Optimization of cell density and LPS concentration for the evaluation of nitric oxide production on BV-2 cells in a Griess assay

Production of nitric oxide (NO) is one of the main responses elicited by a variety of immune cells such as macrophages (e.g. microglia, resident macrophages of brain), during inflammation. Evaluation of NO levels in the inflammatory milieu is considered important to the understanding of the intensity of an immune response; and has been performed using different methods including the Griess assay. To assay NO in culture, an appropriate number of cells are stimulated into an inflammatory phenotype. Common stimuli include lipopolysaccharide (LPS), IFN-γ and TNF-α. However, overt stimulation could cause cell cytotoxicity therefore an ideal concentration of LPS should be used. Objective: To set-up a model of BV-2 cell activation that allows the assay of detectable levels of NO. Optimization of BV-2 microglia cell density and LPS concentrations after stimulation by bacterial lipopolysaccharide (LPS) for the Griess assay is demonstrated in this study. Methods:BV-2 microglia were cultured at different cell densities, and treated with LPS at three concentrations (1, 5, 10 μg/ml). NO production in culture supernatants were then measured at 18, 24, 48 and 72 hours. Moreover, methyl tetrazolium assay (MTT) was also performed to ensure that NO measurement is performed at no-cytotoxic concentrations of LPS. Results and Conclusions: NO production follows a temporal pattern. The density of 25000 cells/well was the ideal seeding density for NO evaluation in BV-2 cells. BV-2 stimulation by LPS is dose dependent, and NO levels are increased proportional to the LPS concentration up to 1.0μg/ml, whereas the higher LPS concentrations are associated with decreased cell viability may be caused by the high toxic levels of LPS or NO. Although Griess assay has been commonly used by the scientists, however, optimization of its parameters on BV-2 cells will be useful for the experiments which will be performed on this particular cell line. The optimized pattern of Griess assay on BV-2 cells was achieved in this study, hence easier and more practical for the future scientists to perform Griess assay on BV-2 cells

An overview of foodborne illness and food safety in Malaysia

Foodborne disease has been associated with microorganisms like bacteria, fungi, viruses and parasites. Most commonly, the outbreaks take place due to the ingestion of pathogenic bacteria like Salmonella Typhi, Escherichia coli, Staphylococcus aureus, Vibrio cholera, Campylobacter jejuni, and Listeria monocytogenes. The disease usually happens as a result of toxin secretion of the microorganisms in the intestinal tract of the infected person. Usually, the level of hygiene in the food premises reflect the quality of the food item, hence restaurant or stall with poor sanitary condition is said to be the contributor to food poisoning outbreak. In Malaysia, food poisoning cases are not rare because the hot and humid climate of this country is very suitable for the growth of the foodborne bacteria. The government is also implementing strict rules to ensure workers and owners of food premises prioritize the cleanliness of their working area. Training programme for food handlers can also help them to implement hygiene as a routine in a daily basis. A lot of studies have been done to reduce foodborne diseases. The results can give information about the types of microorganisms, and other components that affect their growth. The result is crucial to determine how the spread of foodborne bacteria can be controlled safely and the outbreak can be reduced

Molecular characterization of nosocomial methicillin resistant staphylococci aureus by Rep-PCR.

Nosocomial methicillin-resistance Staphylococcus aureus (MRSA) poses problem to clinicians and hospital administrators for its management. In the present study isolation, identification molecular characterizations of methicillin-resistance Staphylococcus aureus were performed at Hospital Kuala Lumpur, Malaysia during December 2005 to April 2007. Twenty seven MRSA positive samples were identified based on cultural, biochemical and antibiotic sensitivity assay. These were, 51% from blood samples, 33.3% from tracheal aspirate and 11,1% from nasal swab and pus. Molecular method rep-PCR was used to characterize the MRSA positive strains. Results of rep-PCR showed 5 different pattern of bands based on their genomic nature. Thus, rep-PCR was proved to be potential tool to determine genomic differences of nosocomial MRSA in resource limited setting

A high incidence of antimicrobial resistance in Group B Streptococcus (GBS) clinical isolates with poor biofilm forming capacity

