Evaluation of housekeeping genes in Listeria monocytogenes as potential internal control references for normalizing mRNA expression levels in stress adaptation models using real-time PCR

Listeria monocytogenes is an important food-borne pathogen that can tolerate a wide range of stress conditions. However, its stress adaptation processes are still poorly understood. Real-time-based quantitative RT-PCR (qRT-PCR) provides a tool to probe gene expression changes underlying stress adaptation. But, a limitation to study mRNA levels by real-time qRT-PCR is that validated reference genes are required for normalization. Such genes are currently lacking for experimental models that may be applied to evaluate stress-related gene expression changes in L. monocytogenes. Therefore, five housekeeping genes (HKG) were studied as potential reference genes. Their expression stability was evaluated across 16 L. monocytogenes strains. Three experimental models designed to assess gene expression changes induced by cold, acid and high NaCl concentration stress adaptation were applied. The 16S rRNA gene was consistently the most stably expressed HKG across the different L. monocytogenes strains under all the experimental conditions. While the expressions of β-glucosidase (bglA), Glyceraldehyde-3P-dehydrogenase (gap), RNA polymerase beta subunit (rpoB) and Ribosomal protein L4 (rplD) was stable amongst the different L. monocytogenes strains, they were prone to significant variations under the different stress adaptation model

The contribution of transcriptomic and proteomic analysis in elucidating stress adaptation responses of Listeria monocytogenes

The foodborne transmission of Listeria monocytogenes requires physiological adaptation to various conditions, including the cold, osmotic, heat, acid, alkaline, and oxidative stresses, associated with food hygiene, processing, and preservation measures. We review the current knowledge on the molecular stress adaptation responses in L. monocytogenes cells as revealed through transcriptome, proteome, genetic, and physiological analysis. The adaptation of L. monocytogenes to stress exposure is achieved through global expression changes in a large number of cellular components. In addition, the cross-protection of L. monocytogenes exposed to different stress environments might be conferred through various cellular machineries that seem to be commonly activated by the different stresses. To assist in designing L. monocytogenes mitigation strategies for ready-to-eat food products, further experiments are warranted to specifically evaluate the effects of food composition, additives, preservatives, and processing technologies on the modulation of L. monocytogenes cellular components in response to specific stresses

16S rRNA gene based analysis of Enterobacter sakazakii strains from different sources and development of a PCR assay for identification

BACKGROUND: E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive [Image: see text] glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated. RESULTS: By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains. CONCLUSION: By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation

Listeria monocytogenes Cold Shock Proteins: Small Proteins with A Huge Impact

Listeria monocytogenes has evolved an extensive array of mechanisms for coping with stress and adapting to changing environmental conditions, ensuring its virulence phenotype expression. For this reason, L. monocytogenes has been identified as a significant food safety and public health concern. Among these adaptation systems are cold shock proteins (Csps), which facilitate rapid response to stress exposure. L. monocytogenes has three highly conserved csp genes, namely, cspA, cspB, and cspD. Using a series of csp deletion mutants, it has been shown that L. monocytogenes Csps are important for biofilm formation, motility, cold, osmotic, desiccation, and oxidative stress tolerance. Moreover, they are involved in overall virulence by impacting the expression of virulence-associated phenotypes, such as hemolysis and cell invasion. It is postulated that during stress exposure, Csps function to counteract harmful effects of stress, thereby preserving cell functions, such as DNA replication, transcription and translation, ensuring survival and growth of the cell. Interestingly, it seems that Csps might suppress tolerance to some stresses as their removal resulted in increased tolerance to stresses, such as desiccation for some strains. Differences in csp roles among strains from different genetic backgrounds are apparent for desiccation tolerance and biofilm production. Additionally, hierarchical trends for the different Csps and functional redundancies were observed on their influences on stress tolerance and virulence. Overall current data suggest that Csps have a wider role in bacteria physiology than previously assumed

Evaluation of three reference genes of Escherichia coli for mRNA expression level normalization in view of salt and organic acid stress exposure in food

Escherichia coli can adapt to various stress conditions encountered in food through induction of stress response genes encoding proteins that counteract the respective stresses. To understand the impact and the induction of these genes under food-associated stresses, changes in the levels of their mRNA expression in response to such stresses can be analysed. Relative quantification of mRNA levels by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) requires normalization to reference genes with stable expression under the experimental conditions being investigated. We examined the validity of three housekeeping genes (cysG, hcaT and rssA) among E. coli strains exposed to salt and organic acid stress. The rssA gene was shown to be the most stably expressed gene under such stress adaptation experimental models. The cysG gene was the least stable, whereas the hcaT gene showed similar interstrain variability as rssA but lower expression stability in the different stress adaptation model

