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Background/purpose: Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) continue to be a problem for clinicians worldwide. The objective of this study was to determine the changes in antibiograms of MRSA and their genotypic characteristics., Methods: The antibiograms of 162 MRSA isolates (52 from 2003 and 110 from 2008) from a tertiary hospital were analyzed by antimicrobial susceptibility tests, the Panton-Valentine leukocidin (PVL) and staphylococcal cassette chromosome mec (SCCmec) types were determined by polymerase chain reaction, and genetic relatedness by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)., Results: All the isolates were sensitive to vancomycin. Resistance to ciprofloxacin, clindamycin, erythromycin, and gentamicin remained high throughout the study period, although a small decrease was observed in 2008 for ciprofloxacin (96% to 90%) and gentamicin (90% to 83%). Similarly, a slight decrease in resistance toward fusidic acid (10% to 9%), linezolid (2% to 1%), rifampicin (8% to 4%), and teicoplanin (4% to 0%) was observed between 2003 and 2008. In contrast, there was a significant increase (p < 0.05) in resistance rates toward trimethoprim-sulfamethoxazole, netilmicin, and tetracycline between 2003 and 2008. Ninety-six percent of the isolates from both 2003 and 2008 were multidrug resistant. Three SCCmec types (SCCmec type III, 90%; SCCmec type IV, 9%; SCCmec V, 1%) were observed. SCCmec type IV (n = 15) and pvl gene (n = 3) were detected in 2008 isolates but not in 2003 isolates. Most of the SCCmec type IV isolates (12 of 15) belonged to sequence type 22 (ST22) and were resistant to erythromycin and ciprofloxacin, with 11 being multidrug resistant. Most of the isolates were genetically related (F > 0.8) as determined by PFGE. Some isolates from 6 years apart shared similar PFGE profiles, indicating the persistence of a particular genotype. Five STs (ST239, ST772, ST22, ST6, and ST1178) were identified among the 2008 isolates but only one ST (ST239) was observed in 2003 isolates., Conclusion: Vancomycin remains the most active agent in vitro against S. aureus infection followed by linezolid and teicoplanin. The prevalence of resistance to fluoroquinolones, aminoglycosides (netilmicin), and tetracyclines had increased over the years. The Malaysian multidrug-resistant MRSA isolates were mostly SCCmec type III and ST239, although SCCmec type IV: ST22 is gaining importance. There was a correlation between resistotypes and PFGE profiles., (Copyright © 2013. Published by Elsevier B.V.)