Background: Group B Streptococcus (GBS) or commonly known as Streptococcus agalactiae are the leading cause of bacterial meningitis and bacterial sepsis in newborns. One of the virulence factors is the biofilm forming capacity of the isolates which may promote adherence and survivability leading to invasion of GBS at the infected sites. In a previous study conducted by Kaur et al., (2009), biofilm formation has been associated with virulence level among the GBS clinical isolates. We had previously characterized a collection of GBS clinical isolates from invasive and colonizing site for serotypes and antimicrobial susceptibility pattern (Emir et al., 2014). In this study, we extended the study by looking into biofilm formation capability of the GBS isolates and analysed the phenotypic pattern in relation to isolation site, serotypes and antibiotics susceptibility pattern for a potential association. Materials and Methods: A total of 51 viable GBS isolates were available in our collection with pre-determined data for serotypes (by multiplex PCR) and antimicrobial susceptibility pattern (by disk diffusion) for erythromycin, clindamycin, trimethoprim sulfamethoxazole, tetracycline, chloramphenicol and penicillin (Emir et al., 2014). The isolates were previous collected from three different hospitals in Klang Valley (n=51). A total of 29 isolates were from colonizing site and another 23 isolates from invasive sites. In this study, the capacity of biofilm formation of GBS was determined by OD-based biofilm assay as described by Kaur et al., (2009). The GBS isolates were considered as good biofilm former based on ODA550 reading >1.0, moderate biofilm former at ODA550 value between 0.5-1.0 and poor biofilm former of ODA550 value <0.5. The tabulation of the categories was compared in relation to the isolation site and previously determined phenotypic and genotypic properties. Results and Conclusion: Based on the OD-based criteria, none of the 51 isolates had good biofilm formation while 11 had moderate and the rest were poor biofilm forming isolates. The proportions of colonizing and invasive isolated were about equal among the moderate and poor biofilm former (45-55%). In relation to antibiotic susceptibility pattern, a majority of those resistant to all tested antibiotics were poor biofilms formers. That in relation to penicillin was not known as there was only one isolates with penicillin intermediate. Six isolates of poor biofilm former and one isolates of moderate biofilm former were identified as multidrug resistant strains (MDR) which are resistant to at least three antibiotics of different classes. Nonetheless, chi square analysis showed none of the categorical tabulation had a significant association. This could be due to the limited number of representative isolates in the categories although obvious differences based on percentage (%) were observed for some of the antibiotics. This limitation also applied in relation to serotype due to its variability which resulted in low representative for some of the serotypes include (serotype II, type III, type V, type VI and type VIII) among the 51 isolates. Serotype Ia had the highest prevalence (n=22) but interestingly 20 of them were poor biofilm formers. For the other respective serotypes, the distribution rate in relation to moderate and poor biofilm were about equal. As far as this study is concerned, there is no specific preference for colonizing and invasive GBS isolates to have certain advantage in term of biofilm capacity. In a study conducted by Talat et al., (2012), serotype Ia and IV were associated with tendency to form biofilm which was in contrast with this study. The tendency of antibiotic resistance to present more among poor biofilm formers in this study are also of the interest to be further elucidated. Further studies also required to define more precisely the effect of antibiotic resistance in biofilm forming GBS isolates

Approaches of learning among medical undergraduates of Faculty of Medicine and Health Sciences, Universiti Putra Malaysia in 2016

Introduction: Challenge arises for medical undergraduates as the subject of medicine is intricate and extensive. Although students come from the same pool of excellent academic background, the medical undergraduates are still prone to failure in exams, resulting in them repeating the year of study or even having the thought of changing to other courses. In order to cope with the programme, students may adopt learning approaches that would help them to go through the programme. Therefore, it would be interesting to explore the learning approaches of medical students in Universiti Putra Malaysia. Methods: The purposes of this study were to determine the learning approach of medical undergraduates of Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (FMHS, UPM) together with its associated factors, which were socio-demographic characteristics and learning environment. This research was a cross-sectional study where the sample size calculated was 554. Self-administered questionnaires were given to the respondents chosen by simple random sampling. The socio-demographic characteristics were analysed using descriptive statistics such as frequency and percentage. Chi-square test was used to analyse the association between the variables. Results: The response rate was 83% (460 respondents agreed to participate). The majority of the respondents were females (73.7%), Malay (67%), and in their clinical years of study (58.7%). Overall, most students preferred deep approach (DA) of learning (49.6%), followed by strategic approach (29.1%) and surface apathetic approach (21.3%) of learning. There were statistically significant associations between learning approach and gender (p=0.005), as well as between learning approach and year of study (p=0.037). Conclusion: Our study showed an association between learning approaches and year of study and gender. DA of learning was the preferred learning approach in medical students at FMHS, UPM. This approach of learning, where students learn to understand the subject matter, may result in students become effective learners. Their understanding about the subject matter will be applicable to their clinical practice in the future

The exoproteomes of clonally related Staphylococcus aureus strains are diverse

Several studies have shown that protein expression patterns vary in unrelated bacterial strains due to genomic plasticity and gene regulation, resulting in enhanced heterogeneity in the infection potential. However, exoprotein expression patterns of closely related clonal strains have not been well characterized. Here, we used medium-range (pH 4–7) immobilized pH gradient–two-dimensional gel electrophoresis to investigate the exoproteome from closely related Staphylococcus aureus clonal isolates. Interestingly, we found that, under identical in vitro experimental conditions, a number of protein spots were uniquely present in samples from each clonal isolate irregardless of the similarity of the genotype and the same virulence gene profile. Only a few abundant invariant proteins were found among identical genotypic isolates. Our results clearly shown that heterogeneity in the exoproteome was present even among clonally related strains. We suggest that this heterogeneity may contribute to the degree of virulence even within one clonal genotype. The heterogeneity in the exoproteome of closely related S. aureus strains observed in the current study postulates that pre-existing antibodies are not very protective during recurrent infection with the same strain. Therefore, our findings underscore the importance of taking all clonally related strains into account during proteome analyses

