Genomic Evaluation of Multidrug-Resistant Extended-Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli from Irrigation Water and Fresh Produce in South Africa: A Cross-Sectional Analysis.

Escherichia coli, both commensal and pathogenic, can colonize plants and persist in various environments. It indicates fecal contamination in water and food and serves as a marker of antimicrobial resistance. In this context, 61 extended-spectrum ß-lactamase (ESBL)-producing E. coli from irrigation water and fresh produce from previous studies were characterized using whole genome sequencing (Illumina MiSeq). The Center for Genomic Epidemiology and Galaxy platforms were used to determine antimicrobial resistance genes, virulence genes, plasmid typing, mobile genetic elements, multilocus sequence typing (MLST), and pathogenicity prediction. In total, 19 known MLST groups were detected among the 61 isolates. Phylogroup B1 (ST58) and Phylogroup E (ST9583) were the most common sequence types. The six ST10 (serotype O101:H9) isolates carried the most resistance genes, spanning eight antibiotic classes. Overall, 95.1% of the isolates carried resistance genes from three or more classes. The blaCTX-M-1, blaCTX-M-14, and blaCTX-M-15 ESBL genes were associated with mobile genetic elements, and all of the E. coli isolates showed a >90% predicted probability of being a human pathogen. This study provided novel genomic information on environmental multidrug-resistant ESBL-producing E. coli from fresh produce and irrigation water, highlighting the environment as a reservoir for multidrug-resistant strains and emphasizing the need for ongoing pathogen surveillance within a One Health context.

Microbiological quality of irrigation water on highly diverse fresh produce smallholder farms: elucidating environmental routes of contamination.

AIM: To evaluate the microbiological safety, potential multidrug-resistant bacterial presence and genetic relatedness (DNA fingerprints) of Escherichia coli isolated from the water-soil-plant nexus on highly diverse fresh produce smallholder farms. METHODS AND RESULTS: Irrigation water (n = 44), soil (n = 85), and fresh produce (n = 95) samples from six smallholder farms with different production systems were analysed for hygiene indicator bacterial counts and the presence of shigatoxigenic E. coli and Salmonella spp. using standard microbiological methods. Identities of isolates were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and the genetic relatedness of the E. coli isolates determined using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) analysis. Irrigation water E. coli levels ranged between 0 and 3.45 log MPN/100 ml-1 with five farms having acceptable levels according to the World Health Organization limit (3 log MPN/100 ml-1). Fresh produce samples on four farms (n = 65) harboured E. coli at low levels (<1 log CFU/g-1) except for one sample from kale, spring onion, green pepper, onion, and two tomato samples, which exceeded international acceptable limits (100 CFU/g-1). Only one baby carrot fresh produce sample tested positive for Salmonella spp. Of the 224 samples, E. coli isolates were identified in 40% (n = 90) of all water, soil, and fresh produce types after enrichment. Additionally, the DNA fingerprints of E. coli isolates from the water-soil-plant nexus of each respective farm clustered together at high similarity values (>90%), with all phenotypically characterized as multidrug-resistant. CONCLUSIONS: The clustering of E. coli isolated throughout the water-soil-plant nexus, implicated irrigation water in fresh produce contamination. Highlighting the importance of complying with irrigation water microbiological quality guidelines to limit the spread of potential foodborne pathogens throughout the fresh produce supply chain.

Influence of Soil Phosphate on Rhizobacterial Performance in Affecting Wheat Yield.

As a primary nutrient in agricultural soils, phosphorus plays a crucial but growth-limiting role for plants due to its complex interactions with various soil elements. This often results in excessive phosphorus fertilizer application, posing concerns for the environment. Agri-research has therefore shifted focus to increase fertilizer-use efficiency and minimize environmental impact by leveraging plant growth-promoting rhizobacteria. This study aimed to evaluate the in-field incremental effect of inorganic phosphate concentration (up to 50 kg/ha/P) on the ability of two rhizobacterial isolates, Lysinibacillus sphaericus (T19), Paenibacillus alvei (T29), from the previous Breedt et al. (Ann Appl Biol 171:229-236, 2017) study on maize in enhancing the yield of commercially grown Duzi® cultivar wheat. Results obtained from three seasons of field trials revealed a significant relationship between soil phosphate concentration and the isolates' effectiveness in improving wheat yield. Rhizospheric samples collected at flowering during the third season, specifically to assess phosphatase enzyme activity at the different soil phosphate levels, demonstrated a significant decrease in soil phosphatase activity when the phosphorus rate reached 75% for both isolates. Furthermore, in vitro assessments of inorganic phosphate solubilization by both isolates at five increments of tricalcium phosphate-amended Pikovskaya media found that only isolate T19 was capable of solubilizing tricalcium at concentrations exceeding 3 mg/ml. The current study demonstrates the substantial influence of inorganic phosphate on the performance of individual rhizobacterial isolates, highlighting that this is an essential consideration when optimizing these isolates to increase wheat yield in commercial cultivation.

