Membrane modification as a survival mechanism through gastric fluid in non-acid adapted enteropathogenic Escherichia coli (EPEC).

In bacterial cells, the cytoplasmic membrane forms a barrier between the environment and the cell's cytoplasm. This barrier regulates which substances (and the amount) that leave and enter the cell, to maintain homeostasis between the cytoplasm and the external environment. One of the mechanisms employed to maintain structure and functionality during exposure to environmental stress is adaptation of the membrane lipids. The aim of this study was to investigate membrane alteration as a possible survival method of non-acid adapted enteropathogenic Escherichia coli (E. coli) (EPEC) (as could be found in contaminated water or unprocessed food) through simulated gastric fluid (SGF). Enteropathogenic E. coli was grown in nutrient-rich media and then exposed to SGF of various pH (1.5, 2.5, 3.5, or 4.5) for 180 min. Flow cytometry was utilised to examine membrane integrity; and morphological changes were investigated using transmission electron microscopy (TEM). Gas chromatography-mass spectrometry (GC-MS) was used to assess the membrane lipid composition. The results of this study showed that SGF treatment caused membrane damage, as well as cell wall thickening and irregular plasma membranes. The morphological changes were accompanied by membrane lipid changes indicative of decreased membrane fluidity and increased rigidity. The findings suggest that non-acid adapted EPEC can perceive pH change in the environment and adapt accordingly.

Acid-happy: Survival and recovery of enteropathogenic Escherichia coli (EPEC) in simulated gastric fluid.

BACKGROUND: Gastric fluid pH serves an important function as an ecological filter to kill unwanted microbial taxa that would otherwise colonise the intestines, thereby shaping the diversity and composition of microbial communities found in the gut. The typical American-based diet causes the gastric pH to increase to pH 4 to 5, and it takes ∼2 h to return to pH 1.5 (normal). This window of increased gastric pH may allow potential pathogens to negotiate the hostile environment of the stomach. Another factor to consider is that in developing countries many people experience hypochlorhydria related to malnutrition and various gastric diseases. Enteropathogenic E. coli (EPEC) is a leading cause of infantile diarrhoea and has a high incidence in the developing world. The aim of this study was to assess the survival and recovery of non-acid adapted EPEC exposed to simulated gastric fluid (SGF) over a period of 180 min. RESULTS: EPEC were grown in nutrient-rich medium and acid challenged in SGF at pH 1.5, 2.5, 3.5 and 4.5. Culturability was evaluated using a standard plate count method, and metabolic viability was assessed via cellular energy (adenosine triphosphate [ATP] assay) and respiratory activity (3-bis(2-methyloxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide [XTT] assay), and recovery and proliferation by means of optical density in liquid cultures. Sampling was performed at 0, 30, 60, 120, and 180 min post-SGF exposure. The results of this study showed that EPEC is remarkably acid resistant and was able to survive a simulated gastric environment for up to 3 h (180 min) at various pH (1.5, 2.5, 3.5, and 4.5). EPEC was culturable at all pH (1.5, 2.5, 3.5 and 4.5) at the higher inoculum size of 5.4-7.1 × 106 CFU/ml, and at all pH except pH 1.5 at the lower inoculums of 5.4-7.1 × 103 CFU/ml or 5.4-7.1 × 101 CFU/ml. The organism remained metabolically viable at pH 1.5, 2.5, 3.5, and 4.5 and was able to recover and proliferate once placed in a neutral, nutrient-rich environment. CONCLUSION: In this study, EPEC demonstrated remarkable acid resistance and recovery at low pH without prior acid adaptation, which could prove to be problematic even in healthy people. In individuals with decreased gastric acidity, there is a higher probability of pathogen colonization and a resulting change in the gut microbiome. The results highlight the potential increase of food- and waterborne diseases in persons with compromised gastric function, or who are malnourished or immunocompromised. The data herein may possibly help in calculating more precisely the risk associated with consuming bacterial contaminated food and water in these individuals.

Adaptations in the physiological heterogeneity and viability of Shigella dysenteriae, Shigella flexneri and Salmonella typhimurium, after exposure to simulated gastric acid fluid.