Metagenome: differences in the gut microbiota among healthy, obese and type 2 diabetes adults

The association between gut microbiota composition with pathogenesis of metabolic diseases namely obesity and type 2 diabetes are increasingly recognized. The aim of the study was to identify the diversity of gut microbiota phylum and families in the gut of healthy, obese and type 2 diabetes adults with metagenomic approach. Six healthy subjects, five obese subjects and five type 2 diabetes subjects of similar inclusion and exclusion criteria were recruited. The different bacterial phyla and families in the stool sample were analyzed with metagenomic analysis. The median (IQR)% of relative abundance for each phylum and families were analyzed. The obese subjects had higher Bacteroidetes 63.50(21.55)% with lower Firmicutes 27.00(13.55)%, meanwhile, the type 2 diabetes subjects also had higher Bacteroidetes 66.50(39.00)% with lower Firmicutes 27.70(19.35)%. These findings shows that there are differences in the gut microbiota composition in the healthy, obese and type 2 diabetes adults which may influence the development of obesity and type 2 diabetes

Comparative analysis of inflammatory markers produced by macrophages inoculated with invasive and colonizing strains of Streptococcus agalactiae (group B streptococcus) and evaluation of patients' clinical data

Introduction: Group B Streptococcus (GBS), infection and recurrence in newborns and pregnant women can lead to chronic medical illness resulting in significant morbidity, and mortality. Pathogenesis of GBS may be due to reasons such as activation of the immune system, followed by the production of inflammatory markers and toxic components by immune cells including macrophages. Methods: The studies on invasive and colonizing GBS strains inoculated either with peripheral or brain macrophages, the expression of nitric oxide (NO), cell viability, and CD40 were also measured by Griess assay, methyl tetrazolium assay (MTT), and flow cytometry, respectively. Furthermore, the clinical manifestations of the selected patients were also assessed for this study. Results: Outcome of inflammatory markers studies, after GBS inoculation indicated that, invasive GBS strains induced higher inflammatory markers in comparison to colonizing GBS strains. Furthermore, patients’ clinical data showed that patients with invasive GBS infections had severe condition unlike among patients with colonizing GBS strains. The fatality rate in patients with invasive GBS strain were 30.8% while there was no death among carriers. Conclusion: This study, aimed to understand the immune response to GBS, and strengthen the knowledge on GBS pathogenesis. It was concluded that invasive GBS strains not only showed higher expression of inflammatory markers on immune cells but also had higher pathogenesis effect in comparison to colonizing GBS strains

Post-partum invasive group B streptococcus infection with fatal outcome: a case report

Group B streptococcus (GBS) is generally known to cause severe disease in the neonate and immunocompromised adults. GBS in the pregnant mother is rare and can potentially be fatal. Clinical presentation can be as mild as an uncomplicated urinary tract infection or serious invasive disease in the form of bacteremia, chorioamnionitis, endometritis and septic abortion. We report a case of a 46-year-old Para 3 lady, post-partum day 12, whom was found dead at home. Prior to her death, she had intermittent fever and abnormal lochia. Autopsy findings indicate GBS endometritis and bacteraemia. She was never screened for GBS. The cost-effectiveness of universal GBS screening needs to be explored to reduce maternal and neonatal morbidity due to GBS

Antimicrobial susceptibility profiles, serotype distribution and virulence determinants among invasive, non-invasive and colonizing Streptococcus agalactiae (group B streptococcus) from Malaysian patients

A total of 103 group B streptococci (GBS) including 22 invasive, 21 non-invasive, and 60 colonizing isolates were collected in a Malaysian hospital (June 2010–October 2011). Isolates were characterized by conventional and molecular serotyping and analyzed for scpB, lmb, hylB, cylE, bac, bca and rib gene content. Antimicrobial susceptibility to penicillins, macrolides, lincosamides, quinolones and tetracyclines was determined using disk diffusion and the MICs for penicillin were determined by E-test. Molecular serotyping for all eight serotypes (Ia, Ib, II–VII) was in full accordance with conventional serotyping. Overall, taking CS and MS together, serotype VI was the most common capsular type (22.3 %) followed by VII (21.4 %), III (20.4 %), Ia (17.5 %), V (9.7 %), II (7.7 %) and IV (1 %). Susceptibility to beta-lactam antimicrobials was prevalent (100 %). Resistance rates for erythromycin, clindamycin and tetracycline were 23.3 %, 17.5 % and 71.8 %, respectively. PCR-virulence gene screening showed the presence of cylE, lmb, scpB and hylB in almost all the isolates while rib, bca, and bac genes were found in 29.1 %, 14.6 % and 9.7 % of the isolates. Certain genes were significantly associated with specific serotypes, namely, rib with serotypes Ia, II, III and VI; bca and bac with serotypes II and III. Furthermore, serotype Ia was significantly more common among patients with invasive infections (p < 0.01) and serotype VI isolates were significantly more common among carriers (p < 0.05). In summary, serotype distribution correlates with virulence gene content will be useful in epidemiological studies and design of vaccines