The implementation of sub-typing techniques to determine the diversity of L. monocytogenes strains adapted to the food processing environment and their association with human listeriosis cases

Philosophiae Doctor - PhDListeria monocytogenes has been established as a food-borne pathogen since the early 1980s and has become a big concern for the food industry and Public Health authorities (Doyle 2001; Oliveira et al. 2003; Capita et al. 2005; Conly and Johnston 2008). It is a Gram-positive, opportunistic facultative intracellular bacterium which is frequently present in nature and may be found in any food environment (Liu 2006; Chen et al. 2007; Conly and Johnston 2008). Of the six species of Listeria, L. monocytogenes is the only one capable of causing listeriosis, a severe food-borne illness in humans (de Vasconcelos et al. 2008). For the average healthy person, although the incidence of infection is low, symptoms of febrile gastroenteritis may be presented (Gianfranceschi et al. 2007; Kersting et al. 2010). In immunocompromised individuals however, the hospitalization and mortality rates are amongst the highest for pathogenic organisms (Tran and Kathariou 2002; Lin et al. 2006; Schuppler and Loessner 2010). Illnesses such as septicaemia and central nervous system infections may also occur in these individuals (Roberts et al. 2006; Schuppler and Loessner 2010). Pregnant women and their fetus are also largely at risk where pre-term delivery and birth defects may occur as a result of listeriosis (Doyle 2001; Garrido et al. 2008). The epidemiological surveillance systems for the reporting of listeriosis are poor as it is a non-notifiable disease in many countries. Therefore, the incidence of infection that is regarded as low must be reconsidered (Mammina et al. 2009; Pinto et al. 2010). Listeria monocytogenes can reproduce in a wide variety of reservoirs within food processing plants, thereby contaminating the food which then poses a risk for food-borne illness. It can be transmitted from infected animals to humans and also through the consumption of foods from animal origin (Kalender 2003; Kersting et al. 2010). Animals are infected by Listeria spp. found in the environment; the organism is then transmitted through the blood, milk and excrement of the animal back into the environment where manure, soil, feed and water can become contaminated again (Akpolat et al. 2004; Kersting et al. 2010). Poultry products and ready-to-eat (RTE) food that support the growth of L. monocytogenes, including soft cheeses, unpasteurized milk, hotdogs, deli meats, vegetables and fruits have been linked to cases of listeriosis (Rørvik et al. 2003; Chen et al. 2007; Conly and Johnston 2008; Ford 2010; Kersting et al. 2010). Regardless of HACCP systems that are in place in the food processing plants, listeriosis outbreaks still occur as a result of the ingestion of these food products. Serotyping, based on the serological reaction between somatic (O) and flagellar (H) antigens and their corresponding sera, has identified 13 L. monocytogenes serotypes (Nadon et al. 2001; Wiedmann 2002; Kérouanton et al. 2010). Of the 13 serotypes of L. monocytogenes, 1/2a, 1/2b and 4b are responsible for more than 95% of listeriosis infections in humans (Mereghetti et al. 2002; Moorhead et al. 2003; Borucki et al. 2004;de Vasconcelos et al. 2008). L. monocytogenes serotypes 1/2a and 1/2b are mainly associated and isolated sporadically from food and 4b is responsible for the major human epidemic cases (Gilbreth et al. 2005). L. monocytogenes serotypes 1/2a and 1/2b are also responsible for sporadic cases of human illness (Wiedmann 2002).South Afric

Sample preparation methods and molecular based detection for the rapid isolation and identification of Listeria monocytogenes in food samples

Magister Scientiae - MScListeria monocytogenes is a Gram-positive bacterium responsible for listeriosis, a food-borne disease, which may result in severe illness and possible death. The importance of L. monocytogenes as a food-borne pathogen has been recognized since the 1980's when a correlation between the cunsumption of contaminated foodstuffs and human listeriosis outbreaks was observed. Listeriosis occurs with the ingestion of contaminated foods. The aim of this study involved developing DNA based methods to aid the food industry for the fast detection of L. monocytogenes in food products. Therefore assays were developed in such a way that they will have potential applications in the food idustry.South Afric

Carbapenem resistance in <i>Enterobacterales</i> from agricultural, environmental and clinical origins: South Africa in a global context

