB. Different mgrB mutations cause different amounts of Arn protein production, which correlated with colistin MICs. Disruption of phoP in an mgrB mutant had a reciprocal effect to direct activation of PhoQ in a wild-type background, but the regulated proteins showed almost total overlap. Disruption of pmrD or pmrA slightly reduced Arn protein production in an mgrB mutant, but production was still high enough to confer colistin resistance; disruption of phoP conferred wild-type Arn production and colistin MIC. Activation of PhoPQ directly or through mgrB mutation did not significantly activate PmrAB or PmrC production, but direct activation of PmrAB by mutation was able to do this, and also activated Arn production and conferred colistin resistance. There was little overlap between the PmrAB and PhoPQ regulons. We conclude that under the conditions used for colistin susceptibility testing, PhoPQ-PmrD-PmrAB cross-regulation is not significant and that independent activation of PhoPQ or PmrAB is the main reason that Arn protein production increases above the threshold required for colistin resistance.

scholarly journals Emergence and Evolution of Multidrug-Resistant Klebsiella pneumoniae with both blaKPC and blaCTX-M Integrated in the Chromosome

2017 ◽  
Vol 61 (7) ◽  
Author(s):  
Weihua Huang ◽  
Guiqing Wang ◽  
Robert Sebra ◽  
Jian Zhuge ◽  
Changhong Yin ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
De Novo ◽  
Genomic Analysis ◽  
Multidrug Resistant ◽  
Comparative Genomic ◽  
Type I ◽  
Content Type ◽  
Antimicrobial Resistance Genes

ABSTRACT The extended-spectrum-β-lactamase (ESBL)- and Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae represent serious and urgent threats to public health. In a retrospective study of multidrug-resistant K. pneumoniae, we identified three clinical isolates, CN1, CR14, and NY9, carrying both bla CTX-M and bla KPC genes. The complete genomes of these three K. pneumoniae isolates were de novo assembled by using both short- and long-read whole-genome sequencing. In CR14 and NY9, bla CTX-M and bla KPC were carried on two different plasmids. In contrast, CN1 had one copy of bla KPC-2 and three copies of bla CTX-M-15 integrated in the chromosome, for which the bla CTX-M-15 genes were linked to an insertion sequence, ISEcp1, whereas the bla KPC-2 gene was in the context of a Tn4401a transposition unit conjugated with a PsP3-like prophage. Intriguingly, downstream of the Tn4401a-bla KPC-2-prophage genomic island, CN1 also carried a clustered regularly interspaced short palindromic repeat (CRISPR)-cas array with four spacers targeting a variety of K. pneumoniae plasmids harboring antimicrobial resistance genes. Comparative genomic analysis revealed that there were two subtypes of type I-E CRISPR-cas in K. pneumoniae strains and suggested that the evolving CRISPR-cas, with its acquired novel spacer, induced the mobilization of antimicrobial resistance genes from plasmids into the chromosome. The integration and dissemination of multiple copies of bla CTX-M and bla KPC from plasmids to chromosome depicts the complex pandemic scenario of multidrug-resistant K. pneumoniae. Additionally, the implications from this study also raise concerns for the application of a CRISPR-cas strategy against antimicrobial resistance.

scholarly journals Phage Selective Pressure Reduces Virulence of Hypervirulent Klebsiella pneumoniae Through Mutation of the wzc Gene

2021 ◽  
Vol 12 ◽  
Author(s):  
Lingjie Song ◽  
Xianggui Yang ◽  
Jinwei Huang ◽  
Xiaokui Zhu ◽  
Guohui Han ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Genomic Analysis ◽  
Resistant Bacteria ◽  
Comparative Genomic ◽  
Wild Type ◽  
Bacteriophage Therapy ◽  
Deletion Mutations ◽  
Invasive Infections

Hypervirulent Klebsiella pneumoniae (hvKp), one of the major community-acquired pathogens, can cause invasive infections such as liver abscess. In recent years, bacteriophages have been used in the treatment of K. pneumoniae, but the characteristics of the phage-resistant bacteria produced in the process of phage therapy need to be evaluated. In this study, two Podoviridae phages, hvKpP1 and hvKpP2, were isolated and characterized. In vitro and in vivo experiments demonstrated that the virulence of the resistant bacteria was significantly reduced compared with that of the wild type. Comparative genomic analysis of monoclonal sequencing showed that nucleotide deletion mutations of wzc and wcaJ genes led to phage resistance, and the electron microscopy and mucoviscosity results showed that mutations led to the loss of the capsule. Meanwhile, animal assay indicated that loss of capsule reduced the virulence of hvKp. These findings contribute to a better understanding of bacteriophage therapy, which not only can kill bacteria directly but also can reduce the virulence of bacteria by phage screening.

scholarly journals Complete Nucleotide Sequences ofblaKPC-4- andblaKPC-5-Harboring IncN and IncX Plasmids from Klebsiella pneumoniae Strains Isolated in New Jersey

