The emergence of Acinetobacter baumannii ST-6 multidrug resistant: A resilient biofilm producer, Malaysia

Aims: The aim of this research is to explore the presence of multidrug-resistance (MDR) Acinetobacter baumannii strains isolated from hospitalized patients in a tertiary-care center, Subang Jaya, Selangor, Malaysia and to compare their genotypic and phenotypic characteristics. Methodology and results: Clonal relationships were determined by multilocus sequence typing (MLST) and biofilm forming ability was evaluated by using 2, 3 - bis (2 - methoxy - 4 - nitro - 5-sulfophenyl) - 5 - [(phenylamino) carbonyl] - 2H-tetrazolium hydroxide (XTT) reduction assay in microplates and Congo red agar method (CRA). Four virulence genes coding for A. baumannii pilus usher-chaperone assembly protein, csuE gene; outer membrane protein, ompA gene; biofilm poly-β-1, 6-Nacetylglucosamine (PNAG) synthesis protein, pgaA gene; and acinetobactin-mediated iron acquisition protein, bauA gene were searched for in a collection of strains. Antimicrobial resistance against 11 antibiotics were studied by broth microdilution method. Seventeen A. baumannii clinical strains were isolated and MLST showed that the strains belonged to 5 distinct sequence types (STs), namely, ST-6, ST-265, ST-324, ST-325 and ST-432. Fiftythree percent of the strains were resistant to 4 or more antibiotics. Twelve strains produced biofilm and out of them, 4 were strong biofilm producer, besides, these strong biofilm producers were MDR strains and belongs to ST-6. In addition, all strains were ompA positive, biofilm producing strains were csuE and pgaA positive and only strong biofilm producing strains were bauA positive. Conclusion, significance and impact study: Our study demonstrates that the ST-6 strains in Malaysia could represent MDR, capable of forming strong biofilm and possess csuE, ompA, pgaA and bauA genes, virulence characteristics that probably help the bacteria to persist and cause infection.

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.

Childhood invasive pneumococcal disease: a hospital-based study from Malaysia.

AIM: New conjugate vaccine for Streptococcus pneumoniae has been introduced in Malaysia recently. Information on infection due to S. pneumoniae in Malaysian children is scarce. We conducted a retrospective chart review of childhood invasive pneumococcal disease (IPD) presented to a single centre in Malaysia. METHODS: A retrospective review of 5 years and 4 months of all cases of IPD in children younger than 14 years of age (May 1999-August 2004) seen at the University of Malaya Medical Centre (UMMC), Kuala Lumpur, was conducted. Cases were identified from the record of Department of Medical Microbiology, UMMC. RESULTS: IPD was identified in 50 children (median age 1.1 years, range 2 weeks-14 years) during the study period. Seventy-six per cent of the cases were younger than 2 years of age. Pattern of infections noted include definite pneumonia (n = 8), probable pneumonia (n = 33), meningitis (n = 4), bacteraemia without focus (n = 4) and septic arthritis (n = 1 each). Pre-morbid diseases were present in 28% of all cases. Complications (n = 12, 24%) due to IPD were seizures (n = 5), pleural effusion/empyema (n = 4), cerebral palsy (n = 2) and deafness (n = 1). No deaths were attributed to IPD. Sixty-two per cent of the pneumococcal isolates were penicillin non-susceptible and were detected throughout the study period. CONCLUSIONS: IPD is associated with high morbidity, particularly among young children. Majority of the isolates were penicillin-non-susceptible strains. Additional information on the serotype of S. pneumoniae isolated is necessary to assess the potential impact of immunisation on preventing pneumococcal infection in Malaysia.

Ethnicity and H. pylori as risk factors for gastric cancer in Malaysia: A prospective case control study.

OBJECTIVE: To determine the risk factors for gastric cancer (GCA), with particular emphasis on ethnicity in our multiracial population. METHODS: A prospective case control study with ratio of cancer:controls of 1:2. Diagnosis of H. pylori was made by serology using the ELISA technique. Dietary intake was assessed by dietary recall over the preceding 6 months. RESULTS: Eighty-seven cases of GCA were enrolled. The cancers were predominantly distal in location and of the intestinal type. Risk factors identified following multiple logistic regression analysis were: Chinese race (OR 10.23 [2.87-36.47]), H. pylori (OR 2.54 [1.16-5.58]), low level of education (OR 9.81 [2.03-47.46]), smoking (OR 2.52 [1.23-5.15]), and high intake of salted fish and vegetables (OR 5.18 [1.35-20.00]) were identified as significant independent risk factors for GCA, while high intake of fresh fruits and vegetables was protective for GCA (OR 0.15 [0.04-0.64]). Chili intake was not a significant protective factor following multivariate analysis. CONCLUSIONS: Chinese race was a strong independent predictor of GCA. H. pylori was an important predictor of GCA with a 2.5-fold greater risk in our patients. Despite a high prevalence of H. pylori, the prevalence of GCA among Indians was low and this paradox can be appropriately called the "Indian enigma."

High prevalence of antimicrobial resistance among clinical Streptococcus pneumoniae isolates in Asia (an ANSORP study).

