Risk assessment of Escherichia coli O157:h7, Listeria monocytogenes, Salmonella enterica subsp. Enterica serovar typhimurium and Staphylococcus aureus within a stone fruit production environment

In recent years there has been a global increase in fresh produce associated foodborne disease outbreaks caused by Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium and Staphylococcus aureus. Fruit and vegetables can become contaminated at various points along the supply chain. Therefore there is a great need for food safety assurance of fresh fruit and vegetables produced on farm. South Africa is a large exporter of peaches and plums and a lack of an effective food safety assurance system could hamper market access and eventually result in a barrier to trade. The aim of this study was to determine the level of risk associated with peaches and plums produced in South Africa. In order to address the overall aim of the study an effective foodborne pathogen molecular detection system in a form of a multiplex PCR was developed and used in monitoring water, fruit and environmental samples. This detection system formed part of the developed framework to assess the possible exposure of fresh produce and to identify the presence of hazards on farm. An aspect of the study was to determine effectiveness of on-farm horticultural safety management system (HSMS) and to make recommendations to primary producers that would allow them to increase food safety assurance. Following the semi-quantitative risk assessment conducted the overall findings showed that peaches and plums were considered low risk commodities, due to the low incidence of fruit contamination in the sample size, as well as the pathogens inability to grow and survive on fruit through a simulated export chain to titres that would exceed the infectious dose of the organism. In conclusion, peaches and plums in this study that were bound for export were found to be safe for consumption, with the risk to the international consumer being low.Thesis (PhD)--University of Pretoria, 2013.Microbiology and Plant PathologyUnrestricte

Effect of temperature and nutrient concentration on survival of foodborne pathogens in deciduous fruit processing environments for effective hygiene management

Temperature and good sanitation practices are important factors for controlling growth of microorganisms. Fresh produce is stored at various temperatures to ensure quality and to prolong shelf life. When foodborne pathogens survive and grow on fresh produce at storage temperatures, then additional control strategies are needed to inactivate these pathogens. The aim of this study was to determine how temperatures associated with deciduous fruit processing and storage facilities (0.5, 4, and 21°C) affect the growth and/or survival of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica subsp. enterica serovar Typhimurium, and Staphylococcus aureus under different nutrient conditions (nutrient rich and nutrient poor) and on simulated contact surfaces (vinyl coupons). Information on the growth and survival of foodborne pathogens at specific deciduous fruit processing and storage temperatures (0.5°C) is not available. All pathogens except E. coli O157:H7 were able to survive on vinyl coupons at all temperatures. L. monocytogenes proliferated under both nutrient conditions independent of temperature. S. aureus was the pathogen least affected by nutrient conditions. The survival of foodborne pathogens on the vinyl coupons, a model system for studying surfaces in fruit preparation and storage environments, indicates the potential for cross-contamination of deciduous fruit products under poor sanitation conditions. Foodborne pathogens that can proliferate and survive at various temperatures under different nutrient conditions could lead to fruit cross-contamination. Temperature mismanagement, which could allow pathogen proliferation in contaminated fruit packing houses and storage environments, is a concern. Therefore, proper hygiene and sanitation practices, removal of possible contaminants, and proper food safety management systems are needed to ensure food safety

Assessment of foodborne pathogen presence in the peach supply chain and its potential risk to the end consumer

Peaches are popular, nutritious and widely consumed. Being a tree crop, it is considered a low risk fruit, with no direct water contact, and no previous foodborne disease outbreaks associated with its consumption. However, in 2014 the pioneer association between stone fruit and a foodborne illness was reported, linking Listeria monocytogenes to stone fruit. This highlights the need for better understanding of risk associated with contaminated fresh stone fruit, in order to implement adequate preventative measures. No information is available on the presence of foodborne pathogens on peaches in the supply chain. A case study approach was therefore followed to assess foodborne pathogen presence on the farm, focusing on the impact of irrigation water, facility sanitation and hygiene by collecting various fruit and environmental samples (n ¼ 428). This study demonstrates the effectiveness of integrating basic microbial testing with safety management and risk assessment tools that can be collectively used to improve the food safety management system. No Salmonella Typhimurium was detected from samples, however, Escherichia coli O157:H7, Listeria spp. and Staphylococcus aureus were detected on fruit and environmental samples. Despite the Global G.A.P. certification status of the farm, livestock frequented water sources which lead to E. coli O157:H7 contamination. This conclusion was based on positive detection of foodborne pathogens from the water sources and subsequent removal of livestock which resulted in a definite decrease in pathogen detection. A number of E. coli O157:H7 and S. aureus were detected during the second year of monitoring from environmental samples and it was observed that the personal hygiene and facility sanitation was not adequately enforced. Based on feedback given to the farmer, enforcement was improved and a definite decrease in foodborne pathogens was observed in the following sampling cycle. Areas of risk that were still identified following the fourth year of monitoring included the water source used for irrigation and poor sanitation in the production and processing facilities. Limited foodborne pathogen prevalence on peaches over the full study period as well as the extended export supply chain at controlled temperatures resulted in low-to-medium calculated consumer risk. The correct and meticulous implementation of integrated and holistic pre- and post-harvest food safety management systems is therefore essential to prevent produce contamination, reduce the consumer risk and therefore ensure overall product safety

