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.

Occurrence of Hybrid Diarrhoeagenic Escherichia coli Associated with Multidrug Resistance in Environmental Water, Johannesburg, South Africa.

This study was undertaken to determine the virulence and antibiotic resistance profiles of diarrhoeagenic Escherichia coli (DEC) in environmental waters of Johannesburg, South Africa. Samples were collected and cultured on selective media. An 11-plex PCR assay was used to differentiate five DEC, namely: enteroaggregative (EAEC), enterohaemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC) and enterotoxigenic (ETEC). The antibiotic resistance profile of isolates was determined using the VITEK®-2 automated system. The virulence profiles of 170 E. coli tested showed that 40% (68/170) were commensals and 60% (102/170) were pathogenic. EPEC had a prevalence of 19.2% (32/170), followed by ETEC 11.4% (19/170), EAEC 6% (10/170) and EHEC 3% (5/170). Hybrid DEC carrying a combination of simultaneously two and three pathogenic types was detected in twenty-eight and nine isolates, respectively. The antibiotic susceptibility testing showed isolates with multidrug resistance, including cefuroxime (100%), ceftazidime (86%), cefotaxime (81%) and cefepime (79%). This study highlighted the widespread occurrence of DEC and antibiotic resistance strains in the aquatic ecosystem of Johannesburg. The presence of hybrid pathotypes detected in this study is alarming and might lead to more severe diseases. There is a necessity to enhance surveillance in reducing the propagation of pathogenic and antibiotic-resistant strains in this area.

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.

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.

Rapid Quantification of the Total Viable Bacterial Population on Human Hands Using Flow Cytometry with SYBR® Green I.

BACKGROUND: Monitoring the efficiency of hand washing typically relies on determining the presence or absence of targeted organisms on the hands, not necessarily determining if the method is working. The results focus on the organism studied and do not give a true reflection of the efficiency of the hygiene intervention. To obtain a better picture, this study tested flow cytometry with SYBR® Green I to quantify bacterial populations on the hands of participants in a healthcare training clinic at a University in South Africa. METHODS: Participants "washed" both hands in a buffer solution in a sterile bag, and the total and viable bacterial populations numbers were determined using flow cytometry and compared with standard culture-based methods, testing for total coliforms, E. coli and heterotrophic organisms (IDEXX Colilert®-18 Quanti-Trays™ and SimPlates®) and fastidious organisms with hemolytic activity (Blood Agar). RESULTS: Compared to the culture-based method, flow cytometry rapidly distinguished total, viable, and dead bacterial populations in all samples. As expected, the culture-based methods gave lower bacterial counts and were limited by the detection limit of each test, requiring further testing with additional costs. Although the exact bacterial species in the population could not be identified with flow cytometry, viable counts from flow cytometric analysis were two times more precise than the data obtained with any of the culture-based methods tested. CONCLUSIONS: Flow cytometry was found to be suitable for immediate, accurate, and automatic detection of bacteria, and because it describes the viable bacterial population, it is ideal to monitor hand hygiene interventions. © 2019 International Clinical Cytometry Society.

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.

Surviving the acid barrier: responses of pathogenic Vibrio cholerae to simulated gastric fluid.

When bacteria are subjected to low acidic pHs of the gastric environment, they may enter the viable but nonculturable (VBNC) state of survival. In this state, bacteria cannot be cultured on solid media, still exhibit signs of metabolic activity (viability). In this study, the response of pathogenic Vibrio cholerae O1 and O139 to low pH-simulated environments of the human stomach was evaluated for their survival by culturability (plate count) and viability (flow cytometry-FC) assays. Bacteria were acid challenged with simulated gastric fluid (SGF) at pH 1.5, 2.5, 3.5 and 4.5 over a period of 180 min. Exposure to SGF up to 120 min increased acid tolerance of the Vibrios up to pH 3.5 with acid challenge occurring at pH 4.5. Bacteria were culturable from pH 2.5 to 4.5 up to 60 min SGF exposure. The stationary-phase cultures of Vibrio were able to survive SGF at all pHs in an 'injured' state with FC. This could possibly mean that the bacteria have entered the VBNC stage of survival. This is a worrying public health concern due to the fact that once favourable conditions arise (intestines), these Vibrios can change back to an infectious state and cause disease.

The viable but non-culturable state in pathogenic Escherichia coli: A general review.

BACKGROUND: The persistence and pathogenicity of pathogenic bacteria are dependent on the ability of the species to survive in adverse conditions. During the infectious process, the organism may need to pass through certain hostile anatomical sites, such as the stomach. Under various environmental stresses, many bacteria enter into the viable but non-culturable (VBNC) state, where they are 'alive' or metabolically active, but will not grow on conventional media. Escherichia coli bacteria encounter several diverse stress factors during their growth, survival and infection and thus may enter into the VBNC state. OBJECTIVES: This review discusses various general aspects of the VBNC state, the mechanisms and possible public health impact of indicator and pathogenic E. coli entering into the VBNC state. METHOD: A literature review was conducted to ascertain the possible impact of E. coli entering into the VBNC state. RESULTS: Escherichia coli enter into the VBNC state by means of several induction mechanisms. Various authors have found that E. coli can be resuscitated post-VBNC. Certain strains of pathogenic E. coli are still able to produce toxins in the VBNC state, whilst others are avirulent during the VBNC state but are able to regain virulence after resuscitation. CONCLUSION: Pathogenic and indicator E. coli entering into the VBNC state could have an adverse effect on public health if conventional detection methods are used, where the number of viable cells could be underestimated and the VBNC cells still produce toxins or could, at any time, be resuscitated and become virulent again.

Detection of human enteric viruses in Umgeni River, Durban, South Africa.

The prevalence of adenovirus (AdV), rotaviruses (RV) and enteroviruses (EV) in Umgeni River waters of Durban, South Africa was assessed qualitatively and quantitatively during April 2011 to January 2012 using polymerase chain reaction (PCR)/reverse transcription-polymerase chain reaction (RT-PCR), nested PCR and quantitative PCR (qPCR), as well as nested integrated cell culture PCR (nested ICC-PCR). The phylogenetic analysis of the adenovirus and enterovirus amplicons was also performed. The nested PCR results effectively detected the presence of AdV and EV in all water samples. The results of qPCR demonstrated that higher populations of EV and of AdV were widely found in the Umgeni River. Rotavirus could only be detected in the upper Umgeni River, mainly during drier seasons. Nested ICC-PCR further confirmed the presence of infectious AdV and EV particles in 100% of water samples using various cell lines. The present study identifies potential viral hazards of Umgeni River water for domestic water supply and recreational activities.

Microbiological, coliphages and physico-chemical assessments of the Umgeni River, South Africa.

The water quality of Umgeni River in KwaZulu-Natal (South Africa) was investigated from April 2011 to January 2012. Indicator bacterial populations, physico-chemical properties, heavy metal contaminants and the presence of coliphages were determined according to standard protocols. The results showed that all sampling points failed to comply with the set guidelines for turbidity, total coliform, faecal coliform and total heterotrophic counts. Salmonella spp., Shigella spp. and Vibrio cholerae were also detected in all the water samples. The somatic coliphages and F-RNA coliphages were detected more frequently in the lower reaches of the river during summer. Temperature, electrical conductivity and pH were found to have positive relationships with the microbial communities especially in the lower catchment area during spring and summer indicating the impacts of various anthropogenic activities in the surrounding areas.