Methicillin sensitive Staphylococcus aureus is an important bacterial pathogen associated with hospital- and community-acquired infections leading to endocarditis, skin tissue infection and pneumonia. The objective of this study was to determine both the genetic characteristics of methicillin-sensitive S. aureus (MSSA) strains, and the occurrence of virulence factors produced by S. aureus strains isolated from UMMC and healthy students in the University from year 2009. Out of 429 nasal swab samples, 67 were MSSA. The prevalence of 21 different virulence genes among 67 Malaysian clinical and community MSSA strains was determined by PCR, and their genetic features were assessed by PCR-RFLP of coa gene, agr types, spa typing and PFGE. The five predominant virulence genes were ica (79 %), efb and fnbA (61 % each), sdrE (57 %) and hlg (45 %). Toxin genes (enterotoxin, etd and pvl) were significantly more common (P < 0.05) in clinical strains compared to community strains. Three agr genotypes were observed: agr type I (45 %), agr type III (25 %) and agr type II (19 %). All 67 MSSA strains were distinguished into 26 profiles by PCR-RFLP of coa, 55 pulsotypes and 21 spa types. Four novel spa types (t7312, t7581, t7582 and t7583) were observed. In conclusion, different virulence profiles were observed in MSSA strains in Malaysia where toxin genes were more prevalent among clinical strains. No correlation between DNA profiles (coa-RFLP, PFGE and spa) and virulotypes was observed. The Malaysian MSSA strains from clinical and community sources were genetically diverse and heterogeneous.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main bacterial pathogens responsible for nosocomial infections leading to pneumonia, bloodstream, skin, and soft tissue infections. The objective of this study was to investigate the genomic changes of MRSA in a tertiary hospital between the years 2003, 2004, 2007, and 2008. One hundred fifty-four MRSA strains were characterized by multilocus sequence typing (MLST), spa, and mec-associated dru typing. Among the 154 strains, 29 different dru, 15 spa, and 8 MLST types were identified. Seven sequence types (STs) (ST239, ST22, ST5, ST6, ST80, ST573, and ST241) were identified among 2007-08 strains, although only 2 STs (ST239 and ST20) were observed among 2003 strains. Clones ST239-t037-dt13g, ST22-t032-(dt10a and dt10aw), and 28 other MRSA clones being introduced in 2007-2008 have replaced the ST239-t037 (dt13d, 14h, 13i, 13l, 13m, 15m, 15l, and 11al) clones present in 2003. The predominant MLST clone, ST239 (90.3%), was further distinguished into 7 different spa types and 26 different dru types, including 17 novel dru types. Maximum parsimony tree based on dru repeats revealed that 10 dru types (dt11am, dt13j, dt15n, dt13q, dt13n, dt13p, dt13f, dt13ao, dt12j, dt7v) shared the same MLST-spa types with dt13d, suggesting that these MRSA clones might have evolved from ST239-t037-dt13d. In conclusion, our data showed that the ST239-t037-dt13d clone and other MRSA clones in 2003 were replaced by ST239-t037-dt13g and other new emerging spa and dru types., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Staphylococcus aureus is a persistent human pathogen responsible for a variety of infections ranging from soft-tissue infections to bacteremia. It produces a variety of virulence factors which are responsible for specific acute staphylococcal toxaemia syndromes. The objective of this study was to determine the prevalence of a repertoire of toxin genes among Malaysian MRSA strains and their genetic diversity by PCR-RFLP of coa gene. One hundred eighty-eight strains (2003, 2004, 2007 and 2008) of methicillin-resistant S. aureus (MRSA) were screened for 20 genes encoding for extracellular virulence determinant (sea, seb, sec, sed, see, seg, seh, sei, sej, tst, eta, etb, etd) and adhesins (cna, etb, fnbA, fnbB, hlg, ica, sdrE). The genetic relatedness of these strains was determined by PCR-RFLP of coa gene and agr grouping. Majority of the strains were tested positive for efb and fnbA (96% each), ica (78%) and hlg (59%) genes. A total of 101 strains were positive for at least one type of staphylococcal enterotoxin genes with sea being the predominant. Genes for seb, sed, see, seh, sej, eta and etb were not detected in any of the MRSA strains. The prevalence of sea, sec and ica among strains isolated in 2008 was increased significantly (p< 0.05) compared to 2003. Most of the strains were of agr type I (97.5%) followed by agr type II (1.2%) and agr type III (0.6%). All sea, sei and tst gene-positive strains were of agr type I. The only etd positive strain was agr type III. PCR-RFLP of coa produced 47 different patterns. The number of strains with virulence factors (sea, sec and ica) had increased over the years. No direct correlation between PCR-RFLP- coa profiles and virulotypes was observed.

Acinetobacter baumannii, genomic species 3 and 13TU are being increasingly reported as the most important Acinetobacter species that cause infections in hospitalized patients. These Acinetobacter species are grouped in the Acinetobacter calcoaceticus- Acinetobacter baumannii (Acb) complex. Differentiation of the species in the Acb-complex is limited by phenotypic methods. Therefore, in this study, amplified ribosomal DNA restriction analysis (ARDRA) was applied to confirm the identity A. baumannii strains as well as to differentiate between the subspecies. One hundred and eighty-five strains from Intensive Care Unit, Universiti Malaya Medical Center (UMMC) were successfully identified as A. baumannii by ARDRA. Acinetobacter genomic species 13TU and 15TU were identified in 3 and 1 strains, respectively. ARDRA provides an accurate, rapid and definitive approach towards the identification of the species level in the genus Acinetobacter. This paper reports the first application ARDRA in genospecies identification of Acinetobacter in Malaysia.

Tsukamurella spp. are a rare but important cause of intravascular catheter-related bacteremia in immunocompromised patients. The organism is an aerobic, Gram-positive, weakly acid-fast bacillus that is difficult to differentiate using standard laboratory methods from other aerobic actinomycetales such as Nocardia spp., Rhododoccus spp., Gordonia spp., and the rapid growing Mycobacterium spp. We report a case of Tsukamurella tyrosinosolvens catheter-related bacteremia in a 51-year-old haematology patient who responded to treatment with imipenem and subsequent line removal. 16srRNA sequencing allowed for the prompt identification of this organism.