Comparison of biofilm formation and water quality when water from different sources was stored in large commercial water storage tanks

Rain-, ground- and municipal potable water were stored in low density polyethylene storage tanks for a period of 90 days to determine the effects of long-term storage on the deterioration in the microbial quality of the water. Total viable bacteria present in the stored water and the resultant biofilms were enumerated using heterotrophic plate counts. Polymerase chain reaction (PCR) and Colilert-18® tests were performed to determine if the faecal indicator bacteria Escherichia coli was present in the water and in the biofilm samples collected throughout the study. The municipal potable water at the start of the study was the only water source that conformed to the South African Water Quality Guidelines for Domestic Use. After 15 days of storage, this water source had deteriorated microbiologically to levels considered unfit for human consumption. E. coli was detected in the ground- and potable water and ground- and potable biofilms periodically, whereas it was detected in the rainwater and associated biofilms at every sampling point. Imperfections in the UV resistant inner lining of the tanks were shown to be ecological niches for microbial colonisation and biofilm development. The results from the current study confirmed that long-term storage can influence water quality and increase the number of microbial cells associated with biofilms on the interior surfaces of water storage tanks

High prevalence of multidrug resistant Escherichia coli isolated from fresh vegetables sold by selected formal and informal traders in the most densely populated Province of South Africa

Contaminated fresh produce has increasingly been implicated in foodborne disease outbreaks. As microbiological safety surveillance in South Africa is limited, a total of 545 vegetable samples (spinach, tomato, lettuce, cucumber, and green beans) were purchased from retailers, street traders, trolley vendors and farmers' markets. Escherichia coli, coliforms and Enterobacteriaceae were enumerated and the prevalence of Escherichia coli, Salmonella spp. and Listeria monocytogenes determined. E. coli isolates were characterized phenotypically (antibiotic resistance) and genotypically (diarrheagenic virulence genes). Coliforms, E. coli and Enterobacteriaceae counts were mostly not significantly different between formal and informal markets, with exceptions noted on occasion. When compared to international standards, 90% to 98% tomatoes, 70% to 94% spinach, 82% cucumbers, 93% lettuce, and 80% green bean samples, had satisfactory (≤ 100 CFU/g) E. coli counts. Of the 545 vegetable samples analyzed, 14.86% (n = 81) harbored E. coli, predominantly from leafy green vegetables. Virulence genes (lt, st, bfpA, eagg, eaeA, stx1, stx2, and ipaH) were not detected in the E. coli isolates (n = 67) characterized, however 40.30% were multidrug-resistant. Resistance to aminoglycosides (neomycin, 73.13%; gentamycin, < 10%), penicillins (ampicillin, 38.81%; amoxicillin, 41.79%; augmentin, < 10%), sulfonamides (cotrimoxazole, 22.39%), tetracycline (19.4%), chloramphenicol (11.94%), cephalosporins (cefepime, 34.33%), and carbapenemases (imipenem, < 10%) were observed. This study highlights the need for continued surveillance of multidrug resistant foodborne pathogens in fresh produce retailed formally and informally for potential consumer health risks. PRACTICAL APPLICATION: The results indicate that the microbiological quality of different vegetables were similar per product type, regardless of being purchased from formal retailers or informal street traders, trolley vendors or farmers' markets. Although no pathogenic bacteria (diarrheagenic E. coli, Salmonella spp. or L. monocytogenes) were isolated, high levels of multidrug-resistance was observed in the generic E. coli isolates. These findings highlight the importance of microbiological quality surveillance of fresh produce in formal and informal markets, as these products can be a reservoir of multidrug resistant bacteria harboring antibiotic resistance and virulence genes, potentially impacting human health