2012 ◽  
Vol 57 (1) ◽  
pp. 269-276 ◽  
Author(s):  
Liang Chen ◽  
Kalyan D. Chavda ◽  
Henry S. Fraimow ◽  
José R. Mediavilla ◽  
Roberto G. Melano ◽  
...  
Keyword(s):  
New Jersey ◽  
Klebsiella Pneumoniae ◽  
Sequence Similarity ◽  
Genomic Analysis ◽  
Multidrug Resistant ◽  
Comparative Genomic ◽  
Bacterial Strains ◽  
Gene Encoding ◽  
Content Type ◽  
Plasmid Backbone

ABSTRACTKlebsiella pneumoniaecarbapenemase (KPC)-producingEnterobacteriaceaehave emerged as major nosocomial pathogens.blaKPC, commonly located on Tn4401, is found in Gram-negative bacterial strains, with the two most common variants,blaKPC-2andblaKPC-3, identified in plasmids with diverse genetic backgrounds. In this study, we examinedblaKPC-4- andblaKPC-5-bearing plasmids recovered from twoK. pneumoniaestrains, which were isolated from a single New Jersey hospital in 2005 and 2006, respectively. IncN plasmid pBK31551 is 84 kb in length and harborsblaKPC-4,blaTEM-1,qnrB2,aac(3)-Ib,aph(3′)-I,qacF,qacEΔ1,sul1, anddfrA14, which confer resistance to β-lactams, quinolones, aminoglycosides, quaternary ammonium compounds, and co-trimoxazole. The conserved regions within pBK31551 are similar to those of other IncN plasmids. Surprisingly, analysis of the Tn4401sequence revealed a large IS110- and Tn6901-carrying element (8.3 kb) inserted into theistAgene, encoding glyoxalase/bleomycin resistance, alcohol dehydrogenase, andS-formylglutathione hydrolase. Plasmid pBK31567 is 47 kb in length and harborsblaKPC-5,dfrA5,qacEΔ1, andsul1. pBK31567 belongs to a novel IncX subgroup (IncX5) and possesses a highly syntenic plasmid backbone like other IncX plasmids; however, sequence similarity at the nucleotide level is divergent. TheblaKPC-5gene is carried on a Tn4401element and differs from the genetic environment ofblaKPC-5described inPseudomonas aeruginosastrain P28 from Puerto Rico. This study underscores the genetic diversity of multidrug-resistant plasmids involved in the spread ofblaKPCgenes and highlights the mobility and plasticity of Tn4401. Comparative genomic analysis provides new insights into the evolution and dissemination of KPC plasmids belonging to different incompatibility groups.

scholarly journals Colistin Resistance in Carbapenem-Resistant Klebsiella pneumoniae Mediated by Chromosomal Integration of Plasmid DNA

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Astrid V. Cienfuegos-Gallet ◽  
Liang Chen ◽  
Barry N. Kreiswirth ◽  
J. Natalia Jiménez
Keyword(s):  
Klebsiella Pneumoniae ◽  
Plasmid Dna ◽  
Clonal Expansion ◽  
Genetic Alterations ◽  
Sequence Type ◽  
Chromosomal Integration ◽  
Colistin Resistance ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Single Locus Variant

ABSTRACT Here we describe the spread of colistin resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae in Medellín, Colombia. Among 32 isolates collected between 2012 and 2014, 24 showed genetic alterations in mgrB. Nineteen isolates belonged to sequence type 512 (ST512) (or its single locus variant [SLV]) and harbored an 8.1-kb hsdMSR insertion corresponding to ISKpn25, indicating a clonal expansion of the resistant strain. The insertion region showed 100% identity to several plasmids, suggesting that the colistin resistance is mediated by chromosomal integration of plasmid DNA.

scholarly journals Comparison of the Superpolymyxin and ChromID Colistin R Screening Media for the Detection of Colistin-Resistant Enterobacteriaceae from Spiked Rectal Swabs

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Delphine Girlich ◽  
Thierry Naas ◽  
Laurent Dortet
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Confidence Interval ◽  
Enterobacter Cloacae ◽  
Bacterial Isolates ◽  
Colistin Resistance ◽  
Gram Negative ◽  
Content Type ◽  
Rectal Swabs ◽  
Stool Samples

ABSTRACT The dissemination of carbapenemase-producing Enterobacteriaceae (CPE) has led to the increased use of colistin, which has resulted in the emergence of colistin-resistant Enterobacteriaceae worldwide. One of the most threatening scenarios is the dissemination of colistin resistance in CPE, particularly the plasmid-encoded resistance element MCR. Thus, it has now become mandatory to possess reliable media to screen for colistin-resistant Gram-negative bacterial isolates, especially Enterobacteriaceae. In this study, we evaluated the performances of the Superpolymyxin medium (ELITechGroup) and the ChromID Colistin R medium (bioMérieux) to screen for colistin-resistant Enterobacteriaceae from spiked rectal swabs. Stool samples were spiked with a total of 94 enterobacterial isolates (Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Enterobacter cloacae), including 53 colistin-resistant isolates. ESwabs (Copan Diagnostics) were then inoculated with those spiked fecal suspensions, and culture proceeded as recommended by both manufacturers. The sensitivity of detection of colistin-resistant Enterobacteriaceae was 86.8% (95% confidence interval [95% CI] = 74.0% to 94.0%) using both the Superpolymyxin medium and the ChromID Colistin R plates. Surprisingly, the isolates that were not detected were not the same for both media. The specificities were high for both media, at 97.9% (95% CI = 87.3% to 99.9%) for the Superpolymyxin medium and 100% (95% CI = 90.4% to 100%) for the ChromID Colistin R medium. Both commercially available media, ChromID Colistin R and Superpolymyxin, provide useful tools to screen for colistin-resistant Enterobacteriaceae from patient samples (rectal swabs) regardless of the level and mechanism of colistin resistance.