Objectives: Acute appendicitis is a common surgical emergency. The etiology and pathophysiology of appendicitis have been well investigated. Aggregatibacter aphrophilus is a fastidious gram-negative coccobacilli. Detection of this organism in clinical samples and its differentiation from Haemophilus aphrophilus or from Aggregatibacter actinomycetemcomitans in routine microbiology settings could be difficult., Methods: In this rare case, we report the isolation of Aggregatibacter aphrophilus from the appendix of a 14-year-old boy presented with acute appendicitis. The genotypic method using 16S rRNA sequencing was used for identification of the organism at species level., Conclusion: This case highlights the importance of detecting fastidious and rare microorganisms such as Aggregatibacter aphrophilus that could be associated with acute appendicitis.

The resistance phenotypes and genomic diversity of 185 Acinetobacter baumannii isolates obtained from the intensive care unit (ICU) of a local teaching hospital in Kuala Lumpur from 2006 to 2009 were determined using antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Antibiogram analyses showed that the isolates were fully resistant to β-lactam antimicrobials and had high resistance rates to the other antimicrobial agents tested. However, the isolates were susceptible to polymyxin B. Resistance to cefoperazone/sulbactam was only detected in strains isolated from 2007 to 2009. Some environmental isolates and an isolate from the hands of a healthcare worker (HCW) had identical resistance profiles and PFGE profiles that were closely related to patient isolates. Cluster analyses based on the PFGE profiles showed there was a persistent clone of endemic isolates in the ICU environment. The transmission route from HCWs to fomites to patients, which caused a long-term infection in the ICU of the University Malaya Medical Centre, was observed in this study. These data provide a better understanding of A. baumannii epidemiology within the hospital and the possible transmission routes. Knowledge of changes in the resistance rates of A. baumannii in our local hospital will improve antimicrobial therapy.

The genetic diversity and antimicrobial resistance rates of clinical Salmonella isolates (2007-2008) at the University of Malaya Medical Centre, Kuala Lumpur, were investigated and the genetic diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic (REP)-PCR. XbaI-PFGE analysis generated 57 profiles (Dice coefficient, F=0.08-1.00), whereas REP-PCR using the REP primer generated only 35 (F=0.34-1.00). PFGE was therefore the more discriminative and reproducible method for assessing the genetic diversity of salmonellae. The antibiograms of 78 Salmonella isolates were assessed against 19 antimicrobials using the disk diffusion method. Twenty serotypes were identified, with the most common being S. Enteritidis (18%) followed by S. Typhimurium (14%), S. Paratyphi B var Java (9%), S. Weltevreden (9%), and S. Corvallis (9%). A total of 38 resistant profiles were defined, with 53.8% of the isolates being resistant to three or more antimicrobials. The highest resistance rates were observed for cephalothin (55.1%), tetracycline (47.4%), and nalidixic acid (35.9%). The presence of multidrug-resistant Salmonella strains is a cause for concern as it may limit the treatment of severe salmonellosis. One multidrug-resistant S. Enteritidis strain was a putative extended-spectrum beta-lactamase producer, based on a double disk diffusion analysis, and was resistant to ceftriaxone (MIC>32 microg/mL). The data generated by this study will contribute towards epidemiological monitoring and investigations of Salmonella infections in Malaysia.

Mupirocin is used topically to treat skin infection caused by methicillin-resistant Staphylococcus aureus (MRSA). One hundred eighty-eight strains (isolated in 2003, 2004, 2007, and 2008) were tested for mupirocin susceptibility using disk diffusion method and minimum inhibitory concentration (MIC). Mupirocin resistance was detected in 10 (5%) strains with 2 of them showing MIC of 256 mg/l. PCR detection using gene-specific primers showed that all 10 mupirocin-resistant strains harbored ileS2 gene whereas mupA gene was detected in 2 mupirocin-resistant strains with MIC of 256 mg/l. Amplification of agr grouping and SCCmec typing showed that all 10 strains were agr group I and SCCmec type III. Sequence analysis of region X of the spa gene yielded 4 distinct spa types (t037, t363, t421, and t6405) which were clonally related. In conclusion, the rate of mupirocin resistance in Malaysia is still low but is much higher than previous reports in Malaysia.