Stomach acidity is an important barrier of the human body to protect itself from microbial pathogens entering the small intestine and causing infection. This study examined the survival adaptations of non-acid adapted diarrheal Shigella and Salmonella strains in an environment mimicking the human stomach. The bacterial responses to the challenge of acidic simulated gastric fluid were studied using flow cytometry physiological heterogeneity, membrane integrity and survival (culturability) respectively. Flow cytometry showed that bacterial cells, when exposed to gastric fluid, transformed distinctly, into physiologically heterogeneous sub-populations: intact, stressed and damaged cells, when stained with propidium iodide and thiazole orange. Shigella and Salmonella cells became membrane compromised during initial acid shock (0-30 min), and 80% of these cells shifted to the stressed state throughout gastric fluid exposure. Approximately 10-30% of bacterial strains remained culturable after 60 min of gastric fluid exposure at pH 2.5-4.5, with the percentage increasing with an inoculum size of 102 CFU/ml. This ability of non-acid adapted Shigella and Salmonella sp. to adapt and survive low pH gastric fluid, even though the bacterial numbers decreased or changed to a stressed state, further supports the possible risk of infection when consumed.

Application of a Novel Proteomic Microarray Reveals High Exposure to Diarrhoeagenic Escherichia coli among Children in Zambia Participating in a Phase I Clinical Trial.

Diarrhoeagenic E. coli (DEC) significantly contributes to the burden of diarrhoea among children. Currently, there is no approved vaccine against DEC, but several vaccines against the enterotoxigenic E. coli (ETEC) pathotype are in advanced clinical trial stages, including the ETVAX® vaccine, undergoing evaluation in Zambia. This study reports on the reactivity of antibodies from ETVAX® vaccine and placebo recipients in a phase I clinical trial to proteins derived from (DEC) other than ETEC. Plasma samples collected at two time points (prior to any vaccination and post-third dose vaccination) from 16 vaccinated and 4 placebo participants in a phase 1 clinical trial examining the safety, tolerability, and immunogenicity of ETVAX® with dmLT adjuvant were evaluated for IgG response to E. coli antigens other than ETEC using the Pan-DEC protein microarray. This was the first field application of the novel pan-DEC array as a new tool in assessing the antigenic breadth of antibody responses induced by the ETVAX vaccine, as well as to assess early life exposure to DEC pathotypes and other bacterial enteric pathogens. We observed that plasma obtained from ETVAX® and placebo recipients had high antibody reactivity to Ipa, SseC and EspB proteins. These findings suggest that there is high exposure early in life to DEC pathogens, like EPEC, EHEC, EAEC and EIEC in addition to ETEC, in the Zambian population. These immunological observations are consistent with the results of recent epidemiological studies assessing the etiology of diarrheal disease among infants and young children in Zambia.

Health Science Students' Perceptions of Hand Hygiene Education and Practice in a South African University: Introducing the University Hand Hygiene Improvement Model.

Hand hygiene serves as a critical preventative measure against the spread of acquired infections in healthcare facilities and is an integral component of patient safety programs. While healthcare students in training are typically introduced to the principles underlying hand hygiene, the translation of this understanding into practice is often lacking, and compliance has remained low. This study aimed to evaluate health science students' in biomedical sciences, chiropractic and emergency medical care, environmental health, complementary medicine, medical imaging and radiation sciences, nursing, optometry, podiatry, and sports and movement studies perceptions regarding hand hygiene education (knowledge and attitude) and practice at a university in South Africa. Consenting students were asked to complete an online questionnaire that tested their knowledge, practices, and skills in handwashing. The ANOVA analysis results suggested significant differences in hand hygiene scores across departments and years of study. The multiple regression analyses confirmed these findings, suggesting that the department of study significantly influenced all aspects of hand hygiene, while the year of study affected hand hygiene skills, and age group influenced hand hygiene practices. Based on these findings, a conceptual model, the University Hand Hygiene Improvement Model (UHHIM), was proposed to enhance the teaching and learning of hand hygiene at the university level. The model underscores the necessity of targeted education, continuous monitoring, and feedback, and the pivotal roles of hand hygiene facilitators and student participation in enhancing hand hygiene behaviors.

High Burden of Co-Infection with Multiple Enteric Pathogens in Children Suffering with Diarrhoea from Rural and Peri-Urban Communities in South Africa.

