Prevalence and characterisation of antimicrobial resistant Enterobacteriaceae in fresh vegetables from farm to retail in the Gauteng Province of South Africa

Abstract

Contaminated fresh produce has increasingly been implicated in foodborne disease outbreaks with antimicrobial resistance reported as a major emerging health threat. This study aimed to determine the microbiological quality and prevalence of potential pathogenic Escherichia coli and foodborne pathogens (Salmonella spp., and Listeria monocytogenes) in fresh produce retailed formally and informally, as well as two commercial spinach production systems on farm, through processing and up to retail, in Gauteng, the most densely populated province of South Africa (SA). Additionally, the prevalence and molecular characteristics of multidrug resistant extended-spectrum β-lactamase (ESBL) and AmpC-producing Enterobacteriaceae were investigated. A total of 833 samples were analysed. This included 545 spinach, tomatoes, lettuce, cucumber and green beans samples purchased from formal and informal retailers in Gauteng Province. Furthermore, 288 samples were collected from two commercial spinach production scenarios with different irrigation water (river and borehole) sources. From the supply chains, spinach samples were taken at harvest, during processing and from the associated retailers. Irrigation water from each respective farm were taken at the source, storage dams, irrigation pivot point in the field and water used during processing. Lastly, soil at harvest and swab samples from contact surfaces including crates, floors and cutting surfaces throughout the respective production systems were analysed. Coliforms, E. coli and other Enterobacteriaceae enumerated from fresh produce at the point of sale were mostly not significantly different between formal and informal markets, with exceptions noted on occasion. In the spinach production systems, where river water was directly used as overhead irrigation, E. coli was enumerated from spinach at harvest, during processing as well as from the ready-to-eat retail samples. Following selective enrichment and plating onto chromogenic media, potential pathogens were identified using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis. In total, 17,5% (n=146) of the samples harboured E. coli, which included 81 samples from the point-of-sale and 65 samples from the spinach production systems. Except for one stx2 positive E. coli isolate from river irrigation water, no virulence genes (lt, st, bfpA, eagg, eaeA, stx1, stx2, ipaH) were detected in any of the E. coli isolates (n=147) following PCR and sequencing. Salmonella spp. isolates (n=11) were only recovered from river water samples, whilst no Listeria spp. were isolated from any of the samples. Source tracking showed a connection between E. coli in source water and on the irrigated crop using ERIC-PCR analysis within each supply chain. Phenotypic antimicrobial resistance profiles (Kirby-Bauer disk diffusion) revealed multidrug resistance (MDR) in 38,8 % of the generic E. coli isolates (n=147). Overall, 16,4 % (137/833) of the samples were found to be contaminated with ESBL/AmpC-producing Enterobacteriaceae which included 95/545 vegetable samples at the point of sale and 42/288 samples throughout spinach production. Dominant species included E. coli, Enterobacter cloacae, Enterobacter asburiae and K. pneumoniae from vegetables at the point of sale and Serratia fonticola, E. coli and K. pneumoniae from the spinach supply chains. In total, 96.8 % (121/125) of the ESBL/AmpC-producing Enterobacteriaceae isolates were multidrug resistant. With PCR analysis, domination of the CTX-M group 9 ESBL type in isolates from vegetables at the point of sale were seen, while the CTX-M group 1 ESBL type were the most prevalent in Enterobacteriaceae from the spinach supply chains. Selected ESBL/AmpC-producing isolates (n=19) that represented critical priority pathogens listed by the World Health Organisation (WHO) isolated from the spinach supply chains were subjected to whole genome sequencing. In one E. coli and five K. pneumoniae strains, integron In191 were present. Relevant similarites to human pathogens were predicted with PathogenFinder for all 19 strains, with a confidence of 0.635- 0.721 in S. fonticola, 0.852 – 0.931 in E. coli, 0.796 – 0.899 in K. pneumoniae and 0.939 in the S. enterica strain. The presence of MDR ESBL/AmpC-producing E. coli, K. pneumoniae, S. fonticola and S. enterica with confirmed similarities to human pathogens reflect the agricultural production environment link in the emergence and spread of antibiotic resistance genes. The necessity of using clean and safe irrigation water in fresh produce production and the need for standardised microbiological safety parameters for irrigation water and ready-to-eat fresh vegetables was highlighted. For the first time, the presence of multidrug resistant ESBL/AmpC-producing Enterobacteriaceae in formally and informally retailed raw vegetables in Gauteng Province were reported. These results contribute to the global knowledge base regarding the prevalence and characteristics of ESBL/AmpC-producing Enterobacteriaceae in fresh vegetables and the agricultural environment. This will contribute towards data required for future risk analysis, and emphasises the need for mitigation strategies for combatting the spread of multidrug resistant environmental strains.