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.

BACKGROUND AND PURPOSE: 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. CONCLUSION: 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.

Rapid detection of ESBL-producing Klebsiella pneumoniae in blood cultures by fluorescent in-situ hybridization.

Multi-resistant Enterobacteriaceae pose a serious threat of hospital acquired infections and their rapid identification is important for better clinical outcome. This study describes the rapid identification of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae of the sulphydryl variable-type by fluorescent in-situ hybridization. The method which rapidly identifies the target genes within 1 h could be a potentially rapid bacterial diagnostic tool.

Extended-spectrum beta-lactam resistance due to AmpC hyperproduction and CMY-2 coupled with the loss of OMPK35 in Malaysian strains of Escherichia coli and Klebsiella pneumoniae.

In this report, we describe the detection of AmpC and CMY-2 beta-lactamases with the loss of OmpK35 porin among seven sporadic strains of ceftazidime-resistant Klebsiella pneumoniae and ceftazidime-resistant Escherichia coli. Cefoxitin, which was used as a marker of resistance toward 7-alpha-methoxy-cephalosporins, exhibited high minimum inhibitory concentration (MIC) values ranging between 128 microg/ml and >256 microg/ml in all the strains. The presence of hyperproducing AmpC enzymes was indicated by the positive three-dimensional test. Isoelectric focusing (IEF) study confirmed the presence of AmpC enzymes in all the strains. The ampC gene was detected by PCR in all the strains and confirmed by DNA sequencing. Large plasmids in all the strains, ranging from 60 kb to 150 kb in size, most likely encode the ampC gene. Two E. coli strains out of the seven strains showed positive amplification of the bla(CMY-2) gene, an AmpC variant, and was confirmed by DNA sequence analyses. DNA hybridization confirmed the bla(CMY-2) gene to be plasmid-mediated in both of these strains. However, one of these two strains also mediated a chromosomal CMY gene. All the strains showed an absence of OmpK35 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and was confirmed by western blot analyses using raised polyclonal anti-OmpK35 antiserum. This suggests that, apart from CMY production, absence of OmpK35 porin also contributed to cefoxitin resistance resulting in extended-spectrum beta-lactam resistance among these isolates.

beta-Lactam resistance phenotype determination in Escherichia coli isolates from University Malaya Medical Centre.

beta-Lactamases have been identified as the major cause of antimicrobial resistance to beta-lactam antibiotics in Escherichia coli. The activities of ampicillin-sulbactam and amoxicillin-clavulanate as well as a range of beta-lactam antibiotics were studied with 87 clinical E. coli isolates from patients of the University Malaya Medical Center using the disc diffusion technique. Susceptible, intermediate and resistant categories were established based on the diameter of zones of inhibition set by the National Committee for Clinical Laboratory Standards (NCCLS). The isolates were then classified into 6 phenotypes according to the criteria stated in the methodology: S (susceptible to all beta-lactams); TL (resistant to aminopenicillins; amoxicillin-clavulanate susceptible and susceptible or intermediate to ampicillin-sulbactam); TI (resistant to aminopenicillins and ampicillin-sulbactam; susceptible to amoxicilin-clavulanate); TH-IRT (resistant to aminopenicillins; intermediate or resistant to amoxicillin-clavulanate; resistant to ampicillin-sulbactam); ESBL (resistant to aminopenicillins and oxyimino cephalosporins; positive results with the double-disc diffusion test); and CP (resistant to aminopenicillins, beta-lactam-beta-lactamase inhibitor combinations, oxyimino cephalosporins and cephamycins). Results showed that the TL phenotype was the commonest (40.2% of the isolates) followed by S (31%), TH-IRT (16.1%), ESBL and CP (3.4% each) and TI (2.3%). One isolate showed both ESBL and CP phenotypes while two isolates were classified as inconclusive. Representatives from each phenotype were further analysed for the presence of beta-lactamases which revealed a predominance of TEM and SHV enzyme producers. PCR-SSCP analysis of the SHV gene from all the ESBL and CP isolates revealed the predominance of SHV 5-type enzyme which was concurrent with our previous studies.

Carbapenem-resistant Pseudomonas aeruginosa in malaysia producing IMP-7 beta-lactamase.

We have isolated and identified a carbapenem-resistant Pseudomonas aeruginosa strain from Malaysia that produces an IMP-7 metallo-beta-lactamase. This isolate showed high-level resistance to meropenem and imipenem, the MICs of which were 256 and 128 micro g/ml, respectively. Isoelectric focusing analyses revealed pI values of >9.0, 8.2, and 7.8, which indicated the possible presence of IMP and OXA. DNA sequencing confirmed the identity of the IMP-7 determinant.