Factors associated with delayed entry into HIV medical care among HIV positive people who are aware of their status in Bulawayo Zimbabwe

Using non-experimental descriptive exploratory survey, this study sought to find out factors associated with delayed entry into HIV medical care among HIV positive people who are aware of their status. A quantitative design was used and a structured questionnaire was used as the data collection instrument. Respondents were patients in a hospital’s Opportunistic Infection Department whose hospital records indicated that they were HIV positive during the time of the study. The study found out that as much as 71.6% (n =43) first entered HIV medical care more than 12 months after testing HIV positive while 40% (n = 24) did so as a result of illness. Low education levels, unemployment and being single are associated with delayed entry into HIV medical care. A percentage of the population uses and trusts non-biomedical approaches to dealing with HIV/AIDS. Being diagnosed HIV positive is therefore not necessarily a strong reason enough for one to immediately enter into medical care. Intensive health education needs to be done at work places, health facilities, schools, through print and electronic media, churches and other community settings to equip the population with knowledge of the advantages of early entry into HIV care.Health StudiesM.A. (Public Health

Market Orientation in MSME: A Qualitative Case Study of MSME in Zimbabwe

In different sectors and countries, the importance of market orientation has been widely recognised in improving organisational performance in large organisations. However the concept of market orientation is being misunderstood by the Micro Small and Medium Enterprises particularly from developing countries context. These misunderstandings led to this study which mainly focused on examining the understanding of market orientation, antecedents and consequences of market orientation by Micro Small and Medium Enterprises operating in the wood and metal furniture manufacturing industry of Zimbabwe. The research was more qualitative in nature and used face to face interviews to gather insights into the market orientation practices, antecedents and consequences in Micro Small and Medium Enterprises. The study’s results revealed that Micro Small and Medium Enterprises lack greater understanding of the market orientation concept, its antecedences and consequences. This has been revealed by the majority of MSME’s top management not having full support of market orientation and not being risk takers. MSMEs confessed not having marketing budgets, research and development teams, marketing plans and they do not see its importance to the success of their businesses. These enterprises are operating more informally and concentrate on methods of increasing products purchase, increasing market share and customer retention. Basing on these findings, the paper provides some managerial implications, limitations of the study and recommendations for future researches. Keywords: Market orientation, Qualitative, Micro Small Medium Enterprises, Zimbabw

HIV-related stigma in rural areas: a case of citrus farm workers based in Addo community in Eastern Cape

While South Africa has recently joined the rest of the world in the race to achieve an AIDS free generation by the year of 2030, little has been done to develop the scientific interventions that address HIV-related stigma, which is one of the major barriers to the fight against HIV. To the researcher’s knowledge, this is the first study in the South African context that seeks to assess the forms of HIV- related stigma in a local context and in a rural area in particular, where HIV related stigma research and interventions remains scant. This study acknowledges the dynamic, unique, multidimensional and sensitive nature of HIV-related stigma and, therefore, an explanatory mixed approach enabled the researcher to provide a comprehensive assessment of HIV-related stigma amongst the citrus farm workers who are based in Addo, Eastern Cape. Quantitative data was collected from 200 participants across five farms, whereas 50 employees across the 5 farms were also selected to take part in the focus group discussions. An assessment of the reliability and validity of local HIV-related stigma scales (Kalichman et al., 2005 personal stigma scale-English, isiXhosa and Afrikaans version and Visser et al. 2008 personal and attributed stigma scale-English version) and the UNAIDS (2012) HIV knowledge scale was conducted. The assessment showed that the reliability of these scales of measurement tend to vary according to context. In this study, the Kalichman et al., (2005) personal stigma scale (English version) scored a weaker reliability score (alpha=0.58). Thus, as the scale is not reliable for the rural context, it was not considered. Furthermore, quantitative findings show that citrus farm workers are characterised by lack of/ little knowledge about HIV, which results in the stigmatisation of people who are HIV positive in the form of symbolic, instrumental, personal and attributed stigma, as well as a fear of HIV disclosure. Findings also revealed that participants who display a high personal stigma score are likely to display a high attributed stigma score (r=0.47, p<0.00). This confirm the assumption of social identity theory that people tend to use stigma as a ‘protective function’ to protect their identity by labelling other people as ‘’deviant’ or ‘stigmatising’. This finding is particularly relevant in that it reveals that, while many people are aware that stigmatising people with HIV is not morally acceptable, they rather attribute stigmatising attitudes to their communities. Thus future studies need to consider emphasising the assessment of attributed stigma in a rural context. Quantitative findings also revealed that the effect of the following demographic variables, namely race (F=20.1, p=0.00), marital status (F=3.58, p=0.00), religion (F=7.17, p=0.03) and education (F=2.63, p=0.03) on HIV-related stigma was statistically significant. This provides proof that HIV-related interventions that were developed for the Addo community need to consider the above-mentioned demographics variables. The qualitative findings provided an in-depth analysis of the key quantitative findings. A lack of knowledge about HIV among participants was further confirmed by the following themes that were common during focus group discussions: misconceptions about HIV transmission and prevention; false beliefs that healthy looking people cannot have HIV; HIV symptoms are clearly visible; and HIV can be transmitted through contact with people who are HIV positive. Possible causes of HIV-related stigma as revealed by the qualitative findings include; fear of death; the belief that HIV is a punishment for bad behaviour; and the fear of being isolated and rejected. The qualitative approach revealed other forms of HIV-related stigma that were not common in the quantitative studynamely; healthcare, employment and verbal stigma. The focus group discussions revealed the major reasons why a large number of the farm works may be reluctant to disclose their status, namely fear of the unknown and attributed stigma. The study concluded by offering recommendations for the designing and tailoring of HIV-related interventions in the rural context and in the citrus sector in particular

