Serotype Distribution, Antimicrobial Resistance, Virulence Genes, and Genetic Diversity of Salmonella spp. Isolated from small-scale Leafy Green Vegetable Supply Chains in South Africa.

Salmonella have been implicated in foodborne disease outbreaks globally and is a pressing concern in the South African small-scale sector due to inadequate hygiene standards and limited regulatory oversight, leading to a higher risk of foodborne diseases. By investigating irrigation water and leafy green vegetables produced by small-scale growers and sold through unregulated supply chains, this study was able to determine the presence, serotype distribution, virulence gene profiles, antibiotic resistance, and genetic diversity of Salmonella isolated from these sources. From 426 samples, 21 Salmonella-positive samples were identified, providing 53 Salmonella isolates. Of these, six different Salmonella serotypes and sequence types (STs) were identified, including Salmonella II 42:r: ST1208 (33.96%; n = 18), Salmonella Enteritidis: ST11 (22.64%; n = 12), Salmonella II 42:z29: ST4395 (16.98%; n = 9), Salmonella Havana: ST1524 (15.09%; n = 8), Salmonella Typhimurium: ST19 (9.43%; n = 5), and Salmonella IIIb 47:i:z: ST7890 (1.89%; n = 1). A total of 92.45% of the isolates were found to be multidrug-resistant, showing high rates of resistance to aztreonam (88.68%; n = 47), ceftazidime (86.79%; n = 46), nalidixic acid (77.36%; n = 41), cefotaxime (75.47%; n = 40), cefepime (71.70%; n = 38), and streptomycin (69.81%; n = 37). All isolates possessed the aac(6')-Iaa antimicrobial resistance gene, with a range of between 9 and 256 virulence genes. Eleven cluster patterns were observed from Enterobacterial Repetitive Intergenic Consensus sequence analyses, demonstrating high diversity among the Salmonella spp., with water and fresh produce isolates clustering, suggesting water as a potential contamination source. Plasmid replicon types were identified in 41.51% (n = 22) of the isolates, including Col(pHAD28) in Salmonella Havana (5.66%; n = 3), Col156 in Salmonella II 42:z29:- (1.89%; n = 1) and both IncFIB(S) and IncFII(S) in Salmonella Enteritidis (22.64; n = 12), Salmonella Typhimurium (9.43%; n = 5), and Salmonella Havana (1.89%; n = 1). This study highlights the presence of multidrug-resistant and multivirulent Salmonella spp. in the small-scale leafy green vegetable supply chains, underscoring the need for the development of a "fit-for-purpose" food safety management system within this system.

Microbiological quality assessment of fresh produce: Potential health risk to children and urgent need for improved food safety in school feeding schemes.

About 388 million school-going children worldwide benefit from school feeding schemes, which make use of fresh produce to prepare meals. Fresh produce including leafy greens and other vegetables were served at 37% and 31% of school feeding programs, respectively, in Africa. This study aimed at assessing the microbiological quality of fresh produce grown onsite or supplied to South African schools that are part of the national school feeding programs that benefit over 9 million school-going children. Coliforms, Escherichia coli, Enterobacteriaceae, and Staphylococcus aureus were enumerated from fresh produce (n = 321) samples. The occurrence of E. coli, Listeria monocytogenes, Salmonella spp., and extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae was determined. Presumptive pathogens were tested for antimicrobial resistance. E. coli was further tested for diarrheagenic virulence genes. Enterobacteriaceae on 62.5% of fresh produce samples (200/321) exceeded previous microbiological guidelines for ready-to-eat food, while 86% (276/321 samples) and 31.6% (101/321 samples) exceeded coliform and E. coli criteria, respectively. A total of 76 Enterobacteriaceae were isolated from fresh produce including E. coli (n = 43), Enterobacter spp. (n = 15), and Klebsiella spp. (n = 18). Extended-spectrum ß-lactamase production was confirmed in 11 E. coli, 13 Enterobacter spp., and 17 Klebsiella spp. isolates. No diarrheagenic virulence genes were detected in E. coli isolates. However, multidrug resistance (MDR) was found in 60.5% (26/43) of the E. coli isolates, while all (100%; n = 41) of the confirmed ESBL and AmpC Enterobacteriaceae showed MDR. Our study indicates the reality of the potential health risk that contaminated fresh produce may pose to school-going children, especially with the growing food safety challenges and antimicrobial resistance crisis globally. This also shows that improved food safety approaches to prevent foodborne illness and the spread of foodborne pathogens through the food served by school feeding schemes are necessary.

