Vasanthakumari Neela

Predominance and Emergence of Clones of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus in Malaysia

ABSTRACT We define the epidemiology of predominant and sporadic methicillin-resistant Staphylococcus aureus (MRSA) strains in a central teaching and referral hospital in Kuala Lumpur, Malaysia. This is done on the basis of spa sequencing, multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, and virulence gene profiling. During the period of study, the MRSA prevalence was 44.1%, and 389 MRSA strains were included. The prevalence of MRSA was found to be significantly higher in the patients of Indian ethnicity (P < 0.001). The majority (92.5%) of the isolates belonged to ST-239, spa type t037, and possessed the type III or IIIA SCCmec. The arginine catabolic mobile element (ACME) arcA gene was detected in three (1.05%) ST-239 isolates. We report the first identification of ACME arcA gene-positive ST-239. Apart from this predominant clone, six (1.5%) isolates of ST-22, with two related spa types (t032 and t4184) and a singleton (t3213), carrying type IVh SCCmec, were detected for the first time in Asia. A limited number of community-acquired (CA) MRSA strains were also detected. These included ST-188/t189 (2.1%), ST-1/t127 (2.3%), and ST-7/t091 (1%). Panton-Valentin leukocidin (PVL) was detected in all ST-1 and ST-188 strains and in 0.7% of the ST-239 isolates. The majority of the isolates carried agr I, except that ST-1 strains were agr III positive. Virulence genes seg and sei were seen only among ST-22 isolates. In conclusion, current results revealed the predominance of ST-239-SCCmec III/IIIA and the penetration of ST-22 with different virulence gene profiles. The emergence in Malaysia of novel clones of known epidemic and pathogenic potential should be taken seriously.

Prevalence of ST9 methicillin-resistant Staphylococcus aureus among pigs and pig handlers in Malaysia

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) of sequence type 398 (ST398) has frequently been detected in pigs and pig handlers. However, in Malaysia, sampling 360 pigs and 90 pig handlers from 30 farms identified novel ST9-spa type t4358-staphylococcal cassette chromosome mec type V MRSA strains that were found to transiently colonize more than 1% of pigs and 5.5% of pig handlers.

First report on methicillin-resistant Staphylococcus aureus of Spa type T037, Sequence type 239, SCCmec type III/IIIA in Malaysia

Methicillin-resistant Staphylococcus aureus (MRSA) from Malaysia were shown to possess staphylococcal cassette chromosome mec (SCCmec)-III and IIIA. Spa sequencing and multi-locus sequence typing (MLST) documented t037 and ST 239 (CC8) for 83.3% of the isolates. This confirms observations in several other Far Eastern countries and corroborates the epidemicity of this clone.

Methicillin-susceptible Staphylococcus aureus from clinical and community sources are genetically diverse.

Despite the association of methicillin-susceptible S. aureus (MSSA) with several life-threatening diseases, relatively little is known about their clinical epidemiology in Malaysia. We characterized MSSA isolates (n=252) obtained from clinical and community (carriage) sources based on spa sequencing and multilocus sequence typing (MLST). The prevalence of several important virulence genes was determined to further define the molecular characteristics of MSSA clones circulating in Malaysia. Among the 142 clinical and 110 community-acquired MSSA isolates, 98 different spa types were identified, corresponding to 8 different spa clonal clusters (spa-CCs). In addition, MLST analysis revealed 22 sequence types (STs) with 5 singletons corresponding to 12 MLST-CCs. Interestingly, spa-CC084/085 (MLST-CC15) (p=0.038), spa-non-founder 2 (MLST-ST188) (p=0.002), and spa-CC127 (MLST-CC1) (p=0.049) were identified significantly more often among clinical isolates. spa-CC3204 (MLST-CC121) (p=0.02) and spa-CC015 (MLST-CC45) (p=0.0002) were more common among community isolates. Five dominant MLST-CCs (CC8, CC121, CC1, CC45, and CC5) having clear counterparts among the major MRSA clones were also identified in this study. While the MSSA strains are usually genetically heterogeneous, a relatively high frequency (19/7.5%) of ST188 (t189) strains was found, with 57.8% of these strains carrying the Panton-Valentine leukocidin (PVL). Analysis of additional virulence genes showed a frequency of 36.5% and 36.9% for seg and sei and 0.8% and 6.3% for etb and tst genes, respectively. Arginine catabolic mobile element (ACME) was detected in 4 community isolates only. These represent the first isolates harbouring this gene in an Asian region. In conclusion, MSSA from the Malaysian community and their clinical counterparts are genetically diverse, but certain clones occur more often among clinical isolates than among carriage isolates and vice versa.

In vitro antibacterial effects of Cinnamomum extracts on common bacteria found in wound infections with emphasis on methicillin-resistant Staphylococcus aureus.