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.

Fungal microbiome shifts on avocado fruit associated with a combination of postharvest chemical and physical interventions.

AIM OF THE STUDY: The aim was to characterize the baseline microbial population of the avocado carposphere and understand shifts in community structure from the harvest to ready-to-eat stages. METHODS AND RESULTS: The changes in surface or stem-end (SE) fungal microbiomes at the postharvest stage of avocado fruit were studied using next-generation sequencing of the internal transcribed spacer region. Avocado fructoplane and SE pulp fungal richness differed significantly between postharvest stages with a decline following prochloraz dip treatments. Known postharvest decay-causing genera, Colletotrichum, Fusarium, Alternaria, Epicoccum, Penicillium and Neofusicoccum were detected, with Papiliotrema, Meyerozyma and Aureobasidium confirmed as the most dominant potentially beneficial genera. Postharvest interventions such as prochloraz had a negative non-target effect on the presence of Papiliotrema flavescens on the avocado fructoplane. CONCLUSION: Our findings reveal a core community of beneficial and pathogenic taxa in the avocado fructoplane and further highlight the reduction of pathogenic fungi as a consequence of fungicide use. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides important baseline data for further exploration of fungal population shifts in avocado fruit driven by chemical (fungicide) as well as physical (cold storage) interventions.

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.

Occurrence, fate and toxic effects of the industrial endocrine disrupter, nonylphenol, on plants - A review.

Nonylphenol (NP) and its detrimental effects on the environment, humans, wildlife, fish and birds is an increasingly important global research focus. The number of investigations on the toxicity and metabolic fate of NP in plants is however limited. This paper reviews the prevalence and source of NP in plants and the effect it has on its morphological, physiological and ultrastructural status. Fruit and vegetables have been found to contain levels of NP that is twenty-fold exceeding the no observable effect level (NOEL) of freshwater algae. Apart from the potential risk this poses to the health of consumers, it can overburden the plant's natural defence system, leading to growth disorders. Plants exposed to NP show signs of overall growth reduction, changes in organelle structure and oxidative damage. These adverse effects may exacerbate the food security dilemma faced by many countries and impede their progress towards attaining the sustainable development goals.

Impact of Postharvest Storage on the Infection and Colonization of Penicillium digitatum and Penicillium expansum on Nectarine.

Very few studies have investigated the host-pathogen interaction of Penicillium spp. on nectarine. Penicillium digitatum was identified as pathogenic and highly aggressive on nectarine. A strong association was made with host age/ripeness. This points to a new mechanism or life strategy used by P. digitatum to infect and colonize previously thought nonhosts. The aim of this study was to determine the effect of postharvest storage of nectarine on the infection and colonization of P. digitatum and Penicillium expansum at molecular and physical (firmness and pH) levels. The impact of environmental conditions (cold storage) and pathogen pressure (inoculum load) was also investigated. Although disease incidence was much lower, lesions caused by P. digitatum were similar in size to those caused by P. expansum on freshly harvested nectarine. Disease incidence and lesion diameter significantly increased (larger than P. expansum) on longer stored fruit. Cold storage had the largest effect on P. digitatum. Inoculum load had a meaningful effect on both Penicillium spp. Storage significantly affected pH modulation and gene expression. The pathogens not only decreased but also, increased and maintained (similar to initial pH of the host) pH of infected tissue. The polygalacturonase (PG) gene and creA were upregulated by P. digitatum on 7-day postharvest fruit (other genes were unaffected). It partly explains the larger lesions on older or riper fruit. A different expression profile was observed from P. expansum: strong downregulation in PG and slight upregulation in pacC. Very different life strategies were used by the two Penicillium spp. when infecting nectarine. Unlike what is known on citrus, P. digitatum showed an opportunistic lifestyle that takes advantage of specific host and environmental conditions. It is largely still unclear (gene expression) what specifically triggers the increase in disease incidence (infection) and lesion diameter (colonization) of P. digitatum on older or riper fruit. The differences between in vivo and in vitro studies make it difficult to directly correlate results. Additional research is still needed to differentiate and understand the infection and colonization of these pathogens on the same host.