Multidrug resistant Escherichia coli from fresh produce sold by street vendors in South African informal settlements.

The aim of this study was to assess the prevalence of commensal and pathogenic Escherichia coli on informally sold fresh produce in South Africa, who harbour and express antimicrobial resistance genes and therefore pose indirect risks to public health. The majority (85.71%) of E. coli isolates from spinach, apples, carrots, cabbage and tomatoes, were multidrug resistant (MDR). Resistance to Aminoglycoside (94.81%), Cephalosporin (93.51%), Penicillin (93.51%) and Chloramphenicol (87.01%) antibiotic classes were most prevalent. Antibiotic resistance genes detected included blaTEM (89.29%), tetA (82.14%), tetB (53.57%), tetL (46.43%), sulI (41.07%), sulII (51.79%), aadA1a (58.93%) and strAB (51.79%). A single isolate was found to harbour eae virulence factor. Moreover, E. coli isolates were grouped into the intra-intestinal infectious phylogenetic group E (28.57%), the rare group C (26.79%), the generalist group B1 (21.43%) and the human commensal group A (16.07%). Presence of MDR E. coli represents a transmission route and significant human health risk.

Characterization of Multidrug-Resistant Escherichia coli Isolated from Two Commercial Lettuce and Spinach Supply Chains.

ABSTRACT: Leafy green vegetables have increasingly been reported as a reservoir of multidrug-resistant pathogenic Enterobacteriaceae, with Shiga toxin-producing Escherichia coli frequently implicated in disease outbreaks worldwide. This study examined the presence and characteristics of antibiotic resistance, diarrheagenic virulence genes, and phylogenetic groupings of E. coli isolates (n = 51) from commercially produced lettuce and spinach from farms, through processing, and at the point of sale. Multidrug resistance was observed in 33 (64.7%) of the 51 E. coli isolates, with 35.7% (10 of 28) being generic and 100% (23 of 23) being extended-spectrum ß-lactamase/AmpC producing. Resistance of E. coli isolates was observed against neomycin (51 of 51, 100%), ampicillin (36 of 51, 70.6%), amoxicillin (35 of 51, 68.6%), tetracycline (23 of 51, 45%), trimethoprim-sulfamethoxazole (22 of 51, 43%), chloramphenicol (13 of 51, 25.5%), Augmentin (6 of 51, 11.8%), and gentamicin (4 of 51, 7.8%), with 100% (51 of 51) susceptibility to imipenem. Virulence gene eae was detected in two E. coli isolates from irrigation water sources only, whereas none of the other virulence genes for which we tested were detected. Most of the E. coli strains belonged to phylogenetic group B2 (25.5%; n = 13), B1 (19.6%; n = 10), and A (17.6%; n = 9), with D (5.9%; n = 3) less distributed. Although diarrheagenic E. coli was not detected, antibiotic resistance in E. coli prevalent in the supply chain was evident. In addition, a clear link between E. coli isolates from irrigation water sources and leafy green vegetables through DNA fingerprinting was established, indicating the potential transfer of E. coli from irrigation water to minimally processed leafy green vegetables.