ETHNOPHARMACOLOGICAL RELEVANCE Cinnamomum species have been widely used in many traditional systems of medicine around the world. In the Malaysian traditional system of medicine, the leaves, stem bark and stem wood of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum have been used to treat wound infections. To study the antibacterial effects of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum against common bacteria found in wound infections with primary focus on methicillin-resistant Staphylococcus aureus (MRSA). MATERIALS AND METHODS The crude extracts from the leaves, stem-bark and stem-wood of Cinnamomum iners, Cinnamomum porrectum, Cinnamomum altissimum and Cinnamomum impressicostatum were obtained using sequential extraction with hexane, ethylacetate, methanol and water. The volatile oils were obtained by hydro-distillation. The antibacterial activities of extracts were investigated using disk diffusion assays and broth microdilution assays. RESULTS The volatile oils obtained from the stem-bark of Cinnamomum altissimum, Cinnamomum porrectum and Cinnamomum impressicostatum have shown significant antibacterial activity against a wide range of Gram positive and Gram negative bacteria including MRSA. A few test extracts have shown better activity against MRSA as compared to methicillin sensitive Staphylococcus aureus (MSSA). Amongst all the test extracts, Cinnamomum impressicostatum stem-bark water extract produced the largest inhibition zone of 21.0mm against MRSA while its inhibition zone against MSSA was only 8.5mm. The minimum inhibitory concentration (MIC) of this extract against MRSA was 19.5 μg mL(-1) and the corresponding minimum bactericidal concentration (MBC) was 39.0 μg mL(-1). CONCLUSIONS This study has scientifically validated the traditional use of Cinnamomum species in treating wound infections. Of high scientific interest was the observation that the antibacterial effect of Cinnamomum impressicostatum stem-bark crude water extract against MRSA was significantly higher than its effect against MSSA, suggesting that the extract contains a compound(s) with higher specific neutralising activity against the drug resistance markers of MRSA.

Stenotrophomonas maltophilia in Malaysia: molecular epidemiology and trimethoprim-sulfamethoxazole resistance.

OBJECTIVES Stenotrophomonas maltophilia is a recently identified nosocomial pathogen in Malaysia. Despite limited pathogenicity, its rate of isolation has increased in recent years. The aim of this study was to investigate the antibiotic susceptibility patterns, antibiotic resistance determinants, and the epidemiology of S. maltophilia at the largest tertiary care hospital in Malaysia. METHODS This study was carried out from January to December 2008. Sixty-four S. maltophilia isolates were investigated for their antibiotic susceptibility patterns by disk diffusion test and E-test. The antibiotic resistance mechanism for trimethoprim-sulfamethoxazole (TMP-SMX) was assessed by PCR for sul1, sul2, qac/smr, and class 1 integrons in general. Epidemiological relatedness among isolates was determined by pulsed-field gel electrophoresis (PFGE). RESULTS The highest number of S. maltophilia infections was observed in the intensive care unit (ICU) (n=13; 20.3%), while the lowest number of infections was seen in the neurology, psychiatric, and dermatology wards (each n=1; 1.6%). All isolates were susceptible to minocycline. One isolate was resistant to TMP-SMX with a minimum inhibitory concentration (E-test) >32 mg/l. The strain carried the sul1 gene and class 1 integron. None of the isolates were positive for the qac/smr genes. Although the data suggest the potential for patient to patient transmission, most of the S. maltophilia strains showed unrelated PFGE patterns and were considered to be genetically diverse. CONCLUSION The increasing number of S. maltophilia isolates seen in the ICU, their resistance to mainstay antibiotics, their genetically diverse nature, and possible cross-transmission within the hospital, strongly underscores the need for continuous surveillance for S. maltophilia in the hospital setting.

Comparative Exoproteomics and Host Inflammatory Response in Staphylococcus aureus Skin and Soft Tissue Infections, Bacteremia, and Subclinical Colonization

ABSTRACT The exoproteome of Staphylococcus aureus contains enzymes and virulence factors that are important for host adaptation. We investigated the exoprotein profiles and cytokine/chemokine responses obtained in three different S. aureus-host interaction scenarios by using two-dimensional gel electrophoresis (2-DGE) and two-dimensional immunoblotting (2D-IB) combined with tandem mass spectrometry (MS/MS) and cytometric bead array techniques. The scenarios included S. aureus bacteremia, skin and soft tissue infections (SSTIs), and healthy carriage. By the 2-DGE approach, 12 exoproteins (the chaperone protein DnaK, a phosphoglycerate kinase [Pgk], the chaperone GroEL, a multisensor hybrid histidine kinase, a 3-methyl-2-oxobutanoate hydroxymethyltransferase [PanB], cysteine synthase A, an N-acetyltransferase, four isoforms of elongation factor Tu [EF-Tu], and one signature protein spot that could not be reliably identified by MS/MS) were found to be consistently present in more than 50% of the bacteremia isolates, while none of the SSTI or healthy-carrier isolates showed any of these proteins. By the 2D-IB approach, we also identified five antigens (methionine aminopeptidase [MetAPs], exotoxin 15 [Set15], a peptidoglycan hydrolase [LytM], an alkyl hydroperoxide reductase [AhpC], and a haptoglobin-binding heme uptake protein [HarA]) specific for SSTI cases. Cytokine and chemokine production varied during the course of different infection types and carriage. Monokine induced by gamma interferon (MIG) was more highly stimulated in bacteremia patients than in SSTI patients and healthy carriers, especially during the acute phase of infection. MIG could therefore be further explored as a potential biomarker of bacteremia. In conclusion, 12 exoproteins from bacteremia isolates, MIG production, and five antigenic proteins identified during SSTIs should be further investigated for potential use as diagnostic markers.