Prevalence of extended-spectrum β-lactamase producing Enterobacterales in Africa's water-plant-food interface: a meta-analysis (2010–2022)

Background: Multidrug-resistant extended-spectrum β-lactamase (ESBL)-producing Enterobacterales is regarded as a critical health issue, yet, surveillance in the water-plant-food interface remains low, especially in Africa. Objectives: The objective of the study was to elucidate the distribution and prevalence of antimicrobial resistance in clinically significant members of the Enterobacterales order isolated from the water-plant-food interface in Africa. Methods: A literature search was conducted using six online databases according to the PRISMA guidelines. All available published studies involving phenotypic and genotypic characterization of ESBL-producing Enterobacterales from water, fresh produce or soil in Africa were considered eligible. Identification and characterization methods used as well as a network analysis according to the isolation source and publication year were summarized. Analysis of Escherichia coli, Salmonella spp. and Klebsiella pneumoniae included the calculation of the multiple antibiotic resistance (MAR) index according to isolation sources and statistical analysis was performed using RStudio. Results: Overall, 51 studies were included for further investigation. Twelve African countries were represented, with environmental AMR surveillance studies predominantly conducted in South Africa. In 76.47% of the studies, occurrence of antimicrobial resistant bacteria was investigated in irrigation water samples, while 50.98% of the studies included fresh produce samples. Analysis of bacterial phenotypic antimicrobial resistance profiles were reported in 94.12% of the studies, with the disk diffusion method predominantly used. When investigating the MAR indexes of the characterized Escherichia coli, Klebsiella pneumoniae and Salmonella spp., from different sources (water, fresh produce or soil), no significant differences were seen across the countries. The only genetic determinant identified using PCR detection in all the studies was the blaCTX − M resistance gene. Only four studies used whole genome sequence analysis for molecular isolate characterization. Discussion: Globally, AMR surveillance programmes recognize ESBL- and carbapenemase-producing Enterobacterales as vectors of great importance in AMR gene dissemination. However, in low- and middle-income countries, such as those in Africa, challenges to implementing effective and sustainable AMR surveillance programmes remain. This review emphasizes the need for improved surveillance, standardized methods and documentation of resistance gene dissemination across the farm-to-fork continuum in Africa

Internalisation potential of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica subsp. enterica serovar Typhimurium and Staphylococcus aureus in lettuce seedlings and mature plants

The internalisation potential of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhimurium in lettuce was evaluated using seedlings grown in vermiculite in seedling trays as well as hydroponically grown lettuce. Sterile distilled water was spiked with one of the four human pathogenic bacteria (105 CFU/mL) and used to irrigate the plants. The potential for pathogen internalisation was investigated over time using light microscopy, transmission electron microscopy and viable plate counts. Additionally, the identities of the pathogens isolated from internal lettuce plant tissues were confirmed using polymerase chain reaction with pathogen-specific oligonucleotides. Internalisation of each of the human pathogens was evident in both lettuce seedlings and hydroponically grown mature lettuce plants. To our knowledge, this is the first report of S. aureus internalisation in lettuce plants. In addition, the levels of background microflora in the lettuce plants were determined by plate counting and the isolates identified using matrix-assisted laser ionisation–time of flight (MALDI–TOF). Background microflora assessments confirmed the absence of the four pathogens evaluated in this study. A low titre of previously described endophytes and soil inhabitants, i.e., Enterobacter cloacae, Enterococcus faecalis, Lysinibacillus fusiformis, Rhodococcus rhodochrous, Staphylococcus epidermidis and Staphylococcus hominis were identified

Microbiological safety of spinach throughout commercial supply chains in Gauteng Province, South Africa and characterization of isolated multidrug-resistant Escherichia coli

Please read abstract in the article.Water Research Commission; South African Agency for Science and Technology Advancement; Partnerships for Enhanced Engagement in Research United States Agency for International Development; Department of Science and Innovation–National Research Foundation (NRF).http://www.wileyonlinelibrary.com/journal/jamhj2023Plant Production and Soil Scienc