Infectious diarrhoea contributes to high morbidity and mortality in young children from sub-Saharan Africa. The aim of this study was to assess the prevalence of single and multiple diarrhoeal-causing pathogen combinations in children suffering from diarrhoea from rural and peri-urban communities in South Africa. A total of 275 diarrhoea stool specimens were collected between 2014 and 2016 from Hospitals and Primary Health Care clinics. The BioFire® FilmArray® Gastrointestinal panel was used to simultaneously detect 22 diarrhoea pathogens (viruses, bacteria, parasites) known to cause diarrhoea. A total of 82% (226/275) enteric pathogens were detected in the stool specimens. The two most detected bacterial, viral and parasitic pathogens each included: EAEC (42%), EPEC (32%), Adenovirus F40/41 (19%), Norovirus (15%), Giardia (8%) and Cryptosporidium (6%), respectively. Single enteric pathogen infections were recorded in 24% (65/275) specimens with EAEC, and Norovirus was found in 26% (17/65) and 14% (9/65) of the specimens, respectively. Multiple enteric pathogen combinations were recorded in 59% (161/275) of the stool specimens with 53% (85/161) containing two pathogens, 22% (35/161) containing three pathogens and 25% (41/161) containing four or more pathogens. The results from this study demonstrated the complex nature of pathogen co-infections in diarrhoeal episodes which could have an impact on treatment effectiveness.

Prevalence of Diarrhoeagenic Escherichia coli among Children Aged between 0-36 Months in Peri-Urban Areas of Lusaka.

Diarrhoea is a major contributor to childhood morbidity and mortality in developing countries, with diarrhoeagenic Escherichia coli being among the top aetiological agents. We sought to investigate the burden and describe the diarrhoeagenic E. coli pathotypes causing diarrhoea among children in peri-urban areas of Lusaka, Zambia. This was a facility-based surveillance study conducted over an 8-month period from 2020 to 2021. Stool samples were collected from children aged 0-3 years presenting with diarrhoea at five peri-urban health facilities in Lusaka. Stool samples were tested for diarrhoeagenic E. coli using the Novodiag bacterial GE+® panel, a platform utilising real-time PCR and microarray technology to detect bacterial pathogens. Of the 590 samples tested, diarrhoeagenic E. coli were detected in 471 (76.1%). The top three pathogens were enteropathogenic E. coli 45.4% (n = 268), enteroaggregative E. coli 39.5% (n = 233), and enterotoxigenic E. coli 29.7% (n = 176). Our results revealed that 50.1% of the diarrhoeagenic E. coli positive samples comprised multiple pathotypes of varying virulence gene combinations. Our study demonstrates a high prevalence of diarrhoeagenic E. coli in childhood diarrhoea and the early exposure (<12 months) of children to enteric pathogens. This calls for the early implementation of preventive interventions for paediatric diarrhoea.

Knowledge, Attitudes, and Perceptions Towards Hand Hygiene of Optometry Students Pre- and Peri-COVID-19 at a Tertiary Institution in Johannesburg, South Africa.

Purpose: Limited research has been completed relating to the knowledge, attitudes and practices (KAP) towards hand hygiene in optometry. The necessity of identifying possible gaps in the cycle of the optometric examination that may have an impact on standard hygiene practices is essential, especially seen in the context of the COVID-19 pandemic. The purpose of the study was to determine if optometry students' KAP towards hand hygiene changed pre- and peri-COVID-19 to minimize the risk of possible infection it may have for their patients, family, and themselves. Patients and Methods: A cross-sectional purposeful sample study was completed among optometry students at a training institution in Johannesburg, South Africa, pre- (2019) and peri-COVID-19 (2022). The WHO hand hygiene knowledge and perception questionnaires for health care workers were adapted and used in the current study. Statistical analyses were performed to test for significant changes between the two groups. Results: There was a significant change (p < 0.01) in the use of alcohol-based hand rub (ABHR) in 2022 (87.2%) compared to 2019 (46.5%), although only 41% of students peri-COVID-19 (2022), were aware that a minimum of 20 seconds is required to effectively clean hands. Students, both pre-COVID-19 (63.8%) and peri-COVID-19 (81.8%) perceived performing a hand hygiene regime during an optometric examination to be problematic. A significant peri-COVID-19 (2022) change in perception (p < 0.01) regarding the importance of completing required hand hygiene practices in front of a patient during examination was seen. Conclusion: The COVID-19 pandemic provided a unique opportunity to research a possible change in KAP towards hand hygiene practices in optometry students that have not been investigated before. Students were more aware of the impact of hand hygiene practices and the perception thereof, especially by patients and fellow students during the peri-COVID-19 (2022) period. An important finding was the difficulty that students experienced to perform a hand hygiene regime during an optometric examination.