scholarly journals Genomic Analysis of Multiresistant Staphylococcus capitis Associated with Neonatal Sepsis

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Glen P. Carter ◽  
James E. Ussher ◽  
Anders Gonçalves Da Silva ◽  
Sarah L. Baines ◽  
Helen Heffernan ◽  
...  
Keyword(s):  
Neonatal Sepsis ◽  
Neonatal Intensive Care ◽  
Genomic Analysis ◽  
Bloodstream Infections ◽  
Multidrug Resistant ◽  
Comparative Genomic ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Staphylococcus Capitis ◽  
Hospital Infection Control

ABSTRACT Coagulase-negative staphylococci (CoNS), such as Staphylococcus capitis, are major causes of bloodstream infections in neonatal intensive care units (NICUs). Recently, a distinct clone of S. capitis (designated S. capitis NRCS-A) has emerged as an important pathogen in NICUs internationally. Here, 122 S. capitis isolates from New Zealand (NZ) underwent whole-genome sequencing (WGS), and these data were supplemented with publicly available S. capitis sequence reads. Phylogenetic and comparative genomic analyses were performed, as were phenotypic assessments of antimicrobial resistance, biofilm formation, and plasmid segregational stability on representative isolates. A distinct lineage of S. capitis was identified in NZ associated with neonates and the NICU environment. Isolates from this lineage produced increased levels of biofilm, displayed higher levels of tolerance to chlorhexidine, and were multidrug resistant. Although similar to globally circulating NICU-associated S. capitis strains at a core-genome level, NZ NICU S. capitis isolates carried a novel stably maintained multidrug-resistant plasmid that was not present in non-NICU isolates. Neonatal blood culture isolates were indistinguishable from environmental S. capitis isolates found on fomites, such as stethoscopes and neonatal incubators, but were generally distinct from those isolates carried by NICU staff. This work implicates the NICU environment as a potential reservoir for neonatal sepsis caused by S. capitis and highlights the capacity of genomics-based tracking and surveillance to inform future hospital infection control practices aimed at containing the spread of this important neonatal pathogen.

scholarly journals High-Level Aminoglycoside Resistance in Human Clinical Klebsiella pneumoniae Complex Isolates and Characteristics of armA-Carrying IncHI5 Plasmids

2021 ◽  
Vol 12 ◽  
Author(s):  
Xueya Zhang ◽  
Qiaoling Li ◽  
Hailong Lin ◽  
Wangxiao Zhou ◽  
Changrui Qian ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Genomic Analysis ◽  
Comparative Genomic Analysis ◽  
Conjugative Plasmid ◽  
Comparative Genomic ◽  
Aminoglycoside Resistance ◽  
Life Threatening ◽  
Aminoglycoside Resistance Genes ◽  
High Level

Aminoglycosides are important options for treating life-threatening infections. However, high levels of aminoglycoside resistance (HLAR) among Klebsiella pneumoniae isolates have been observed to be increasing frequently. In this study, a total of 292 isolates of the K. pneumoniae complex from a teaching hospital in China were analyzed. Among these isolates, the percentage of HLAR strains was 13.7% (40/292), and 15 aminoglycoside resistance genes were identified among the HLAR strains, with rmtB being the most dominant resistance gene (70%, 28/40). We also described an armA-carrying Klebsiella variicola strain KP2757 that exhibited a high-level resistance to all aminoglycosides tested. Whole-genome sequencing of KP2757 demonstrated that the strain contained one chromosome and three plasmids, with all the aminoglycoside resistance genes (including two copies of armA and six AME genes) being located on a conjugative plasmid, p2757-346, belonging to type IncHI5. Comparative genomic analysis of eight IncHI5 plasmids showed that six of them carried two copies of the intact armA gene in the complete or truncated Tn1548 transposon. To the best of our knowledge, for the first time, we observed that two copies of armA together with six AME genes coexisted on the same plasmid in a strain of K. variicola with HLAR. Comparative genomic analysis of eight armA-carrying IncHI5 plasmids isolated from humans and sediment was performed, suggesting the potential for dissemination of these plasmids among bacteria from different sources. These results demonstrated the necessity of monitoring the prevalence of IncHI5 plasmids to restrict their worldwide dissemination.

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