Investigation of toxin genes among methicillin-resistant Staphylococcus aureus strains isolated from a tertiary hospital in Malaysia.

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.

ermA, ermC , tetM and tetK are essential for erythromycin and tetracycline resistance among methicillin-resistant Staphylococcus aureus strains isolated from a tertiary hospital in Malaysia.

The objective of this study was to determine the expression and transferability of tetracycline and erythromycin resistance among 188 MRSA strains from a Malaysian tertiary hospital. The minimum inhibitory concentrations (MICs) for oxacillin, erythromycin, tetracycline and ciprofloxacin ranged from 4 to 512 µg/ml, 0.25 to 256 µg/ml, 0.5 to 256 µg/ml and 0.5 to 512 µg/ml, respectively. Tetracycline-resistant strains showed co-resistance towards ciprofloxacin and erythromycin. There was a significant increase (P<0.05) of high-level tetracycline (≥MIC 256 µg/ml) and erythromycin (≥MIC 128 µg/ml) resistant strains in between the years 2003 and 2008. All erythromycin-resistant strains harboured ermA or ermC gene and all tetracycline-resistant strains harboured tetM or tetK gene. The blaZ was detected in all MRSA strains, whereas ermA, tetM, ermC, tetK and msrA genes were detected in 157 (84%), 92 (49%), 40 (21%), 39 (21%) and 4 (2%) MRSA strains, respectively. The blaZ, tetM, ermC and tetK genes were plasmid-encoded, with ermC gene being easily transmissible. Tn5801-like transposon was present in 78 tetM-positive strains. ermA and tetM genes were the most prevalent erythromycin and tetracycline resistance determinants, respectively, in MRSA strains. The association of resistance genes with mobile genetic elements possibly enhances the spread of resistant traits in MRSA.

Tsukamurella tyrosinosolvens intravascular catheter-related bacteremia in a haematology patient: a case report.

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.

Isolation of Aggregatibacter aphrophilus from a patient with acute appendicitis.

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.

Application of amplified ribosomal DNA restriction analysis in identification of Acinetobacter baumannii from a tertiary teaching hospital, Malaysia.

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.

Application of amplified ribosomal DNA restriction analysis in identification of Acinetobacter baumannii from a Tertiary Teaching Hospital, Malaysia

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.

Genomic species identification of Acinetobacter of clinical isolates by 16S rDNA sequencing.

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.

Antibiotic susceptibility and REP-PCR fingerprints of Acinetobacter spp. isolated from a hospital ten years apart.

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.

Combined use of ribotyping, PFGE typing and IS431 typing in the discrimination of nosocomial strains of methicillin-resistant Staphylococcus aureus.

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.

Pulsed-field gel electrophoresis of chromosomal DNA of methicillin-resistant Staphylococcus aureus associated with nosocomial infections.

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.

Characterization of methicillin-resistant Staphylococcus aureus associated with nosocomial infections in the University Hospital, Kuala Lumpur.

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.

Methicillin-resistant Staphylococcus aureus (MRSA)--phage-typing of Malaysian and international isolates.

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 in surgical patients: Malaysian experience.

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.

Nosocomial infections in an intensive care unit.

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.