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.

Awakening from the listeriosis crisis: Food safety challenges, practices and governance in the food retail sector in South Africa.

Background: The recent listeriosis outbreak in South Africa brought food safety concerns to the fore in terms of both policy and practice. These concerns encompass both health and nutrition aspects, as well as the economy, because the food system in South Africa contributes significantly to economic growth and food security. However, the food sector is challenged with food safety risks, such as foodborne diseases, food fraud and a general lack of effective enforcement of regulation. The inability of government to effectively regulate the food sector is a contributing factor to increased food safety risks. Focusing on the formal sector, which is subject to regulation, this review provides an overview of the current state of food safety policies and regulations, food safety challenges, and food safety practices in the food system, after the listeriosis crisis of 2017 and 2018. Method: This study used a systematic process to review three sets of data in South Africa: food safety related public policies and regulations, company reports (2013-2018) and media articles (May 2017-May 2018). Food safety policies were selected from a food system policy database created by the research team. The company reports were retrieved from their websites. Factivia was used to search for the media articles. The data were thematically analyzed. The analysis framework was informed by the Food and Agriculture Organization's (FAO) food safety risk analysis. Activities related to food safety risk analysis: risk assessment, risk management, and risk communication were searched for in each material included in the study. Results: Seventy-four documents made up of 13 policies, 47 media articles and 15 company reports were reviewed. Food safety is regulated by three governments departments: Department of Health (DOH), Department of Agriculture, Forestry and Fisheries (DAFF), and Department of Trade and Industry (DTI) through bylaws and regulations. The departments are directly (DAFF) or indirectly (DOH through municipal or metro Environmental Health Professional) involved in food safety enforcement, surveillance, and education. The enforcement of different regulatory processes is often poorly coordinated. Responding to this regulatory environment, food safety activities of the food retail industry include a self-regulatory system reliant on internal and third-party food audits, worker training, external testing, and consumer education. Given this fragmented framework and the lack of interaction, it is clear that the governance of the South African food safety system is not "fit for purpose" in that there is a gap in the effectiveness of government regulation and the self-regulation of the formal sector, and a growing risk from an inability to regulate the large informal sector. Food safety challenges identified in our analysis included disease outbreaks, concerns over mislabeling, and lack of regulation for food handling and distribution. Conclusion: The findings suggest that there should be a combination of responsibility from all levels of stake-holders in the food retail sector in order to improve food safety and prevent food safety breaches. In addition, strong governance of the food safety system is required to enable effective legislation and enforcement.

Relative proportions of E. coli and Enterococcus spp. may be a good indicator of potential health risks associated with the use of roof harvested rainwater stored in tanks.

A total of 285 water samples were collected from 71 roof harvested rainwater tanks from four villages in different provinces over a two-year (2013-2014) period during the early (October to December) and late (January to March) rainy season. Water quality was evaluated based on Escherichia coli, faecal coliforms and Enterococcus spp. prevalence using the IDEXX Quanti-Tray quantification system. Real-Time PCR was used to analyse a subset of 168 samples for the presence of Shigella spp., Salmonella spp. and E. coli virulence genes (stx1, stx2 and eaeA). Escherichia coli were detected in 44.1% of the samples, Enterococcus spp. in 57.9% and faecal coliforms in 95.7%. The most prevalent E. coli concentrations in harvested rainwater were observed in 29.1% of samples and 22.5% for Enterococcus spp. and, were within 1-10 cfu/100 ml and 10-100 cfu/100 ml, respectively, whereas those for faecal coliforms (36.6%) were within 100-1000 cfu/100 ml. On average 16.8% of the samples had neither E. coli nor Enterococcus spp. detected, while 33.9% had only Enterococcus spp. and 23.7% had only E. coli. E. coli and Enterococcus spp. were detected together in 25.5% of the samples. Evaluation of samples for potential pathogenic bacteria showed all tested samples to be negative for the Shigella spp. ipaH gene, while five tested positive for Salmonella ipaB gene. None of the samples tested positive for the stx1 and stx2 genes, and only two tested positive for the eaeA gene. These findings are potentially useful in the development of a simplified risk assessment strategy based on the concentrations of indicator bacteria.

