scholarly journals Amino Acid Substitutions of CrrB Responsible for Resistance to Colistin through CrrC in Klebsiella pneumoniae

2016 ◽  
Vol 60 (6) ◽  
pp. 3709-3716 ◽  
Author(s):  
Yi-Hsiang Cheng ◽  
Tzu-Lung Lin ◽  
Yi-Tsung Lin ◽  
Jin-Town Wang
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Site Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Missense Mutations ◽  
Colistin Resistance ◽  
Two Component System ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Elevated Expression

Colistin is a last-resort antibiotic for treatment of carbapenem-resistantKlebsiella pneumoniae. A recent study indicated that missense mutations in the CrrB protein contribute to colistin resistance. In our previous study, mechanisms of colistin resistance were defined in 17 of 26 colistin-resistantK. pneumoniaeclinical isolates. Of the remaining nine strains, eight were highly resistant to colistin. In the present study,crrABsequences were determined for these eight strains. Six separate amino acid substitutions in CrrB (Q10L, Y31H, W140R, N141I, P151S, and S195N) were detected. Site-directed mutagenesis was used to generatecrrBloci harboring individual missense mutations; introduction of the mutated genes into a susceptible strain, A4528, resulted in 64- to 1,024-fold increases in colistin MICs. ThesecrrBmutants showed increased accumulation ofH239_3062,H239_3059,pmrA,pmrC, andpmrHtranscripts by quantitative reverse transcription (qRT)-PCR. Deletion ofH239_3062(but not that ofH239_3059) in the A4528crrB(N141I) strain attenuated resistance to colistin, andH239_3062was accordingly namedcrrC. Similarly, accumulation ofpmrA,pmrC, andpmrHtranscripts induced bycrrB(N141I) was significantly attenuated upon deletion ofcrrC. Complementation ofcrrCrestored resistance to colistin and accumulation ofpmrA,pmrC, andpmrHtranscripts in acrrB(N141I) ΔcrrCstrain. In conclusion, novel individual CrrB amino acid substitutions (Y31H, W140R, N141I, P151S, and S195N) were shown to be responsible for colistin resistance. We hypothesize that CrrB mutations induce CrrC expression, thereby inducing elevated expression of thepmrHFIJKLMoperon andpmrC(an effect mediated via the PmrAB two-component system) and yielding increased colistin resistance.

scholarly journals VIM-19, a Metallo-β-Lactamase with Increased Carbapenemase Activity from Escherichia coli and Klebsiella pneumoniae

2009 ◽  
Vol 54 (1) ◽  
pp. 471-476 ◽  
Author(s):  
Jose-Manuel Rodriguez-Martinez ◽  
Patrice Nordmann ◽  
Nicolas Fortineau ◽  
Laurent Poirel
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Class 1 Integron ◽  
Carbapenem Resistant ◽  
Class 1

ABSTRACT Two carbapenem-resistant isolates, one Escherichia coli isolate and one Klebsiella pneumoniae isolate, recovered from an Algerian patient expressed a novel VIM-type metallo-β-lactamase (MBL). The identified bla VIM-19 gene was located on a ca. 160-kb plasmid and located inside a class 1 integron in both isolates. VIM-19 differed from VIM-1 by the Asn215Lys and Ser228Arg substitutions, increasing its hydrolytic activity toward carbapenems. Site-directed mutagenesis experiments showed that both substitutions were necessary for the increased carbapenemase activity of VIM-19. This study indicates that MBLs with enhanced activity toward carbapenems may be obtained as a result of very few amino acid substitutions.

scholarly journals Colistin Resistance Mechanisms in Klebsiella pneumoniae Strains from Taiwan

2015 ◽  
Vol 59 (5) ◽  
pp. 2909-2913 ◽  
Author(s):  
Yi-Hsiang Cheng ◽  
Tzu-Lung Lin ◽  
Yi-Jiun Pan ◽  
Yu-Ping Wang ◽  
Yi-Tsung Lin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Resistance Mechanisms ◽  
Single Amino Acid ◽  
Colistin Resistance ◽  
Amino Acid Mutation ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Resistant Strains ◽  
Sequence Types

