Determination of antibacterial activity and minimum inhibitory concentration of larval extract of fly via resazurin-based turbidometric assay.

BACKGROUND: Antimicrobial resistance is currently a major global issue. As the rate of emergence of antimicrobial resistance has superseded the rate of discovery and introduction of new effective drugs, the medical arsenal now is experiencing shortage of effective drugs to combat diseases, particularly against diseases caused by the dreadful multidrug-resistant strains, such as the methicillin-resistant Staphylococcus aureus (MRSA). The ability of fly larvae to thrive in septic habitats has prompted us to determine the antibacterial activity and minimum inhibitory concentrations (MICs) of larval extract of flies, namely Lucilia cuprina, Sarcophaga peregrina and Musca domestica against 4 pathogenic bacteria [Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa and Escherichia coli] via a simple and sensitive antibacterial assay, resazurin-based turbidometric (TB) assay as well as to demonstrate the preliminary chemical profile of larval extracts using gas chromatography-mass spectrophotometry (GC-MS). RESULTS: The resazurin-based TB assay demonstrated that the L. cuprina larval extract was inhibitory against all tested bacteria, whilst the larval extract of S. peregrina and M. domestica were only inhibitory against the MRSA, with a MIC of 100 mg ml-1. Subsequent sub-culture of aliquots revealed that the larval extract of L. cuprina was bactericidal against MRSA whilst the larval extracts of S. peregrina and M. domestica were bacteriostatic against MRSA. The GC-MS analysis had quantitatively identified 20 organic compounds (fatty acids or their derivatives, aromatic acid esters, glycosides and phenol) from the larval extract of L. cuprina; and 5 fatty acid derivatives with known antimicrobial activities from S. peregrina and M. domestica. CONCLUSION: The resazurin-based turbidometric assay is a simple, reliable and feasible screening assay which evidently demonstrated the antibacterial activity of all fly larval extracts, primarily against the MRSA. The larval extract of L. cuprina exerted a broad spectrum antibacterial activity against all tested bacteria. The present study revealed probable development and use of novel and effective natural disinfectant(s) and antibacterial agent(s) from flies and efforts to screen more fly species for antibacterial activity using resazurin-based TB assay should be undertaken for initial screening for subsequent discovery and isolation of potential novel antimicrobial substances, particularly against the multi-drug resistant strains.

Community-acquired necrotising pneumonia caused by Panton-Valentine leucocidin-producing methicillinresistant Staphylococcus aureus

A 61-year-old male presented with community-onsetpneumonia not responding to treatment despite givenappropriate antibiotics. Computed tomography scan of thethorax showed large multiloculated pleural effusion withmultiple cavitating foci within collapsed segments; lesionswhich were suggestive of necrotising pneumonia. Drainageof the effusion and culture revealed methicillin-resistantStaphylococcus aureus, which had the same antibioticprofile with the blood isolate and PVL gene positive.

Resistotype of Helicobacter pylori isolates: the impact on eradication outcome.

Antibiotic resistance is increasing worldwide, and it has been regarded as the main factor reducing the efficacy of Helicobacter pylori therapy. The aim of this study was to determine the phenotype and genotype of antibiotic-resistant strains of H. pylori in the Malaysian population and to evaluate the impact of antibiotic resistance to eradication outcome. One hundred and sixty-one H. pylori isolates were analysed in this study. Metronidazole, clarithromycin, fluoroquinolone, amoxicillin and tetracycline susceptibilities were determined by Etest. PCR followed by DNA sequencing was carried out to determine mutations. The medical records of the patients infected with resistant strains were reviewed to determine the eradication outcome. Metronidazole resistance was encountered in 36.6 % of H. pylori isolates, whereas clarithromycin and fluoroquinolone resistance was observed in 1.2  and 1.9 % of isolates, respectively. All strains tested were susceptible to amoxicillin and tetracycline. Frameshift and nonsense mutations in rdxA and frxA genes resulting in stop codons contributed to metronidazole resistance, which leads to reduced eradication efficacy. A2142G and A2143G mutations of 23S rRNA were identified as causing failure of the eradication therapy. Mutation at either codon 87 or 91 of the gyrA gene was identified in fluoroquinolone-resistant strains. However, the effect of resistance could not be assessed. This study showed that frameshift and nonsense mutations in rdxA or frxA genes and point mutations in the 23S rRNA affected the efficacy of H. pylori eradication therapy.