Oral vaccination with Lactococcus lactis expressing the Vibrio cholerae Wzm protein to enhance mucosal and systemic immunity

A gene associated with lipopolysaccharide (LPS) transport was cloned from a local clinical Vibrio cholerae O1 strain of the Ogawa serotype by using the Lactococcus lactis nisin-controlled expression (NICE) system. The V. cholerae wzm gene, which codes for an integral membrane transporter protein, was expressed and targeted to the cytoplasmic membrane, and was crudely isolated through simple centrifugation and SDS solubilization. To examine seroreactivity of this construct, rabbits were orally fed with 10(9) cfu/ml of live, recombinant L. lactis carrying the wzm gene, induced with nisin prior to administration. Recombinant plasmids were retrieved from L. lactis cultured directly from stool samples of inoculated rabbits. Reverse-transcriptase PCR of wzm using the retrieved plasmids confirmed transcription of this gene, indicating viability and stability of the recombinants in vivo. The L. lactis-Wzm construct elicited substantial levels of IgG and sIgA, and challenge with virulent V. cholerae O1 evoked severe diarrhoea in the naive, non-immunised control group, but not in those fed with either recombinant or non-recombinant L. lactis. Oral administration with recombinant L. lactis expressing the V. cholerae wzm gene increases both systemic and mucosal immunity, whereas L. lactis itself appears capable of protecting against the diarrhoeal symptoms caused by V. cholerae. Wzm is a conserved membrane protein associated with the LPS endotoxin, and together with the food-grade L. lactis, represent an attractive target for the development of a safer, live anti-infective therapy against V. cholerae.

Extracellular enzyme profiling of Stenotrophomonas maltophilia clinical isolates

Stenotrophomonas maltophilia, which is still defined as an organism of limited pathogenicity, has risen prominently as a nosocomial pathogen. Despite the increase in the spectrum of clinical syndromes associated with S. maltophilia, very little is known about the extracellular enzymes profile, pigment production and motility patterns which may have potential roles in pathogenesis. In this study, we screened and compared an array of extracellular enzymes in S. maltophilia collected from invasive and non-invasive clinical specimens by substrate plate assays. We also grouped the isolates as device related and non-device related and compared the enzyme profile. Our study showed all clinical isolates irrespective of source produced substantial levels of enzymes assayed, produced melanin and exhibited swimming and swarming motility pattern. These data suggests clinical isolates of S. maltophilia is a reservoir for pathogenic potential enzymes.

Incidence of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in patients with urinary tract infection

CONTEXT AND OBJECTIVES Resistant bacteria are emerging worldwide as a threat to favorable outcomes from treating common infections in community and hospital settings. The present investigation was carried out to study the incidence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients with urinary tract infection in different seasons of the year, in order to determine the prevalence of the genes blaTEM, blaSHV and blaCTX-M, which are responsible for ESBL production among ESBL-producing K. pneumoniae, in three cities in Iran, and to investigate the antimicrobial susceptibility pattern of K. pneumoniae in different seasons. DESIGN AND SETTING Retrospective study carried out among patients with urinary tract infections in five hospitals in Iran. METHOD Two hundred and eighty-eight clinical isolates of K. pneumoniae were collected between March 2007 and April 2008 from five hospitals in three cities in Iran. ESBLs were identified by phenotypic and genotypic methods. ESBL-producing Klebsiella pneumoniae were evaluated against non-beta-lactam antibiotics. Genes coding for ESBLs (blaSHV, TEM and CTX-M) were screened. RESULTS Among the 288 clinical isolates of K. pneumoniae, 37.7%, 46.7% and 15.6% were obtained from hospitals in Ilam, Tehran and Tabriz, respectively, of which 39.4%, 50.7% and 45.8% were ESBL-producing K. pneumoniae in Ilam, Milad and Emam Reza hospitals, respectively. CONCLUSION According to the results from this study, resistance to third-generation cephalosporins is higher during the cold months than during the warm months.