The effect of a hand hygiene intervention on the behaviour, practices and health of parents of preschool children in South Africa.

INTRODUCTION: Diarrhoea and upper respiratory diseases are a leading cause of child mortality in children under 5 years of age both in South Africa and worldwide. Hand hygiene (HH) interventions play a critical role in reducing HH-related diseases, and the inclusion of all stakeholders in such interventions has improved the success of such interventions. The purpose of this study is to determine the effect of an HH intervention on the behaviour, practices, and health of parents of preschool children. METHODOLOGY: Seventeen preschools were randomly selected and placed into intervention (IG = 8) and control groups (CG = 9). Parents (N = 191) were requested to complete questionnaires both pre- and postintervention. An intervention was applied to IG preschool respondents. The data were analysed and compared pre- and postintervention between IG and CG. RESULTS: Parents of IG showed a significant difference pre- and postintervention in HH practices such as washing hands after coughing and sneezing, and after using the toilet while parents in CG also indicated significant differences in HH practices of washing hands after coughing and sneezing, and after wiping children's noses. Postintervention, IG families reporting runny tummies were significantly less than pre-intervention and a decrease in doctor's visits. There was a 5% improvement of all HH practices in both IG and CG. CONCLUSION: Over 90% of parents in both groups washed hands after using the toilet, both pre- and postintervention. All HH practices for both groups showed increases both pre- and postintervention. By making use of available resources and regular communication with parents of preschoolers they are able to make the small changes necessary to improve their HH and that of their families.

A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid.

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a "stressed state" due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.

Adaptation of the WHO Hand Hygiene Observation Form for Application in Optometry Training Facilities.

BACKGROUND: The link between hand and surface hygiene and the spread of disease has been reported by various studies and understanding the appropriate use of hand hygiene is important in healthcare. The World Health Organization (WHO) introduced the "My Five Moments for Hand Hygiene" model to educate healthcare workers about the importance of hand hygiene to stop the spread of disease. OBJECTIVE: The objectives of this paper were to determine if the WHO Hand Hygiene Observation Form could be used to monitor for hand and personal hygiene practices of Optometry students, and what their actual hygiene practices are in an Optometry training facility in South Africa. METHODS: This cross-sectional study was performed at an Optometry training facility in South Africa using third (n=32) and fourth year (n=39) students. The Hand Hygiene Knowledge Questionnaire for Health Care Workers by the WHO was used to determine their knowledge regarding hand hygiene. After completing the questionnaire, participants were observed in the clinic while consulting patients to determine the hand and surface hygiene practices they apply. RESULTS: The WHO hand hygiene observation rubric was not appropriate to evaluate hand and surface hygiene in Optometry facilities. The adapted observation rubric created was a better tool to evaluate student hygiene practices and was able to record which hand or surface hygiene were missed when presented with such opportunities. Although 90.1% of the students knew that germs could be transferred to the patients via hands, only 46.5% routinely used an alcohol-based hand sanitizer. CONCLUSION: This observation rubric developed during this study can assist in creating awareness with students about their own hand and surface hygiene deficiencies when interacting with patients.

Co-Existence of Free-Living Amoebae and Potential Human Pathogenic Bacteria Isolated from Rural Household Water Storage Containers.

This study investigated the co-existence of potential human pathogenic bacteria and free-living amoebae in samples collected from stored water in rural households in South Africa using borehole water as a primary water source. Over a period of 5 months, a total of 398 stored water and 392 biofilm samples were collected and assessed. Free-living amoebae were identified microscopically in 92.0% of the water samples and 89.8% of the biofilm samples. A further molecular identification using 18S rRNA sequencing identified Vermamoeba vermiformis, Entamoeba spp., Stenamoeba spp., Flamella spp., and Acanthamoeba spp. including Acanthamoeba genotype T4, which is known to be potentially harmful to humans. Targeted potential pathogenic bacteria were isolated from the water samples using standard culture methods and identified using 16S rRNA sequencing. Mycobacterium spp., Pseudomonas spp., Enterobacter spp., and other emerging opportunistic pathogens such as Stenotrophomonas maltophilia were identified. The results showed the importance of further studies to assess the health risk of free-living amoebae and potential human pathogenic bacteria to people living in rural communities who have no other option than to store water in their homes due to water shortages.