Carbapenem agents are regarded as last-resort antibiotics, however, bacterial resistance towards carbapenems has been reported in both clinical and agricultural settings worldwide. Carbapenem resistance, defined as the resistance of a bacteria towards one or more carbapenem drugs, can be mediated in either of, or a combination of, three mechanisms–although, the mechanism mediated through the production of carbapenemases (β-lactamases that are able to enzymatically degrade carbapenems) is of most significance. Of particular concern is the occurrence of carbapenemase producing Enterobacterales (CPE), with literature describing a dramatic increase in resistance globally. In South Africa, increases of carbapenemase activity occurring in Enterobacter species, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa have recently been reported. CPE can also be found in agricultural environments, as global studies have documented numerous instances of CPE presence in various animals such as pigs, cattle, seafood, horses and dogs. However, most reports of CPE occurrence in agricultural settings come from Northern America, Europe and some parts of Asia, where more extensive research has been conducted to understand the CPE phenomenon. In comparison to clinical data, there are limited studies investigating the spread of CPE in agricultural settings in Africa, highlighting the importance of monitoring CPE in livestock environments and the food chain. Further research is necessary to uncover the true extent of CPE dissemination in South Africa. This review will discuss the phenomenon of bacterial antibiotic resistance (ABR), the applications of the carbapenem drug and the occurrence of carbapenem resistance globally

Invasive carbapenem-resistant Enterobacteriaceae infection at a paediatric hospital: A case series

Background. There are no paediatric reports of invasive infection caused by carbapenem-resistant Enterobacteriaceae (CRE) from Africa.Objectives. To document a series of cases of CRE infections at a tertiary children’s hospital in Cape Town, South Africa, describing the clinical and microbiological findings in these children.Methods. A retrospective, descriptive study was completed using data from a series of children with invasive CRE infection between 2010 and 2015, sourced from their clinical notes and microbiology results.Results. The first of 10 invasive CRE infections during the study period occurred in November 2012. Nine CRE infections were caused by Klebsiella pneumoniae, and one by both K. pneumoniae and Escherichia coli. The median age was 25 months (interquartile range (IQR) 5 - 60). All 10 CRE infections were hospital acquired. The median length of hospitalisation before CRE infection was 28.5 days (IQR 20 - 44). Eight of the children were exposed to carbapenems during the 12-month period prior to invasive CRE infection. Six were treated with colistin and carbapenem combination therapy, of whom 2 died, including 1 of a non-CRE event. The other 4 children received colistin monotherapy. All these children died, including 2 from non-CRE events.Conclusions. Children with invasive CRE infection and severe underlying disease must be treated with combination antibiotic therapy. Strict infection control practice and antibiotic stewardship are necessary to contain the spread of CRE and limit the number of new infections

Emergence of vancomycin-resistant Enterococcus at a tertiary paediatric hospital in South Africa

Background. During 2013, the haematology/oncology unit at a tertiary level paediatric hospital in South Africa experienced the emergence of infection with vancomycin-resistant Enterococcus (VRE).Objective. To describe the clinical and molecular aspects of the cases identified.Methods. VRE isolates identified from blood culture specimens processed at the National Health Laboratory Service were screened for the presence of the vancomycin resistance genes vanA, B and C1, 2 and 3. Further characterisation of these isolates was carried out using pulsed-field gel electrophoresis (PGFE) and multilocus sequence typing (MLST). Clinical records of infected patients were reviewed to identify possible risk factors, while surveillance with rectal swabs was performed to identify VRE-colonised patients.Results. Four patients with haematological malignancies were identified with VRE bloodstream infections. Patients were immunocompromised at the time of the bloodstream infection (BSI), with receipt of vancomycin prior to VRE-BSI, and infections were treated with linezolid. Colonisation with VRE was found in 8 of 55 patients screened. Infected and colonised patients were isolated in the unit during their admission and strict contact precaution infection control practices were instituted. The vanA gene was identified in all of the isolates but one. PFGE and MLST results showed a degree of genetic relatedness between certain isolates obtained from rectal swab and blood culture samples, suggesting possible patient-to-patient transmission or persistence of the isolates in the unit.Conclusion. Strict infection control practices are necessary to prevent infection and transmission of resistant organisms among vulnerable patients

The Effect of Spray Parameters on the Survival of Bacteriophages

There have been numerous studies highlighting the efficacy of various bacteriophages (phages) and phage cocktails in the reduction of pathogens in food. Despite approval from legislative bodies permitting phage use in food processing environments, applied via spray or dip, there is still no information on which spray parameters should be used for successful implementation. The study here investigates phage survival diluted to 1% in distilled water (dH2O) and prepared bottled water (PBW), followed by a subsequent spray application through a fixed nozzle (530 &mu;m) and strainer size (74 &times; 74 &mu;m), with pressures of 3, 5, and 6 Bar. The survival of the phage was determined through sampling the outputs of the spray system and performing double agar overlay plaque assays. PBW decreased the phage concentration (p = 0.18) more than the dH2O (p = 0.73) prior to spray application. It was found that the PBW phage solution was less affected by the various spray parameters (p = 0.045) than the dH2O (p = 0.011). The study showed that unchlorinated water (dH2O), as well as a pressure of 3 Bar, had the highest output phage concentration through the nozzle and strainer, providing valuable information for industrial implementation