Ethnicity association of Helicobacter pylori virulence genotype and metronidazole susceptibility.

AIM: To characterise the cag pathogenicity island in Helicobacter pylori (H. pylori) isolates by analysing the strains' vacA alleles and metronidazole susceptibilities in light of patient ethnicity and clinical outcome. METHODS: Ninety-five H. pylori clinical isolates obtained from patients with dyspepsia living in Malaysia were analysed in this study. Six genes in the cagPAI region (cagE, cagM, cagT, cag13, cag10 and cag67) and vacA alleles of the H. pylori isolates were identified by polymerase chain reaction. The isolates' metronidazole susceptibility was also determined using the E-test method, and the resistant gene was characterised by sequencing. RESULTS: More than 90% of the tested isolates had at least one gene in the cagPAI region, and cag67 was predominantly detected in the strains isolated from the Chinese patients, compared with the Malay and Indian patients (P < 0.0001). The majority of the isolates (88%) exhibited partial deletion (rearrangement) in the cagPAI region, with nineteen different patterns observed. Strains with intact or deleted cagPAI regions were detected in 3.2% and 8.4% of isolates, respectively. The prevalence of vacA s1m1 was significantly higher in the Malay and Indian isolates, whereas the isolates from the Chinese patients were predominantly genotyped as vacA s1m2 (P = 0.018). Additionally, the isolates from the Chinese patients were more sensitive to metronidazole than the isolates from the Malay and Indian patients (P = 0.047). Although we attempted to relate the cagPAI genotypes, vacA alleles and metronidazole susceptibilities to disease outcome, no association was observed. The vacA alleles were distributed evenly among the strains with intact, partially deleted or deleted cagPAI regions. Interestingly, the strains exhibiting an intact cagPAI region were sensitive to metronidazole, whereas the strains with a deleted cagPAI were more resistant. CONCLUSION: Successful colonisation by different H. pylori genotypes is dependent on the host's genetic makeup and may play an important role in the clinical outcome.

Prevalence Study of Candidiasis by Conventional Pap Smear at Sarawak General Hospital, Malaysia / Jurnal Sains Kesihatan Malaysia

This study was conducted to determine the prevalence of candidiasis among women of 20 to 59 years of age at Sarawak General Hospital by examining the conventional Pap smear. The prevalence of this infection was studied among the five main ethnic groups comprising of Iban, Chinese, Malay, Bidayuh and Orang Ulu. Microscopic screening of 300 conventional Pap smears which had been processed and stained with the Papanicolaou staining method was conducted. Based on the information provided in the Pap smear screening form, the clinical manifestations were observed which may provide some indications on the type of infection. The details of the clinical manifestations were very important because in an infection or inflammation, Pap smears would usually demonstrate the cyto-morphological changes of the epithelial cells. The age factor among the different ethnics also plays important role in the assessment of the infection frequency. Candida sp. was detected in 12.7% of the specimens. The infection occurred in all ethnic groups as well as in all age groups. All cases presented with clinical signs and symptoms. The results of this study revealed the importance of undergoing Pap smear screening for candidiasis detection besides screening for the early detection of cervical cancer

The presence of heterogeneous vancomycin-lntermediate Staphylococcus aureus (heteroVISA) in a major Malaysian hospital.

