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Comparison of three different methods for the presumptive detection of ESBL production in ceftazidime-resistant strains of Klebsiella pneumoniae

Palasubramaniam S, Parasakthi N

Malays J Pathol, 2001 Dec;23(2):73-8.
PMID: 12166595

Twenty-eight (28) strains of ceftazidime-resistant Klebsiella pneumoniae were isolated from blood cultures of in-patients from University Hospital, Kuala Lumpur between March 1995 and August 1996. Three methods were used to detect the production of ESBL enzymes by these strains. These three methods include the double-disc synergy test (DDST), inhibitor-potentiated disc-diffusion test (IPDD) and the E-test ESBL method. All strains could be identified as ESBL producers using the DDST method by a minimum of two beta-lactams and these included either a combination of ceftazidime and ceftriaxone with clavulanate respectively or cefotaxime and aztreonam with clavulanate respectively. Similarly using a combination of either cefotaxime and ceftriaxone with clavulanate or ceftriaxone and aztreonam with clavulanate respectively would have detected all strains as ESBL producers. The IPDD method could also detect for ESBL activity based on combinations of beta-lactam antibiotics with clavulanate respectively. All combinations of beta-lactam antibiotics could detect for ESBL activity in all the strains except a combination of either ceftazidime and aztreonam or cefotaxime and ceftriaxone with clavulanate respectively. The E-Test method using ceftazidime alone and in combination with clavulanate was found to be the most effective method in the presumptive identification of ESBL activity in all the strains.
SHV-5 extended-spectrum beta-lactamases in clinical isolates of Escherichia coli in Malaysia

Subramaniam G, Palasubramaniam S, Navaratnam P

Indian J Med Microbiol, 2006 Jul;24(3):205-7.
PMID: 16912441

Escherichia coli isolates resistant to ceftazidime isolated in the University Malaya Medical Center (UMMC) Kuala Lumpur, Malaysia, between the years 1998 and 2000 were studied for extended-spectrum beta-lactamase (ESBL) production. All strains were analysed phenotypically and genotypically and found to be ESBL-producing organisms harbouring SHV-5 beta-lactamase. This was confirmed by PCR-SSCP and nucleotide sequencing of the blaSHV amplified gene. As there was no evidence of ESBL activity in E. coli prior to this, coupled with the fact that there was a predominance of SHV-5 beta-lactamases in Klebsiella pneumoniae isolates in UMMC, we postulate that the E. coli obtained the SHV-5 beta-lactamase genes by plasmid transfer from the ESBL-producing K. pneumoniae.
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

Palasubramaniam S, Muniandy S, Navaratnam P

J Microbiol Immunol Infect, 2009 Apr;42(2):129-33.
PMID: 19597644

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

Palasubramaniam S, Muniandy S, Navaratnam P

J Microbiol Methods, 2008 Jan;72(1):107-9.
PMID: 18054098

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

Palasubramaniam S, Subramaniam G, Muniandy S, Parasakthi N

Microb Drug Resist, 2007;13(3):186-90.
PMID: 17949305

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.
SHV-5 extended-spectrum beta-lactamase from Klebsiella pneumoniae associated with a nosocomial outbreak in a paediatric oncology unit in Malaysia

Palasubramaniam S, Subramaniam G, Muniandy S, Parasakthi N

Int J Infect Dis, 2005 May;9(3):170-2.
PMID: 15840458
Epidemiology and molecular characterization of nosocomially transmitted multidrug-resistant Klebsiella pneumoniae

Parasakthi N, Vadivelu J, Ariffin H, Iyer L, Palasubramaniam S, Arasu A

Int J Infect Dis, 2000;4(3):123-8.
PMID: 11179914

OBJECTIVES: To describe the epidemiology, antimicrobial susceptibility, genomic profiles, and control of a nosocomial outbreak of multidrug-resistant Klebsiella pneumoniae (MRKP) that occurred in the pediatric oncology unit of the University of Malaya Medical Centre in Kuala Lumpur.
MATERIALS AND METHODS: A prospective epidemiologic and microbiologic study was conducted of MRKP isolated from the blood and wound of a boy with necrotizing fasciitis after a 7-day course of ceftazidime and amikacin. In the following 2 weeks, phenotypically similar MRKP were isolated from the blood cultures of four other patients and rectal swabs of another three patients and two liquid soap samples located in the same ward.
RESULTS: Antimicrobial profiles demonstrated that all the isolates were resistant to ceftazidime, sensitive to imipenem and ciprofloxacin, and confirmed to be extended-spectrum beta-lactamase producers. Plasmids of varying molecular weights were present in all isolates. In eight of these isolates, which included four from blood, there were common large molecular weight plasmids ranging from 80 kb to 100 kb. Pulsed-field gel electrophoresis analysis using XbaI demonstrated six different DNA profiles, A to F. Profile A was shared by two blood culture isolates and were related by 91%. Profile B was found in one rectal swab isolate and one isolate from liquid soap and were related by 94%. Profile C was shared by one blood isolate and one liquid soap isolate and showed 100% relatedness. Profiles D, E, and F each were demonstrated by one blood isolate and two rectal swab isolates, respectively. These showed only 65% relatedness.
CONCLUSIONS: The MRKP strains in this outbreak were not clonal in origin. The decline of the outbreak after 4 weeks was attributed to the reemphasis of standard infection control procedures and the implementation of a program that addressed sites of environmental contamination.
Imipenem-resistance in Klebsiella pneumoniae in Malaysia due to loss of OmpK36 outer membrane protein coupled with AmpC hyperproduction

Palasubramaniam S, Karunakaran R, Gin GG, Muniandy S, Parasakthi N

Int J Infect Dis, 2007 Sep;11(5):472-4.
PMID: 17337225
Carbapenem-resistant Pseudomonas aeruginosa in malaysia producing IMP-7 beta-lactamase

Ho SE, Subramaniam G, Palasubramaniam S, Navaratnam P

Antimicrob Agents Chemother, 2002 Oct;46(10):3286-7.
PMID: 12234862

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

Wong JS, Mohd Azri ZA, Subramaniam G, Ho SE, Palasubramaniam S, Navaratnam P

Malays J Pathol, 2003 Dec;25(2):113-9.
PMID: 16196367

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.