Characterization of the new metallo-beta-lactamase VIM-13 and its integron-borne gene from a Pseudomonas aeruginosa clinical isolate in Spain.
During a survey conducted to evaluate the incidence of class B carbapenemase (metallo-beta-lactamase [MBL])-producing Pseudomonas aeruginosa strains from hospitals in Majorca, Spain, five clinical isolates showed a positive Etest MBL screening test result. In one of them, strain PA-SL2, the presence of a new bla(VIM) derivative (bla(VIM-13)) was detected by PCR amplification with bla(VIM-1)-specific primers followed by sequencing. The bla(VIM-13)-producing isolate showed resistance to all beta-lactams (except aztreonam), gentamicin, tobramycin, and ciprofloxacin. VIM-13 exhibited 93% and 88% amino acid sequence identities with VIM-1 and VIM-2, respectively. bla(VIM-13) was cloned in parallel with bla(VIM-1), and the resistance profile conferred was analyzed both in Escherichia coli and in P. aeruginosa backgrounds. Compared to VIM-1, VIM-13 conferred slightly higher levels of resistance to piperacillin and lower levels of resistance to ceftazidime and cefepime. VIM-13 and VIM-1 were purified in parallel as well, and their kinetic parameters were compared. The k(cat)/K(m) ratios for the antibiotics mentioned above were in good agreement with the MIC data. Furthermore, EDTA inhibited the activity of VIM-13 approximately 25 times less than it inhibited the activity of VIM-1. VIM-13 was harbored in a class 1 integron, along with a new variant (Ala108Thr) of the aminoglycoside-modifying enzyme encoding gene aacA4, which confers resistance to gentamicin and tobramycin. Finally, the VIM-13 integron was apparently located in the chromosome, since transformation and conjugation experiments consistently yielded negative results and the bla(VIM-13) probe hybridized only with the genomic DNA.
Juan
Carlos
C
Servicio de Microbiología and Unidad de Investigación, Hospital Son Dureta, Palma de Mallorca, Spain. cjuan@hsd.es
Beceiro
Alejandro
A
Gutiérrez
Olivia
O
Albertí
Sebastián
S
Garau
Margalida
M
Pérez
José L
JL
Bou
Germán
G
Oliver
Antonio
A
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D014644:Genetic Variation; D006801:Humans; D015378:Imipenem / Q000494:pharmacology; D032023:Integrons; D007700:Kinetics; D000077731:Meropenem; D008969:Molecular Sequence Data; D010802:Phylogeny; D011552:Pseudomonas Infections / Q000188:drug therapy / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013030:Spain; D013845:Thienamycins / Q000494:pharmacology; D018440:beta-Lactam Resistance / Q000235:genetics / Q000502:physiology; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D013845:Thienamycins; D015378:Imipenem; C417264:VIM-1 metallo-beta-lactamase; C533914:metallo-beta-lactamase (VIM-13), Pseudomonas aeruginosa; D001618:beta-Lactamases; D000077731:Meropenem