This study was conducted to detect the presence of heterogenous vancomycin-intermediate Staphylococcus aureus (heteroVISA) among MRSA isolates in a major hospital. Forty-three MRSA isolates with vancomycin MIC 2 microg/ml collected in 2009 was screened for heteroVISA using Etest Glycopeptide Resistance Detection (GRD) and confirmed by population analysis profile-area under curve method. The genetic relatedness of heteroVISA strains with other MRSA was examined by pulsed-field gel electrophoresis (PFGE) method. Two isolates were shown to be heteroVISA and derived from the same clone. This showed that heteroVISA strains were already present among our local strains since 2009 and were genetically related to other susceptible strains.

The Presence of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (heteroVISA) in a Major MalaysianHospital

This study was conducted to detect the presence of heterogenous vancomycin-intermediate Staphylococcus aureus (heteroVISA) among MRSA isolates in a major hospital. Forty-three MRSA isolates with vancomycin MIC 2 μg/ml collected in 2009 was screened for heteroVISA using Etest Glycopeptide Resistance Detection (GRD) and confirmed by population analysis profile-area under curve method. The genetic relatedness of heteroVISA strains with other MRSA was examined by pulsed-field gel electrophoresis (PFGE) method. Two isolates were shown to be heteroVISA and derived from the same clone. This showed that heteroVISA strains were already present among our local strains since 2009 and were genetically related to other susceptible strains.

The discrimination of d-tartrate positive and d-tartrate negative S. enterica subsp. enterica serovar Paratyphi B isolated in Malaysia by phenotypic and genotypic methods

Serotyping is not suffi cient to differentiate between Salmonella species that cause paratyphoid fever from the strains that cause milder gastroenteritis as these organisms share the same serotype Salmonella Paratyphi B (S. Paratyphi B). Strains causing paratyphoid fever do not ferment d-tartrate and this key feature was used in this study to determine the prevalence of these strains among the collection of S. Paratyphi B strains isolated from patients in Malaysia. A total of 105 isolates of S. Paratyphi B were discriminated into d-tartrate positive (dT+) and d-tartrate negative (dT-) variants by two lead acetate test protocols and multiplex PCR. The lead acetate test protocol 1 differed from protocol 2 by a lower inoculum size and different incubation conditions while the multiplex PCR utilized 2 sets of primers targeting the ATG start codon of the gene STM3356. Lead acetate protocol 1 discriminated 97.1% of the isolates as S. Paratyphi B dT+ and 2.9% as dT- while test protocol 2 discriminated all the isolates as S. Paratyphi B dT+. The multiplex PCR test identifi ed all 105 isolates as S. Paratyphi B dT+ strains. The concordance of the lead acetate test relative to that of multiplex PCR was 97.7% and 100% for protocol 1 and 2 respectively. This study showed that S. Paratyphi B dT+ is a common causative agent of gastroenteritis in Malaysia while paratyphoid fever appears to be relatively uncommon. Multiplex PCR was shown to be a simpler, more rapid and reliable method to discriminate S. Paratyphi B than the phenotypic lead acetate test.

Occurrence of virulent genes among environmental isolates of Legionella pneumophila serogroup 1 strains from various parts of peninsular Malaysia

Legionella pneumophila are intracellular pathogens, associated with human disease, attributed to the presence and absence of certain virulent genes. In this study, virulent gene loci (lvh and rtxA regions) associated with human disease were determined. Thirty-three cooling tower water isolates, isolated between 2004 to 2006, were analyzed for the presence of these genes by PCR method. Results showed that 19 of 33 (57.5%) of the L. pneumophila serogroup 1 isolates have both the genes. Six (18.2%) of the isolates have only the lvh gene and 2 (6.1%) of the isolates have only the rtxA gene. However, both genes were absent in 6 (18.2%) of the L. pneumophila isolates. The result of our study provides some insight into the presence of the disease causing L. pneumophila serogroup 1 in the environment. Molecular epidemiological studies will provide better understanding of the prevalence of the disease in Malaysia.