Molecular Profiling and Antibiotic Resistance of Salmonella Enterica Subsp. Enterica Isolated from Indigenous Ulam and Poultry Meat

Salmonella enterica subsp. enterica formed the major group that represents nearly 60% of the salmonellae. Salmonella organisms emerged as a public health problem in many countries as salmonellosis has become the most prevalent foodborne disease worldwide. It has been estimated that approximately 1.4 million cases were reported annually in the developed nations such as USA. In Malaysia, of 8,640 cases of food poisoning reported by the Ministry of Health for the year 1999, 811 (9.4%) were due to Salmonella. The purpose of this study was to characterize and study Salmonella enterica subsp. enterica (S. enterica) using multiple antimicrobial resistance and several molecular typing methods including plasmid profiling, PCR-RFLP, RAPD, ERIC-PCR and Multiplex PCR on antibiotic resistant gene. The isolate were recovered from poultry meat (55), four types of indigenous vegetables namely ‘selom’ (Oenanthe stolonifera) (59), ‘pegaga’ (Centella asiatica) (20), ‘kesum’ (Polygonum minus) (41), ‘kangkong’ (Ipomoea aquatica) (14) and processed food (11).Genomic DNA of the 200 S. enterica isolates belonging to 43 different serovars were recovered from poultry meat, various indigenous vegetables and processed food was confirmed by specific and duplex PCR targeting the iroB gene that yielded 443 bp and 606 bp amplicons. The PCR amplification of iroB gene is a rapid and reliable method for distinguishing between S. enterica and other bacterial species. Plasmids of S. enterica varied in sizes from 2 to more than 200 kb. Despite limited knowledge on their function, their presence is frequently used for strain differentiation in epidemiological studies. Plasmid profiling on the 200 S. enterica isolates demonstrated high discriminatory capability for serovars differentiation in this study that was clustered into 70 groups based on the number and pattern of the bands. One of the amplification based techniques used in this study for molecular characterization was PCR-RFLP that incorporated PCR of iroB1, iroB2 and restriction digest with BglII and AluI to determine the relatedness of bacterial strains. Results obtained showed that PCR-RFLP has excellent typeablity but low discriminatory power due to its inability to produce different banding patterns. ERIC sequences are short, highly conserved 126 bp non-coding regions found in the Enterobacteriaceae. Its location in bacterial genomes allows discrimination at the genus, species and serovars levels. RAPD is an amplification-based technique using arbitrary primers to detect changes in the DNA sequence at the sites in the genome and enable the discrimination of samples according to sources and serovars. Dendrogram of RAPD and ERIC-PCR were analyzed and comparisons made using BioNumerics gel analysis software (Applied Maths, Kortrijk, Belgium). Among the 200 isolates of S. enterica, RAPD with arbitrary primers OPAR02, OPAR17 and OPAR19 generated 47 clusters and 13 single isolates whereas ERIC-PCR with primers ERIC-1 and ERIC-2 produced 46 clusters and 12 single isolates at 60% similarity level with discriminatory index (D) of 0.9726 and 0.9606 respectively. Composite analysis of RAPD and ERIC-PCR profiling simultaneously produced 50 clusters and 18 single isolates at 60% similarity level with highest discriminatory index of 0.9824. These results demonstrated that composite analysis of RAPD (OPAR02, OPAR17 and OPAR19) together with ERIC-PCR are a better tool for differentiation and characterization of S. enterica as compared to a single method approach. The multiplex PCR targeted three different antibiotic resistance genes that was used to detect TEM, PSE-1 and cmlA/tetR genes segment encoding resistance towards ampicillin, chloramphenicol and tetracycline, respectively which could reduce labour and cost in analysis of a large number of isolates. Subsequently antimicrobial resistance was performed using disc diffusion method with a selection of 13 different antimicrobial agents. Total of 66 profiles were generated and multiple antimicrobial resistance (MAR) analysis indicated poultry meat still remains as the main reservoir for multi drug resistant Salmonella. In contrast, six isolates from the indigenous vegetables showed the highest MAR index (0.69). This might be due to animal waste fertilizer, irrigation water, contaminated container and improper handling of food by human that contributed to be the sources of Salmonella contamination of vegetables. Further investigations need to be conducted to determine if Salmonella isolates in recovered from indigenous vegetables were gaining more antimicrobial resistance. The characterization of MAR enabled the determination of antimicrobial patterns and trends in Salmonella from poultry meat and indigenous vegetables in Malaysia. As a conclusion, the results from this study could provide valuable information on the epidemiology and drug resistance trends of S. enterica, and hence contribute towards better surveillance and infection control measures as well as improved public health policy