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

AIM: To investigate the microbiological quality, potential foodborne pathogen presence, and to phenotypically (antimicrobial resistance [AMR] profiles) and genotypically (DNA fingerprints and diarrhoeagenic genes) characterize Escherichia coli isolated throughout 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 irrigation water. E. coli was enumerated throughout the chain where river water was directly used for overhead irrigation at levels between 0.00 and 3.22 log colony forming unit (CFU) g-1 . Following enrichment, isolation and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identification, E. coli was isolated from 22.57% (n = 65/288) of all samples. Salmonella spp. were isolated from 3% (n = 9/288) of river and irrigation water samples on one farm, and no Listeria monocytogenes was detected throughout the study. Of the 80 characterized E. coli isolates, one harboured the stx2 virulence gene, while 43.75% (n = 35) were multidrug resistant. Overall, 26.30% of the multidrug-resistant E. coli isolates were from production scenario one that used river irrigation water, and 17.50% from the second production scenario that used borehole irrigation water. A greater percentage of resistance phenotypes were from water E. coli isolates (52.50%), than isolates from spinach (37.50%). E. coli isolates from spinach and irrigation water clustered together at high similarity values (>90%) using enterobacterial repetitive intergenic consensus-polymerase chan reaction analysis. CONCLUSIONS: This study reported the presence of multidrug-resistant environmental E. coli throughout spinach production from farm, during processing and up to retail. Furthermore, the similarity of multi-drug resistant E. coli isolates suggests transfer from irrigation water to spinach in both scenarios, reiterating that irrigation water for vegetables consumed raw, should comply with standardized microbiological safety guidelines. SIGNIFICANCE AND IMPACT OF STUDY: Multidrug-resistant E. coli presence throughout spinach production emphasizes the necessity of increased surveillance of AMR in fresh produce and the production environment within a One Health paradigm to develop AMR mitigation strategies.

Whole Genome Sequencing of Extended-Spectrum- and AmpC- ß-Lactamase-Positive Enterobacterales Isolated From Spinach Production in Gauteng Province, South Africa.

The increasing occurrence of multidrug-resistant (MDR) extended-spectrum ß-lactamase- (ESBL) and/or AmpC ß-lactamase- (AmpC) producing Enterobacterales in irrigation water and associated irrigated fresh produce represents risks related to the environment, food safety, and public health. In South Africa, information about the presence of ESBL/AmpC-producing Enterobacterales from non-clinical sources is limited, particularly in the water-plant-food interface. This study aimed to characterize 19 selected MDR ESBL/AmpC-producing Escherichia coli (n=3), Klebsiella pneumoniae (n=5), Serratia fonticola (n=10), and Salmonella enterica (n=1) isolates from spinach and associated irrigation water samples from two commercial spinach production systems within South Africa, using whole genome sequencing (WGS). Antibiotic resistance genes potentially encoding resistance to eight different classes were present, with bla CTX-M-15 being the dominant ESBL encoding gene and bla ACT-types being the dominant AmpC encoding gene detected. A greater number of resistance genes across more antibiotic classes were seen in all the K. pneumoniae strains, compared to the other genera tested. From one farm, bla CTX-M-15-positive K. pneumoniae strains of the same sequence type 985 (ST 985) were present in spinach at harvest and retail samples after processing, suggesting successful persistence of these MDR strains. In addition, ESBL-producing K. pneumoniae ST15, an emerging high-risk clone causing nosocomical outbreaks worldwide, was isolated from irrigation water. Known resistance plasmid replicon types of Enterobacterales including IncFIB, IncFIA, IncFII, IncB/O, and IncHI1B were observed in all strains following analysis with PlasmidFinder. However, bla CTX-M-15 was the only ß-lactamase resistance gene associated with plasmids (IncFII and IncFIB) in K. pneumoniae (n=4) strains. In one E. coli and five K. pneumoniae strains, integron In191 was observed. Relevant similarities 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 similarities to human pathogens in the agricultural production systems reflects environmental and food contamination mediated by anthropogenic activities, contributing to the spread of antibiotic resistance genes.

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.

Occurrence, Phenotypic and Molecular Characterization of Extended-Spectrum- and AmpC- ß-Lactamase Producing Enterobacteriaceae Isolated From Selected Commercial Spinach Supply Chains in South Africa.

