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 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 Integron-Located oxa-32 Gene Cassette Encoding an Extended-Spectrum Variant of OXA-2 β-Lactamase from Pseudomonas aeruginosa

2002 ◽  
Vol 46 (2) ◽  
pp. 566-569 ◽  
Author(s):  
Laurent Poirel ◽  
Patrick Gerome ◽  
Christophe De Champs ◽  
Jean Stephanazzi ◽  
Thierry Naas ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Substitution ◽  
Gene Cassette ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Class 1 Integron ◽  
Class D ◽  
Class 1

ABSTRACT Pseudomonas aeruginosa clinical isolate CY-1, which was resistant to ceftazidime, harbored a conjugative ca. 250-kb plasmid that contained a class 1 integron with two gene cassettes encoding OXA-32, an OXA-2- type β-lactamase, and the aminoglycoside acetyltransferase AAC(6′)Ib9. OXA-32 differed from OXA-2 by an Leu169Ile amino acid substitution (class D numbering). Site-directed mutagenesis established that Ile169 is responsible for resistance to ceftazidime but not to cefotaxime.

scholarly journals Characterization of a Novel Plasmid-Mediated Cephalosporinase (CMY-9) and Its Genetic Environment in an Escherichia coli Clinical Isolate

2002 ◽  
Vol 46 (8) ◽  
pp. 2427-2434 ◽  
Author(s):  
Yohei Doi ◽  
Naohiro Shibata ◽  
Keigo Shibayama ◽  
Kazunari Kamachi ◽  
Hiroshi Kurokawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Cassette ◽  
Single Amino Acid ◽  
Common Region ◽  
Class 1 Integron ◽  
Class 1 ◽  
The Common ◽  
High Level

ABSTRACT An Escherichia coli strain, HKYM68, which showed resistance to broad-spectrum cephalosporins was isolated from a sputum specimen in Japan. The high-level resistance of the strain to ceftazidime, cefpirome, and moxalactam was carried by a self-transferable plasmid. The β-lactamase gene responsible for the resistance was cloned and sequenced. The deduced amino acid sequence of this gene product, CMY-9, had a single amino acid substitution (E85D), the residue reported to be part of the recognition site for the R1 side chain of β-lactams, compared with the amino acid sequence of CMY-8 and also had 78% identity with the amino acid sequence of CepH, a chromosomal cephalosporinase of Aeromonas hydrophila. A sul1-type class 1 integron containing an aacA1-orfG gene cassette was identified upstream of bla CMY-9 and ended with a truncated 3′ conserved segment. The following 2.1 kb was almost identical to the common region of integrons In6 and In7 and the integron of pSAL-1, except that orf513 encoding a putative transposase was identified instead of orf341 due to addition of a single nucleotide. bla CMY-9 was closely located downstream of the end of the common region. These observations are indicative of the exogenous derivation of bla CMY-9 from some environmental microorganisms such as aeromonads.

scholarly journals First Detection of a Carbapenem-Hydrolyzing Metalloenzyme in Two Enterobacteriaceae Isolates in Spain

2005 ◽  
Vol 49 (8) ◽  
pp. 3492-3494 ◽  
Author(s):  
M. Teresa Tórtola ◽  
Susana Lavilla ◽  
Elisenda Miró ◽  
Juan José González ◽  
Nieves Larrosa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Gene Cassette ◽  
Class 1 Integron ◽  
Class 1 ◽  
First Time

ABSTRACT Two strains of Enterobacteriaceae, Escherichia coli and Klebsiella pneumoniae, producing VIM-1 were isolated for the first time in Spain. In both strains, bla VIM-1 was found to be carried on a gene cassette inserted into a class 1 integron. The bla VIM-1-containing integron was located on a transferable plasmid.

scholarly journals Predicting Evolution by In Vitro Evolution Requires Determining Evolutionary Pathways

2002 ◽  
Vol 46 (9) ◽  
pp. 3035-3038 ◽  
Author(s):  
Barry G. Hall
Keyword(s):  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Dna Shuffling ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
Single Mutation ◽  
Wild Type ◽  
In Vitro Evolution ◽  
Evolutionary Pathway

ABSTRACT In an early example of DNA shuffling, Stemmer (W. P. C. Stemmer, Nature 370:389-390, 1994) demonstrated a dramatic improvement in the activity of the TEM-1 β-lactamase toward cefotaxime as the consequence of six amino acid substitutions. It has been pointed out (B. G. Hall, FEMS Microbiol. Lett. 178:1-6, 1999; M. C. Orencia, J. S. Yoon, J. E. Ness, W. P. Stemmer, and R. C. Stevens, Nat. Struct. Biol. 8:238-242, 2001) that the power of DNA shuffling might be applied to the problem of predicting evolution in nature from in vitro evolution in the laboratory. As a predictor of natural evolutionary processes, that power may be misleading because in nature mutations almost always arise one at a time, and each advantageous mutation must be fixed into the population by an evolutionary pathway that leads from the wild type to the fully evolved sequence. Site-directed mutagenesis was used to introduce each of Stemmer's six substitutions into TEM-1, the best single mutant was chosen, and each of the remaining five substitutions was introduced. Repeated rounds of site-directed mutagenesis and selection of the best mutant were used in an attempt to construct a pathway between the wild-type TEM-1 and Stemmer's mutant with six mutations. In the present study it is shown (i) that no such pathway exists between the wild-type TEM-1 and the supereffective cefotaxime-hydrolyzing mutant that was generated by six amino acid substitutions via DNA shuffling (Stemmer, Nature 370:389-390, 1994) but that a pathway to a fourfold more efficient enzyme resulting from four of the same substitutions does exist, and (ii) that the more efficient enzyme is likely to arise in nature as the result of a single mutation in the naturally occurring TEM-52 allele.

scholarly journals A Single Amino Acid Substitution in a Mannosyltransferase, WbdA, Converts the Escherichia coli O9 Polysaccharide into O9a: Generation of a New O-Serotype Group

2000 ◽  
Vol 182 (9) ◽  
pp. 2567-2573 ◽  
Author(s):  
Nobuo Kido ◽  
Hidemitsu Kobayashi
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Chimeric Genes ◽  
Functional Studies

ABSTRACT wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1. The equivalent structural O polysaccharide in the E. coli O9 andKlebsiella O3 strains is 2-αMan-1,2-αMan-1,2-αMan-1,3-αMan-1,3-αMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdAgenes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.

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