Gynura procumbens: An Overview of the Biological Activities

Gynura procumbens (Lour.) Merr. (Family Asteraceae) is a medicinal plant commonly found in tropical Asia countries such as China, Thailand, Indonesia, Malaysia and Vietnam. Traditionally, it is widely used in many different countries for the treatment of a wide variety of health ailments such as kidney discomfort, rheumatism, diabetes mellitus, constipation and hypertension. Based on the traditional uses of G. procumbens, it seems to possess high therapeutic potential for treatment of various diseases making it a target for pharmacological studies aiming to validate and provide scientific evidence for the traditional claims of its efficacy. Although there has been considerable progress in the research on G. procumbens, to date there is no review paper gathering the reported biological activities of G. procumbens. Hence, this review aims to provide an overview of the biological activities of G. procumbens based on reported in vitro and in vivo studies. In brief, G. procumbens has been reported to exhibit antihypertensive, cardioprotective, antihyperglycemic, fertility enhancement, anticancer, antimicrobial, antioxidant, organ protective and antiinflammatory activity. The commercial applications of G. procumbens have also been summarized in this paper based on existing patents. The data compiled illustrate that G. procumbens is a potential natural source of compounds with various pharmacological actions which can be utilised for the development of novel therapeutic agents

Comparison of PCR fingerprinting techniques for the discrimination of Salmonella enterica subsp. enterica serovar Weltevreden isolated from indigenous vegetables in Malaysia.

Salmonella enterica subsp. enterica (S.) serovar Weltevreden has emerged as a public health problem in many countries. Genomic DNA of S. Weltevreden from indigenous vegetables namely 'selom' (Oenanthe stolonifera), 'pegaga' (Centella asiatica), 'kesum' (Polygonum minus) and 'kangkong' (Ipomoea aquatica) were characterized by duplex-polymerase chain reaction (duplex-PCR), multiplex-polymerase chain reaction (multiplex-PCR), random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results demonstrated that a total of four clusters and three single isolates were generated from ERIC-PCR with primers ERIC-1 and ERIC-2 whereas RAPD with arbitrary primers OPAR2, OPAR17 and OPAR19 discriminated the S. Weltevreden into nine clusters and eight single isolates at a common 65% similarity level with discriminatory index (D) of 0.7443 and 0.9394 respectively. Composite analysis of banding profiles generated from RAPD-PCR and ERIC-PCR showed eight clusters and six single isolates at 65% similarity level with the highest D value that is 0.9508. On the other hand, PCR-RFLP and duplex PCR data exhibited a consistent profile for S. Weltevreden. Multiplex-PCR targeting three different antibiotic resistance genes and a common Salmonella specific gene segment produced two distinguishing profiles among the S. Weltevreden examined. These results demonstrated that the combined analysis of RAPD-PCR and ERIC-PCR is a better tool for characterizing S. Weltevreden than individual methods

miR-205 in situ expression and localization in head and neck tumors - a tissue array study

Background: microRNAs are small non-coding RNA that control gene expression by mRNA degradation or translational inhibition. These molecules are known to play essential roles in many biological and physiological processes. miR-205 may be differentially expressed in head and neck cancers; however, there are conflicting data and localization of expression has yet to be determined. Materials and Methods: miR-205 expression was investigated in 48 cases of inflammatory, benign and malignant tumor tissue array of the neck, oronasopharynx,larynx and salivary glands by Locked Nucleic Acid in situ hybridization (LNA-ISH) technology. Results: miR-205 expression was significantly differentially expressed across all of the inflammatory, benign and malignant tumor tissues of the neck. A significant increase in miR-205 staining intensity (p<0.05) was observed from inflammation to benign and malignant tumors in head and neck tissue array, suggesting that miR-205 could be a biomarker to differentiate between cancer and non-cancer tissues. Conclusions: LNA-ISH revealed that miR-205 exhibited significant differential cytoplasmic and nuclear staining among inflammation, benign and malignant tumors of head and neck. miR-205 was not only exclusively expressed in squamous epithelial malignancy. This study offers information and a basis for a comprehensive study of the role of miR-205 that may be useful as a biomarker and/or therapeutic target in head and neck tumors

Characterization of multiple-antimicrobial-resistant Salmonella enterica Subsp. enterica isolated from indigenous vegetables and poultry in Malaysia.