ABSTRACTColistin is one of the antibiotics of last resort for the treatment of carbapenem-resistantKlebsiella pneumoniaeinfection. This study showed that capsular type K64 (50%) and ST11 (53.9%) are the prevalent capsular and sequence types in the colistin-resistant strains in Taiwan. The interruption of transcripts (38.5%) and amino acid mutation (15.4%) inmgrBare the major mechanisms contributing to colistin resistance. In addition, novel single amino acid changes in MgrB (Stop48Tyr) and PhoQ (Leu26Pro) were observed to contribute to colistin resistance.

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 Rapid Increase in Prevalence of Carbapenem-ResistantEnterobacteriaceae(CRE) and Emergence of Colistin Resistance Genemcr-1in CRE in a Hospital in Henan, China

2018 ◽  
Vol 56 (4) ◽  
Author(s):  
Yi Li ◽  
Qiao-ling Sun ◽  
Yingbo Shen ◽  
Yangjunna Zhang ◽  
Jun-wen Yang ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Carbapenem Resistance ◽  
Clinical Samples ◽  
New Delhi ◽  
Colistin Resistance ◽  
Large Hospital ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Recent Emergence ◽  
Almost All

ABSTRACTThe global spread of carbapenem-resistantEnterobacteriaceae(CRE) is one of the most severe threats to human health in a clinical setting. The recent emergence of plasmid-mediated colistin resistance genemcr-1among CRE strains greatly compromises the use of colistin as a last resort for the treatment of infections caused by CRE. This study aimed to understand the current epidemiological trends and characteristics of CRE from a large hospital in Henan, the most populous province in China. From 2014 to 2016, a total of 7,249Enterobacteriaceaeisolates were collected from clinical samples, among which 18.1% (1,311/7,249) were carbapenem resistant. Carbapenem-resistantKlebsiella pneumoniaeand carbapenem-resistantEscherichia coliwere the two most common CRE species, withKlebsiella pneumoniaecarbapenemases (KPC) and New Delhi metallo-β-lactamases (NDM), respectively, responsible for the carbapenem resistance of the two species. Notably, >57.0% (n= 589) of theK. pneumoniaeisolates from the intensive care unit were carbapenem resistant. Furthermore,blaNDM-5andmcr-1were found to coexist in oneE. coliisolate, which exhibited resistance to almost all tested antibiotics. Overall, we observed a significant increase in the prevalence of CRE isolates during the study period and suggest that carbapenems may no longer be considered to be an effective treatment for infections caused byK. pneumoniaein the studied hospital.

scholarly journals Identification of Genetic Alterations Associated with Acquired Colistin Resistance in Klebsiella pneumoniae Isogenic Strains by Whole-Genome Sequencing

Antibiotics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 374
Author(s):  
Myeongjin Choi ◽  
Kwan Soo Ko
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Whole Genome Sequencing ◽  
Candidate Genes ◽  
Genome Sequencing ◽  
Parental Strain ◽  
Genetic Alterations ◽  
Amino Acid Substitutions ◽  
Whole Genome ◽  
Colistin Resistance

The present study was undertaken to find novel genes associated with colistin resistance in Klebsiella pneumoniae. Five colistin-resistant mutants were derived from four colistin-susceptible parental K. pneumoniae strains belonging to different clones. Whole-genome sequencing was performed for the nine K. pneumoniae strains to screen altered candidate genes. Expression levels of genes with amino acid alterations in derivative strains were determined using quantitative real-time Polymerase chain reaction (PCR). Colistin susceptibility was examined in a parental strain complemented with altered candidate genes. Overall, 13 genetic alterations were identified in five pairs of isogenic K. pneumoniae strains. Genetic alterations related to KP1_3468, including the insertion of an IS5-like element in an intergenic or coding region and amino acid substitutions, were identified in three separate derivative strains. Amino acid substitutions and deletion of PhoQ were determined in one derivative strain. With inactivation of CrrA and substituted CrrB, amino acid substitutions and deletion were identified in a repressor of galETK operon (KP1_0061) and hypothetical protein (KP1_3620), respectively. Decreased colistin susceptibility was observed in a parental strain complemented with KP1-0061, but not a KP1-3620 gene. This study demonstrated diverse genetic paths to colistin resistance in K. pneumoniae. Our results suggest that a repressor of galETK operon may play an important role in colistin resistance in K. pneumoniae.