The increasing occurrence of multidrug-resistant (MDR) extended-spectrum ß-lactamase- (ESBL) and/or AmpC ß-lactamase-producing Enterobacteriaceae in health care systems, the environment and fresh produce is a serious concern globally. Production practices, processing and subsequent consumption of contaminated raw fruit and vegetables represent a possible human transmission route. The purpose of this study was to determine the presence of ESBL/AmpC-producing Enterobacteriaceae in complete spinach supply chains and to characterize the isolated strains phenotypically (antimicrobial resistance profiles) and genotypically (ESBL/AmpC genetic determinants, detection of class 1, 2, and 3 integrons). Water, soil, fresh produce, and contact surface samples (n = 288) from two commercial spinach production systems were screened for ESBL/AmpC-producing Enterobacteriaceae. In total, 14.58% (42/288) of the samples were found to be contaminated after selective enrichment, plating onto chromogenic media and matrix-assisted laser desorption ionization time-of-flight mass spectrometry identity confirmation of presumptive ESBL/AmpC isolates. This included 15.28% (11/72) water and 12.12% (16/132) harvested- and processed spinach, while 25% (15/60) retail spinach samples were found to be contaminated with an increase in isolate abundance and diversity in both scenarios. Dominant species identified included Serratia fonticola (45.86%), Escherichia coli (20.83%), and Klebsiella pneumoniae (18.75%). In total, 48 (81.36%) isolates were phenotypically confirmed as ESBL/AmpC-producing Enterobacteriaceae of which 98% showed a MDR phenotype. Genotypic characterization (PCR of ESBL/AmpC resistance genes and integrons) further revealed the domination of the CTX-M Group 1 ESBL type, followed by TEM and SHV; whilst the CIT-type was the only plasmid-mediated AmpC genetic determinant detected. Integrons were detected in 79.17% (n = 38) of the confirmed ESBL/AmpC-producing isolates, of which we highlight the high prevalence of class 3 integrons, detected in 72.92% (n = 35) of the isolates, mostly in S. fonticola. Class 2 integrons were not detected in this study. This is the first report on the prevalence of ESBL/AmpC-producing Enterobacteriaceae isolated throughout commercial spinach production systems harboring class 1 and/or class 3 integrons in Gauteng Province, South Africa. The results add to the global knowledge base regarding the prevalence and characteristics of ESBL/AmpC-producing Enterobacteriaceae in fresh vegetables and the agricultural environment required for future risk analysis.

Occurrence, Identification, and Antimicrobial Resistance Profiles of Extended-Spectrum and AmpC ß-Lactamase-Producing Enterobacteriaceae from Fresh Vegetables Retailed in Gauteng Province, South Africa.

Extended-spectrum ß-lactamase (ESBL) and AmpC ß-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 ß-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 ß-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.

Viable bacterial population and persistence of foodborne pathogens on the pear carpoplane.

BACKGROUND: Knowledge on the culturable bacteria and foodborne pathogen presence on pears is important for understanding the impact of postharvest practices on food safety assurance. Pear fruit bacteria were investigated from the point of harvest, following chlorine drenching and after controlled atmosphere (CA) storage to assess the impact on natural bacterial populations and potential foodborne pathogens. RESULTS: Salmonella spp. and Listeria monocytogenes were detected on freshly harvested fruit in season one. During season one, chemical drenching and CA storage did not have a significant effect on the bacterial load of orchard pears, except for two farms where the populations were lower 'after CA storage'. During season two, bacterial populations of orchard pears from three of the four farms increased significantly following drenching; however, the bacterial load decreased 'after CA storage'. Bacteria isolated following enumeration included Enterobacteriaceae, Microbacteriaceae, Pseudomonadaceae and Bacillaceae, with richness decreasing 'after drench' and 'after CA storage'. CONCLUSION: Salmonella spp. and L. monocytogenes were not detected after postharvest practices. Postharvest practices resulted in decreased bacterial species richness. Understanding how postharvest practices have an impact on the viable bacterial populations of pear fruit will contribute to the development of crop-specific management systems for food safety assurance. © 2016 Society of Chemical Industry.

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.

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 (10(5) 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.

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.