A total of 685 clinical Streptococcus pneumoniae isolates from patients with pneumococcal diseases were collected from 14 centers in 11 Asian countries from January 2000 to June 2001. The in vitro susceptibilities of the isolates to 14 antimicrobial agents were determined by the broth microdilution test. Among the isolates tested, 483 (52.4%) were not susceptible to penicillin, 23% were intermediate, and 29.4% were penicillin resistant (MICs >/= 2 mg/liter). Isolates from Vietnam showed the highest prevalence of penicillin resistance (71.4%), followed by those from Korea (54.8%), Hong Kong (43.2%), and Taiwan (38.6%). The penicillin MICs at which 90% of isolates are inhibited (MIC(90)s) were 4 mg/liter among isolates from Vietnam, Hong Kong, Korea, and Taiwan. The prevalence of erythromycin resistance was also very high in Vietnam (92.1%), Taiwan (86%), Korea (80.6%), Hong Kong (76.8%), and China (73.9%). The MIC(90)s of erythromycin were >32 mg/liter among isolates from Korea, Vietnam, China, Taiwan, Singapore, Malaysia, and Hong Kong. Isolates from Hong Kong showed the highest rate of ciprofloxacin resistance (11.8%), followed by isolates from Sri Lanka (9.5%), the Philippines (9.1%), and Korea (6.5%). Multilocus sequence typing showed that the spread of the Taiwan(19F) clone and the Spain(23F) clone could be one of the major reasons for the rapid increases in antimicrobial resistance among S. pneumoniae isolates in Asia. Data from the multinational surveillance study clearly documented distinctive increases in the prevalence rates and the levels of antimicrobial resistance among S. pneumoniae isolates in many Asian countries, which are among the highest in the world published to date.

Macrolide resistance and genotypic characterization of Streptococcus pneumoniae in Asian countries: a study of the Asian Network for Surveillance of Resistant Pathogens (ANSORP).

OBJECTIVES: To characterize mechanisms of macrolide resistance among Streptococcus pneumoniae from 10 Asian countries during 1998-2001. METHODS: Phenotypic and genotypic characterization of the isolates and their resistance mechanisms. RESULTS: Of 555 isolates studied, 216 (38.9%) were susceptible, 10 (1.8%) were intermediate and 329 (59.3%) were resistant to erythromycin. Vietnam had the highest prevalence of erythromycin resistance (88.3%), followed by Taiwan (87.2%), Korea (85.1%), Hong Kong (76.5%) and China (75.6%). Ribosomal methylation encoded by erm(B) was the most common mechanism of erythromycin resistance in China, Taiwan, Sri Lanka and Korea. In Hong Kong, Singapore, Thailand and Malaysia, efflux encoded by mef(A) was the more common in erythromycin-resistant isolates. In most Asian countries except Hong Kong, Malaysia and Singapore, erm(B) was found in >50% of pneumococcal isolates either alone or in combination with mef(A). The level of erythromycin resistance among pneumococcal isolates in most Asian countries except Thailand and India was very high with MIC(90)s of >128 mg/L. Molecular epidemiological studies suggest the horizontal transfer of the erm(B) gene and clonal dissemination of resistant strains in the Asian region. CONCLUSION: Data confirm that macrolide resistance in pneumococci is a serious problem in many Asian countries.

Comparison of susceptibility test methods to detect penicillin susceptibility in Streptococcus pneumoniae isolates.

The increasing prevalence of penicillin-resistant Streptococuus pneumoniae urges for fast and accurate susceptibility testing methods. This study evaluated the comparability of three commonly used techniques; disk diffusion, E-test and agar dilution, to detect penicillin susceptibility in clinical isolates of S. pneumoniae. Fifty pneumococcal isolates, obtained from patients at the University of Malaya Medical Centre, were selected to include both penicillin-susceptible strains and those that had decreased susceptibility (resistant and intermediate) to penicillin. The minimum inhibitory concentration (MIC) values of penicillin to serve as the reference was determined by the agar dilution method in which, based on the MIC breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS), 27 strains had decreased susceptibility to penicillin with 17 strains resistant and 10 intermediate. Comparing to the agar dilution method, oxacillin disk diffusion test detected all strains with decreased penicillin susceptibility as such while E-test showed a close agreement of susceptibility (92%) of the isolates to penicillin. This confirmed that oxacillin is a good screening test for S. pneumoniae isolates with decreased susceptibility to penicillin while E-test is very reliable for rapid and accurate detection of penicillin susceptibility.

Penicillin susceptibility and molecular characteristics of clinical isolates of Streptococcus pneumoniae at the University of Malaya Medical Center, Kuala Lumpur, Malaysia.

OBJECTIVE: To determine the prevalence of penicillin resistance and molecular characteristics of pneumococcal isolates at the University of Malaya Medical Center. METHODS: From March 1999 to July 2000, 100 clinical isolates of Streptococcus pneumoniae were obtained from 93 patients of various ages and from various body sites. The minimum inhibitory concentrations (MICs) for penicillin and ceftriaxone were determined by E test, and results were interpreted according to guidelines recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Fifty isolates were further serotyped, and analyzed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) of the penicillin-binding protein (pbp) 2b and 2x genes. RESULTS: The majority of the isolates were from respiratory sites. Thirty-one isolates showed decreased susceptibility to penicillin (PRSP), and many of these also showed decreased susceptibility to ceftriaxone. Twelve serogroup/types (SGTs) were present, with 19F being the most common. PFGE analysis identified two dominant profiles, consisting mainly of PRSPs that had common serotypes (19F) and pbp gene patterns within their respective groups, although PCR-RFLP analysis showed different patterns of pbp genes among the PRSPs as compared to penicillin-susceptible strains, which had a uniform pattern. CONCLUSION: PRSPs were genetically related as shown by PFGE and serotype. The consistency of pbp gene patterns, observed among many of the PRSPs within their respective PFGE profiles, supported their relatedness as established by PFGE.

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.