The development of teachers’ pedagogical content knowledge (PCK) in the mediation of chemical equilibrium: A formative interventionist study

Persistent student errors in understanding chemical equilibrium as shown by poor student achievement in national examinations reflect student difficulties in learning and deficiencies in teaching methodologies. Studies which have been conducted in Namibia have explored the teaching of chemical equilibrium and revealed that teachers seem not to have adequate pedagogical content knowledge (PCK) for mediating chemical equilibrium and therefore there is a need for continuing professional development (CPD). However, it seems the CPD facilitators also find this topic difficult and are unsure of which methods are really effective owing to the disagreements among teachers and researchers. In addition, current CPD practices ostensibly fail to address the teachers’ needs because the facilitators have their own commitments and accountabilities and may ignore teachers’ contexts. It is against this backdrop that I conducted this formative interventionist study to improve teachers’ PCK for mediating chemical equilibrium through expansive learning (learning something that does not yet exist). A blend of the interpretivist and critical paradigm underpinned this study, which assumed a transactional epistemology. The qualitative case study research design was used to gather in-depth information about the multiple realities of the participants, bearing in mind that teaching is idiosyncratic, and the teaching approaches or strategies employed heavily depend on the contexts. Accordingly, the cultural historical activity theory was used to guide the teachers’ activities during the intervention for generating the data which were analysed using the topic-specific pedagogical content knowledge. The study revealed that the participants faced challenges in their teaching, namely: (i) students’ difficulties with comprehension and (ii) teachers’ instructional problems or deficiencies in instructional skills. The findings also revealed that the intervention enabled the participants to collectively transform their practices and therefore address the major challenges in their practices, that is, they expansively learnt how to effectively teach chemical equilibrium.Thesis (MEd) -- Faculty of Education, Education, 202

HIV-related stigma amongst service staff in Grahamstown a comparison of Hi-Tec security guards and Rhodes catering in the Eastern Cape

Despite the acknowledged reality that HIV-related stigma is a major barrier to effective HIV prevention and treatment, and perhaps because it is complex in nature, few local empirical scales have been developed to measure stigma and to be able evaluate the impact of anti-stigma interventions. Whilst the development of two recent South African HIV-related stigma scales can be celebrated as a major breakthrough, the reliability and validity of these scales across contexts remains largely unknown. This research project employs these two local, and competing, HIV-related personal stigma scales - the first developed by Kalichman et al. (2005) and the second developed by Visser, Kershaw, Makin and Forsyth (2008)-to compare the psychometric properties of the scales and to obtain a measure of HIV-related stigma with a sample of 246 service staff employed at either Rhodes University Catering Division or the Hi-Tec Security company, both organisations located in Grahamstown, a small town in the Eastern Cape, South Africa. Both organisations are major employers of semi-skilled workers in this local context. The results suggest that the Visser et al. scale (2008) reports slightly better psychometric properties than the Kalichman et al. (2005) scale for this sample. Furthermore, the security guards appear to be more stigmatising than the caterers, and it is suggested that this might be a consequence of the combined influences of normative occupational roles and workplace context. Results also show that participants who practices safe sex, know someone with HIV and/or who have been tested for HIV show lower levels of HIV-related stigma. Finally, personal stigma scores are generally lower than attributed stigma scores, which might offer a useful point of intervention