The objectives of this study were to determine the antibiotypes, SCCmec subtypes, PVL carriage, and genetic diversity of MRSA strains from a tertiary hospital. Sixtysix MRSA strains were selected randomly (2003, 2004, and 2007) and tested for the Panton-Valentine leukocidin gene, mecA gene, and SCCmec type via a PCR. The antibiograms were determined using a standard disc diffusion method, and the genetic diversity of the isolates was determined by PFGE. Thirty-four antibiograms were obtained, with 55% of the 66 strains exhibiting resistance to more than 4 antimicrobials. All the isolates remained susceptible to vancomycin, and low resistance rates were noted for fusidic acid (11%), rifampicin (11%), and clindamycin acid (19%). The MRSA isolates that were multisensitive (n=12) were SCCmec type IV, whereas the rest (multiresistant) were SCCmec type III. Only two isolates (SCCmec type IV) tested positive for PVL, whereas all the isolates were mecA-positive. The PFGE was very discriminative and subtyped the 66 isolates into 55 pulsotypes (F=0.31-1.0). The multisensitive isolates were distinctly different from the multidrug-resistant MRSA. In conclusion, no vancomycin-resistant isolate was observed. The Malaysian MDR MRSA isolates were mostly SCCmec type III and negative for PVL. These strains were genetically distinct from the SCCmec type IV strains, which were sensitive to SXT, tetracycline, and erythromycin. Only two strains were SCCmec IV and PVL-positive. The infections in the hospital concerned were probably caused by multiple subtypes of MRSA.

We report a 17-year-old Malay boy with cystic fibrosis who over a 14-month period experienced worsening respiratory symptoms and deteriorating lung function. Burkholderia pseudomallei was eventually isolated from his sputum. He improved clinically following treatment for meliodosis and his lung function returned to normal.

Introduction: This study aims to identify Acinetobacter of clinical isolates from the University of Malaya Medical Centre (UMMC), Kuala Lumpur, to the species level by 16S rDNA sequencing., Methods: 12 representative Acinetobacter isolates of the UMMC inpatients were randomly picked and used for the study. The 16S rDNA sequences were determined and phylogenetic relationships to all known Acinetobacter species were established., Results: Based on the 16S rDNA sequences, all the UMMC isolates were identified as Acinetobacter baumannii. The isolates shared a common ancestral lineage with the prototypes Acinetobacter baumannii DSM30007 and DSM30008 with 99-100 percent sequence similarities. The isolates could be differentiated into two groups by a single nucleotide difference (thymine-cytosine) within the 16S rRNA sequence. Three different genotypes, 1, 3 and 4, were recognised using REP-PCR., Conclusion: The previously uncharacterised Acinetobacter isolates from the UMMC were identified by their 16S rDNA sequences as Acinetobacter baumannii. The isolates were distinguished into at least three different genotypes by REP-PCR genotyping. These findings confirmed for the first time the presence of Acinetobacter baumannii of different genotypes among patients at UMMC.

The antibiotic susceptibility profiles and the repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR)-determined genotypes of 109 Acinetobacter strains collected from the University Malaya Medical Center (UMMC), Kuala Lumpur, Malaysia, in 1987 (N=21) and 1996-1998 (N=88) were established. Twelve antibiotic susceptibility profiles of antibiotics used at the UMMC were obtained. In descending order of effectiveness, imipenem, amikacin and ciprofloxacin were the most effective against the Acinetobacter strains. Compared with 1987 isolates, the isolates obtained in 1996-1998 had decreased susceptibility to these antibiotics and were tolerant to the antibiotics up to an MIC90 of > or =256 mg/L. REP-PCR DNA fingerprints of all the isolates revealed the presence of four Acinetobacter spp. lineages; 92% of all the isolates belonged to two dominant lineages (genotypes 1 and 4). Genotype 4 isolates predominant in 1987 showed increased resistance and antibiotic tolerance to imipenem, amikacin and ciprofloxacin compared with the 1996-1998 isolates. In contrast, genotype 1 isolates from 1996-1998 were mainly sensitive to these antibiotics. These findings demonstrate the presence of at least two independent Acinetobacter spp. lineages in the same hospital, and suggest the possibility that genotype 4 Acinetobacter spp. acquired the resistance phenotype in situ, whereas most of the genotype 1 isolates were probably introduced to the hospital in recent years.