scholarly journals A Novel FexA Variant from a Canine Staphylococcus pseudintermedius Isolate That Does Not Confer Florfenicol Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5763-5766 ◽  
Author(s):  
Elena Gómez-Sanz ◽  
Kristina Kadlec ◽  
Andrea T. Feßler ◽  
Myriam Zarazaga ◽  
Carmen Torres ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Recognition ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Base Pairs ◽  
Chloramphenicol Resistance ◽  
Content Type ◽  
Resistance Phenotype ◽  
First Time

ABSTRACTTransposon Tn558integrated in the chromosomalradCgene was detected for the first time inStaphylococus pseudintermedius. It carried a novelfexAvariant (fexAv) that confers only chloramphenicol resistance. The exporter FexAv exhibited two amino acid substitutions, Gly33Ala and Ala37Val, both of which seem to be important for substrate recognition. Site-directed mutagenesis that reverted the mutated base pairs to those present in the originalfexAgene restored the chloramphenicol-plus-florfenicol resistance phenotype.

scholarly journals NDM-12, a Novel New Delhi Metallo-β-Lactamase Variant from a Carbapenem-Resistant Escherichia coli Clinical Isolate in Nepal

2014 ◽  
Vol 58 (10) ◽  
pp. 6302-6305 ◽  
Author(s):  
Tatsuya Tada ◽  
Basudha Shrestha ◽  
Tohru Miyoshi-Akiyama ◽  
Kayo Shimada ◽  
Hiroshi Ohara ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Clinical Isolate ◽  
Urine Sample ◽  
Enzymatic Activities ◽  
Amino Acid Substitutions ◽  
New Delhi ◽  
Content Type ◽  
Carbapenem Resistant

ABSTRACTA novel New Delhi metallo-β-lactamase variant, NDM-12, was identified in a carbapenem-resistantEscherichia coliclinical isolate obtained from a urine sample from a patient in Nepal. NDM-12 differed from NDM-1 by two amino acid substitutions (M154L and G222D). The enzymatic activities of NDM-12 against β-lactams were similar to those of NDM-1, although NDM-12 showed lowerkcat/Kmratios for all β-lactams tested except doripenem. TheblaNDM-12gene was located in a plasmid of 160 kb.

scholarly journals Experimental evolution in morbidostat reveals converging genomic trajectories on the path to triclosan resistance

2021 ◽  
Vol 7 (5) ◽  
Author(s):  
Semen A. Leyn ◽  
Jaime E. Zlamal ◽  
Oleg V. Kurnasov ◽  
Xiaoqing Li ◽  
Marinela Elane ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Experimental Evolution ◽  
Type Species ◽  
Acyl Carrier Protein ◽  
Consumer Products ◽  
Amino Acid Substitutions ◽  
Missense Mutations ◽  
Content Type ◽  
Link Type