Aims: The aims of this communication were to study characterization of serogroups among Salmonella isolates and the relationship of antimicrobial resistance to serogroups. Multiple antimicrobial resistance (MAR) was performed on 189 Salmonella enterica isolates associated with 38 different serovars that were recovered from poultry and four types of indigenous vegetables. Methods and Results: Disc diffusion analysis was performed with a selection of 10 different antimicrobial agents. Isolates recovered from indigenous vegetables showed 100% (134/134) resistant to erythromycin and followed by 42%, 34%, 19% for tetracycline, streptomycin and trimethroprim-sulfamethoxazole respectively. In general, 90·1% (50/55) and 56·7% (76/134) of Salmonella isolated from poultry and indigenous vegetables, respectively, exhibited MAR index more than 0·2. Conclusions: Characterization of Salmonella isolates based on the MAR results indicated that poultry still remains as the main reservoir for multi-drug-resistant Salmonella. Four isolates from the indigenous vegetables showed the highest MAR index in this study. Further investigations need to be conducted to determine if Salmonella isolates recovered from indigenous vegetables were gaining more antimicrobial resistance. Significance and Impact of the Study: The study enabled us to determine antimicrobial patterns and trends in Salmonella from poultry and indigenous vegetables in Malaysia

An overview of the characteristics of the novel avian influenza A H7N9 virus in humans

The novel avian influenza A H7N9 virus which caused the first human infection in Shanghai, China; was reported on the 31st of March 2013 before spreading rapidly to other Chinese provinces and municipal cities. This is the first time the low pathogenic avian influenza A virus has caused human infections and deaths; with cases of severe respiratory disease with pneumonia being reported. There were 440 confirmed cases with 122 fatalities by 16 May 2014; with a fatality risk of ~28%. The median age of patients was 61 years with a male-to-female ratio of 2.4:1. The main source of infection was identified as exposure to poultry and there is so far no definitive evidence of sustained person-to-person transmission. The neuraminidase inhibitors, namely oseltamivir, zanamivir, and peramivir; have shown good efficacy in the management of the novel H7N9 virus. Treatment is recommended for all hospitalized patients, and for confirmed and probable outpatient cases; and should ideally be initiated within 48 h of the onset of illness for the best outcome. Phylogenetic analysis found that the novel H7N9 virus is avian in origin and evolved from multiple reassortments of at least four origins. Indeed the novel H7N9 virus acquired human adaptation via mutations in its eight RNA gene segments. Enhanced surveillance and effective global control are essential to prevent pandemic outbreaks of the novel H7N9 virus

Differential microRNA expression and identification of putative miRNA targets and pathways in head and neck cancers.

MicroRNAs (miRNAs) are small noncoding RNAs that involved in various cancer-related cellular processes. Diverse studies on expression profiling of miRNAs have been performed and the data showed that some miRNAs are up-regulated or down-regulated in cancer. Until now, there are no data published on the miRNA expression in head and neck cancers from Malaysia. Hence, this study aimed to investigate potentially crucial miRNAs in head and neck cancer patients from Malaysian populations. A global miRNA profiling was performed on 12 samples of head and neck cancer tissue using microarray analysis followed by validation using real-time RT-PCR Microarray analysis identified 10 miRNAs that could distinguish malignant head and neck cancer lesions from normal tissues; 7 miRNAs (hsa-miR-181a-2*, hsa-miR-29b-1*, hsa-miR-181a, hsa-miR-181b, hsa-miR-744, hsa-miR-1271 and hsa-miR-221*) were up-regulated while 3 miRNAs (hsa-miR-141, hsa-miR-95 and hsa-miR-101) were down-regulated. These miRNAs may contribute in a simple profiling strategy to identify individuals at higher risk of developing head and neck cancers, thus helping in the elucidation of the molecular mechanisms involved in head and neck cancer pathogenesis