Detection of Free-Living Amoebae and Their Intracellular Bacteria in Borehole Water before and after a Ceramic Pot Filter Point-of-Use Intervention in Rural Communities in South Africa.

Free-living amoebae (FLA) are ubiquitous in nature, whereas amoeba-resistant bacteria (ARB) have evolved virulent mechanisms that allow them to resist FLA digestion mechanisms and survive inside the amoeba during hostile environmental conditions. This study assessed the prevalence of FLA and ARB species in borehole water before and after a ceramic point-of-use intervention in rural households. A total of 529 water samples were collected over a five-month period from 82 households. All water samples were subjected to amoebal enrichment, bacterial isolation on selective media, and molecular identification using 16S PCR/sequencing to determine ARB species and 18S rRNA PCR/sequencing to determine FLA species present in the water samples before and after the ceramic pot intervention. Several FLA species including Acanthamoeba spp. and Mycobacterium spp. were isolated. The ceramic pot filter removed many of these microorganisms from the borehole water. However, design flaws could have been responsible for some FLA and ARB detected in the filtered water. FLA and their associated ARB are ubiquitous in borehole water, and some of these species might be potentially harmful and a health risk to vulnerable individuals. There is a need to do more investigations into the health risk of these organisms after point-of-use treatment.

Soul of the Jukskei River: The Extent of Bacterial Contamination in the Jukskei River in Gauteng Province, South Africa.

River water quality is an important health issue as the water is utilised for drinking, domestic and agricultural use in developing countries. This study aimed to investigate the effect water from a major city has on the water quality of the Jukskei River that daylights in Johannesburg, South Africa. The river water samples were analysed for physio-chemical properties, microbiology, antibiotic resistance of bacterial isolates, genetic markers, and potentially toxic metals. Data analysis revealed increased electrical conductivity, total dissolved solids, and turbidity since 2010. Total Coliform and Escherichia coli detected were above the South African water quality guidelines for domestic, recreational, and irrigation purposes. Additionally, sodium, zinc, nickel, lithium, and lead exceeded the guidelines in domestic, recreational, and irrigation water. Pathogenic strains of E. coli (aEPEC, EHEC, EIEC, and EAEC) were isolated from the water. Various other potentially pathogenic organisms that have been implicated as causes of gastro-intestinal, and a wide range of other diseases, were also detected and demonstrated multiple levels of resistance to antibiotics tested. The results show that the river water is a potential health threat to downstream users. These results will feed into the environmental management action plan for Water for the Future (NGO group).

Effect of a simple intervention on hand hygiene related diseases in preschools in South Africa: research protocol for an intervention study.

INTRODUCTION: Hand hygiene (HH) related illnesses such as diarrhoea and respiratory diseases, contribute to the burden of disease and are included in the top five causes of mortality in children under 5 years in South Africa. Children attending preschools are more susceptible to these infections due to the higher number of children in preschools. HH interventions have shown to reduce HH-related diseases by improving HH practices. In South Africa, there are no documented HH interventions or studies in children under 5 years. The purpose of the study is to determine whether an HH intervention can reduce HH-related diseases among 4-5-year-old preschool children and to improve HH practices in these children, their caregivers and their parents. METHODOLOGY AND ANALYSIS: This is a protocol for a controlled intervention study to be conducted at preschools in Kempton Park, City of Ekurhuleni, Gauteng, South Africa. Preschools will be randomly distributed into control and experimental groups (n=70). The intervention includes interactive simulation learning, educational emails and education and poster reminders obtained from the WHO and the Global Handwashing Day website. Data collection, including the intervention, will take place during the calendar year as this coincides with the school year. Data will be analysed both preintervention and postintervention in the experimental group as well as between the experimental and control group. Data collected by means of questionnaires, observations, disease registers, hygiene inspections, semi-structured interviews and hand swabs will be analysed to determine these outcomes. ETHICS AND DISSEMINATION: Permission has been obtained from the University of Johannesburg Ethics Committee and Ministerial Consent for Non-Therapeutic Research on Minors from the Department of Health National Ethics Research Council. Permissions for use of copyright protected materials has been obtained. Results of the study will be disseminated through peer-reviewed publications, and feedback within relevant structures through conference proceedings.