Understanding the dynamics and mechanisms of acquired drug resistance across major classes of antibiotics and bacterial pathogens is of critical importance for the optimization of current anti-infective therapies and the development of novel ones. To systematically address this challenge, we developed a workflow combining experimental evolution in a morbidostat continuous culturing device with deep genomic sequencing of population samples collected in time series. This approach was applied to the experimental evolution of six populations of Escherichia coli BW25113 towards acquiring resistance to triclosan (TCS), an antibacterial agent in various consumer products. This study revealed the rapid emergence and expansion (up to 100% in each culture within 4 days) of missense mutations in the fabI gene, encoding enoyl-acyl carrier protein reductase, the known TCS molecular target. A follow-up analysis of isolated clones showed that distinct amino acid substitutions increased the drug IC90 in a 3–16-fold range, reflecting their proximity to the TCS-binding site. In contrast to other antibiotics, efflux-upregulating mutations occurred only rarely and with low abundance. Mutations in several other genes were detected at an earlier stage of evolution. Most notably, three distinct amino acid substitutions were mapped in the C-terminal periplasmic domain of CadC protein, an acid stress-responsive transcriptional regulator. While these mutations do not confer robust TCS resistance, they appear to play a certain, yet unknown, role in adaptation to relatively low drug pressure. Overall, the observed evolutionary trajectories suggest that the FabI enzyme is the sole target of TCS (at least up to the ~50 µm level), and amino acid substitutions in the TCS-binding site represent the main mechanism of robust TCS resistance in E. coli . This model study illustrates the potential utility of the established morbidostat-based approach for uncovering resistance mechanisms and target identification for novel drug candidates with yet unknown mechanisms of action.

scholarly journals Emergence of Carbapenem-Resistant Providencia rettgeri and Providencia stuartii Producing IMP-Type Metallo-β-Lactamase in Japan

2020 ◽  
Vol 64 (11) ◽  
Author(s):  
Shu Iwata ◽  
Tatsuya Tada ◽  
Tomomi Hishinuma ◽  
Mari Tohya ◽  
Satoshi Oshiro ◽  
...  
Keyword(s):  
Amino Acid ◽  
Carbapenem Resistance ◽  
The Other ◽  
Amino Acid Substitutions ◽  
Whole Genome ◽  
Providencia Rettgeri ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Hydrolytic Activities ◽  
Providencia Stuartii

ABSTRACT Four Providencia rettgeri isolates and one Providencia stuartii isolate were obtained from urine samples of five patients in 2018 in Japan. All of the isolates were resistant to imipenem and meropenem, and three were highly resistant to both carbapenems, with MICs of 512 μg/ml. The three highly carbapenem-resistant isolates harbored blaIMP-70, encoding a variant of IMP-1 metallo-β-lactamase with two amino acid substitutions (Val67Phe and Phe87Val), and the other two harbored blaIMP-1 and blaIMP-11, respectively. Whole-genome sequencing revealed that an isolate harbored two copies of blaIMP-1 on the chromosome and that the other four harbored a copy of blaIMP-11 or blaIMP-70 in a plasmid. Expression of blaIMP-70 conferred carbapenem resistance in Escherichia coli. Recombinant IMP-70 and an IMP-1 variant with Val67Phe but without Phe87Val had significant higher hydrolytic activities against meropenem than recombinant IMP-1, indicating that an amino acid substitution of Val67Phe affects increased activities against meropenem in IMP-70. These results suggest that Providencia spp. become more highly resistant to carbapenems by acquisition of two copies of blaIMP-1 or by mutation of blaIMP genes with amino acid substitutions, such as blaIMP-70.

scholarly journals Molecular Epidemiology of Colistin-Resistant, Carbapenemase-Producing Klebsiella pneumoniae in Serbia from 2013 to 2016

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Katarina Novović ◽  
Anika Trudić ◽  
Snežana Brkić ◽  
Zorica Vasiljević ◽  
Milan Kojić ◽  
...  
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Molecular Epidemiology ◽  
Gene Transcription ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Amino Acid Substitutions ◽  
Content Type ◽  
Sequence Types

ABSTRACT Twenty-seven colistin-resistant, carbapenemase-producing Klebsiella pneumoniae isolates were identified from hospitals in Serbia. All isolates were bla CTX-M-15 positive; ST101, ST888, ST437, ST336, and ST307 were bla OXA-48 positive; and ST340 was bla NDM-1 positive. ST307 had an insertion, and ST336 had a premature stop codon in the mgrB gene. Amino acid substitutions were detected in PmrAB of isolates ST101, ST888, ST336, and ST307. The mcr-1 and mcr-2 were not detected. An increase in phoP, phoQ, and pmrK gene transcription was detected for all sequence types.

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