We have previously reported the phenotypic characterization of methicillin-resistant Staphylococcus aureus (MRSA) clinical strains isolated in Malaya University Hospital in the period 1987 to 1989 using antibiogram, coagulase typing, plasmid profiles, and phage typing. Here, we report the analysis of the same strains with three genotyping methods; ribotyping, pulsed-field gel electrophoresis (PFGE) typing, and IS431 typing (a restriction enzyme fragment length polymorphism analysis using an IS431 probe). Ribotyping could discriminate 46 clinical MRSA strains into 5 ribotypes, PFGE typing into 22 types, and IS431 typing into 15 types. Since the differences of the three genotyping patterns from strain to strain were quite independent from one another, the combined use of the three genotyping methods could discriminate 46 strains into 39 genotypes. Thus, the powerful discriminatory ability of the combination was demonstrated.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has been endemic in the University Hospital, Kuala Lumpur since the late 1970s. Fifty isolates of MRSA obtained from clinical specimens of patients with nosocomial infections associated with this organism have been studied by pulsed-field gel electrophoresis (PFGE) of its chromosomal DNA fragments to discrimate between strains and to identify the predominant strain. Twenty-one chromosomal patterns were observed which could be further grouped into nine types. The predominant strain was Type 9-b (40% of isolates) found mainly in the Orthopaedic and Surgical Units. Outbreak strains found in the Special Care Nursery were of Type 1, entirely different from those of the surgical ward S2, which were of Type 9-b. Type 8 strains were found mainly at one end of the hospital building where the maternity, paediatric and orthopaedic units were situated. Genomic DNA fingerprinting by PFGE is recommended as a useful and effective tool for the purpose of epidemiological studies of MSRA infections, particularly for nosocomial infections.

Methicillin-resistant Staphylococcus aureus (MRSA) as a hospital pathogen has presented many clinical problems in the University Hospital, Kuala Lumpur, Malaysia since 1978. The need for control of spread of these organisms became evident by 1985 when it was noted that the incidence of MRSA among S. aureus isolated from hospital inpatients had increased from 11.5% in 1979 to 18.8% in 1985. The characteristics of 50 MRSA isolates associated with nosocomial infections in the hospital are described here. The predominant strains produced Type IV coagulase and 84% of isolates studied showed moderate to high resistance to methicillin with MIC values of 25 mg l-1 or higher. All the MRSA isolates that could be phagetyped were susceptible to Group III phages, with 76.6% of the isolates being susceptible to phage 85. At least 10 different patterns were distinguishable by plasmid typing, the majority of isolates harbouring up to four small plasmids.

Twenty-one isolates of methicillin-resistant Staphylococcus aureus (MRSA) from Malaysia (M-MRSA) derived from various sources associated with nosocomial infections were phage-typed and compared with 54 international isolates associated with epidemic and sporadic episodes of infections. It appeared that the majority of M-MRSA were non-typable by the international basic set of phages. Two (9.5%) were typed by phage 85. Phage-typing of MRSA revealed that the strains were almost completely restricted to phage groups III and a lesser portion to phage groups I and III.

Methicillin-resistant Staphylococcus aureus has emerged as an important cause of nosocomial infections in recent years. During 1988 in the Department of Surgery of the University Hospital in Kuala Lumpur, Malaysia, 148 patients were shown to be infected or colonized with these organisms. The patients at risk were those who stay in hospital for greater than 14 days, those over 50 years of age, patients who underwent neurosurgery, cardiothoracic surgery, or were admitted with major burns. Of the 148 patients, 78 (52.7%) were clinically infected, the remaining 70 being colonized. A total of 28 patients died (18.9%) but only five (3.4%) as a direct result of this infection. The estimated annual cost of controlling the organism was found to be approximately MR$250,000. (50,000 pounds). This nosocomial infection therefore represents a serious problem, especially in developing countries where health funding and health facilities are limited.

A total of 676 patients were admitted to the intensive care unit, University Hospital, Kuala Lumpur between January 1989 and March 1990. Fifty-one hospital-acquired infections were recorded, giving a rate of 7.6%. The most frequent site of infection was the respiratory tract (41.2%), followed by the urinary tract (27.5%). Most of the pathogens were gram-negative bacilli (71%). The three most common pathogens were Klebsiella species, Pseudomonas aeruginosa and Staphylococcus aureus.