Occurrence, identification, and antimicrobial resistance profiles of extended-spectrum and ampC β-lactamase-producing enterobacteriaceae from fresh vegetables retailed in Gauteng province, South Africa

Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase-producing Enterobacteriaceae are no longer restricted to the health care system, but represent increased risks related to environmental integrity and food safety. Fresh produce has been increasingly reported to constitute a reservoir of multidrug-resistant (MDR) potential human pathogenic Enterobacteriaceae. This study aimed to detect, identify, and characterize the antimicrobial resistance of ESBL/AmpC-producing Enterobacteriaceae isolates from fresh vegetables at point of sale. Vegetable samples (spinach, tomatoes, lettuce, cucumber, and green beans; n = 545) were purchased from retailers in Gauteng, the most densely populated province in South Africa. These included street vendors, trolley vendors, farmers' market stalls, and supermarket chain stores. Selective enrichment, plating onto chromogenic media, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) confirmation of isolate identities showed that 17.4% (95/545) vegetable samples analyzed were contaminated with presumptive ESBL/AmpC-producing Enterobacteriaceae. Dominant species identified included Escherichia coli, Enterobacter cloacae, Enterobacter asburiae, and Klebsiella pneumoniae. Phenotypic antibiotic resistance analysis showed that 96.1% of 77 selected isolates were MDR, while resistance to aminoglycoside (94.8%), chloramphenicol (85.7%), and tetracycline (53.2%) antibiotic classes was most prevalent. Positive phenotypic analysis for ESBL production was shown in 61 (79.2%) of the 77 isolates, and AmpC production in 41.6% of the isolates. PCR and sequencing confirmed the presence of β-lactamase genes in 75.3% isolates from all vegetable types analyzed, mainly in E. coli, Enterobacter spp., and Serratia spp. isolates. CTX-M group 9 (32.8%) was the dominant ESBL type, while EBC (24.1%) was the most prevalent plasmidic type AmpC β-lactamase. Our findings document for the first time the presence of MDR ESBL/AmpC-producing Enterobacteriaceae in raw vegetables sold at selected retailers in Gauteng Province, South Africa

Microbiological safety of spinach throughout commercial supply chains in Gauteng Province, South Africa and characterisation of isolated multidrug resistant Escherichia coli

Aim: To investigate the microbiological quality, potential human foodborne pathogen presence, and to phenotypically (antimicrobial resistance profiles) and genotypically (DNA fingerprinting and diarrheagenic gene presence) characterise Escherichia coli isolated throughout commercial spinach production systems from farm-to-sale. Methods and Results: Samples (n=288) were collected from two commercial supply chains using either river or borehole water for irrigation. Escherichia coli was enumerated throughout the chain where river water was directly used for overhead irrigation at levels between 0.00-3.22 log CFU.g-1. Mean Enterobacteriaceae and coliform counts of spinach ranged between 3.33-6.57 log CFU.g-1 and 3.33-6.64 log CFU.g-1, respectively. Following enrichment, isolation and MALDI-TOF identification, E. coli was isolated from 22.57% (n=65/288) of all samples, Salmonella spp. from 3% (n=9/288) of all samples, specifically river and irrigation water samples on one farm, and no Listeria monocytogenes was detected throughout the study. Of the 80 characterised E. coli isolates, one harboured the stx2 virulence gene, while 43.75% (n=35) were multidrug resistant. This included 26.30% multidrug resistant E. coli isolates from production scenario one, where river water was used for irrigation, and 17.50% from the second production scenario that used borehole water for irrigation. Overall, a greater percentage of resistance phenotypes were from water E. coli isolates (52.50%), than isolates from spinach (37.50%). Escherichia coli isolates from spinach and irrigation water clustered together at high similarity values (>90%) using ERIC-PCR analysis. Conclusions: The results from this study provide valuable background information regarding the presence of multidrug resistant environmental E. coli throughout spinach production from farm, during processing and up to retail. Furthermore, the similarity of MDR E. coli isolates demonstrated transfer from irrigation water to spinach in both scenarios, reiterating that irrigation water for vegetables consumed raw, should comply with standardised microbiological safety guidelines. Significance and Impact of Study: Multidrug resistant E. coli presence throughout spinach production emphasises the necessity of increased surveillance of antimicrobial resistance in fresh produce and the production environment within a One Health paradigm to develop antimicrobial resistance mitigation strategies