Resistance to extended-spectrum beta-lactams by the emergence of SHV-12 and the loss of OmpK35 in Klebsiella pneumoniae and Escherichia coli in Malaysia.
OBJECTIVE
In addition to beta-lactamase production, loss of porins confers resistance to extended-spectrum beta-lactams in Klebsiella pneumoniae and Escherichia coli infection. This study describes the detection of SHV-12 extended-spectrum beta-lactamase (ESBL) subtype and the loss of OmpK35 porin in 4 strains of K. pneumoniae and E. coli.


METHODS
Isoelectric focusing was performed to detect beta-lactamases in 4 strains of K. pneumoniae and E. coli. The presence of the SHV gene in the 4 isolates was characterized by polymerase chain reaction, DNA sequencing, and DNA hybridization. Loss of porin in these strains was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis.


RESULTS
The strains of K. pneumoniae and E. coli were confirmed to be ESBL producers and were resistant to cefoxitin, with minimal inhibitory concentration values of 512 microg/mL. All 4 strains had beta-lactamases with an isoelectric value of 8.2. The SHV gene from these strains was characterized to be the SHV-12 subtype and was plasmid-borne. The deduced amino acid sequence showed that the SHV-12 beta-lactamase was a derivative of the more common ESBL, SHV-5 subtype. All the strains showed absence of the OmpK35 porin.


CONCLUSIONS
Resistance of the strains towards extended-spectrum beta-lactams was a result of a dual-mechanism - the production of SHV-12 enzymes and loss of the OmpK35 porin.
Palasubramaniam
Selvi
S
Department of Medical Microbiology, University of Malaya, Kuala Lumpur, Malaysia.
Muniandy
Sekaran
S
Navaratnam
Parasakthi
P
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D015153:Blotting, Western; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics*; D006801:Humans; D007525:Isoelectric Focusing; D007710:Klebsiella Infections / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000302:isolation & purification; D008296:Malaysia; D008826:Microbial Sensitivity Tests; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D018272:Porins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D047090:beta-Lactams / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C119261:OmpK35 porin, Klebsiella pneumoniae; C544069:OmpK35 protein, E coli; D018272:Porins; D047090:beta-Lactams; C107198:beta-lactamase SHV-12; D001618:beta-Lactamases