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.

Microbial quality and suitability of roof-harvested rainwater in rural villages for crop irrigation and domestic use.

The study aimed at assessing the microbiological quality and suitability of roof-harvested rainwater (RHRW) for crop irrigation and domestic use. In total, 80 rainwater tanks (246 samples) across three rural villages (Ga-Molepane, Jericho and Luthngele) were visited. Culture-based techniques were used to isolate bacterial microbes and identities were confirmed using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF-MS). Uncultured fungal populations were also identified using pyrosequencing. Salmonella spp. (3%), Listeria monocytogenes (22%), total coliforms (57.7%), Escherichia coli (30.5%), Enterococcus spp. (48.8%), Pseudomonas spp. (21.5%) were detected in RHRW samples after rainfall. Fungal sequences belonging to species known to cause fever, coughing and shortness of breath in humans (Cryptococcus spp.) were identified. This study indicates that RHRW quality can be affected by external factors such as faecal material and debris on rooftops. The use of untreated RHRW could pose a potential health risk if used for irrigation of crops or domestic use, especially in the case of a relative high population of immunocompromised individuals. This study does not dispute the fact that RHRW is an alternative irrigation water source but it recommends treatment before use for domestic purposes or for watering crops.

Antimicrobial Resistance Profiles of Salmonella spp. from Agricultural Environments in Fruit Production Systems.

Foodborne disease outbreaks involving fresh produce have increased in recent years. The risk of infection from contaminated food is worsened by the increased prevalence of antibiotic-resistant strains. This study evaluated the prevalence of antibiotic resistance in Salmonella isolates (n = 263) from agricultural production systems through to the final packed product. Salmonella isolates were preliminarily identified by matrix-assisted laser desorption ionization-time-of-flight mass spectroscopy (MALDI-TOF MS) and API 20E and identities confirmed by invA gene polymerase chain reaction. Antimicrobial susceptibility was performed with 15 antimicrobial agents using the Kirby-Bauer disk diffusion test. Of the 263 Salmonella isolates assessed, 59.3% were resistant to one or more antimicrobials. The most frequently detected resistance was against chloramphenicol and kanamycin (46.7%), trimethoprim-sulfamethoxazole (28%), and streptomycin (14%), and the less frequently detected resistance was toward ampicillin (1.14%), amikacin (0.76%), and amoxicillin-clavulanic acid (0.38%). Multiple antimicrobial resistance (MAR) (resistance to ≥3 antibiotics) was found in 48.7% (76/156) isolates. The most common MAR phenotype was to chloramphenicol and trimethoprim/sulfamethoxazole-kanamycin (43.6%). Resistance to chloramphenicol, kanamycin, or trimethoprim/sulfamethoxazole was only observed in MAR phenotypes. All isolates were susceptible to ceftiofur, cefoxitin, ceftriaxone, ciprofloxacin, nalidixic acid, gentamicin, and tetracycline. This study confirms the importance of fresh produce production environments as potential reservoirs and fresh produce as carriers of antibiotic-resistant Salmonella spp. with significant clinical importance. Further studies to evaluate the actual level of health risk from these pathogens should include characterization of the antibiotic resistance determinant genes among the isolates.

Pathogenicity and Host Susceptibility of Penicillium spp. on Citrus.

Citrus fruit are exposed to numerous postharvest pathogens throughout the fresh produce supply chain. Well-known postharvest citrus fruit pathogens are Penicillium digitatum and P. italicum. Lesser-known pathogens include P. crustosum and P. expansum. This study examined pathogenicity and aggressiveness of Penicillium spp. present in fresh fruit supply chains on various Citrus spp. and cultivars. The impact of different inoculation methods and storage conditions on decay were also assessed. P. digitatum and P. italicum were the most aggressive Penicillium spp. on citrus but aggressiveness varied significantly over the evaluated citrus range. Decay and tissue-response lesions caused by P. crustosum were observed on 'Nules Clementine', 'Nova', 'Owari Satsuma', 'Delta Valencia', 'Cambria Navel', 'Eureka' seeded, and 'Star Ruby' for the first time. Likewise, these lesions caused by P. expansum were noted on Nules Clementine, Owari Satsuma, Delta Valencia, 'Midknight Valencia', and Eureka seeded for the first time. Tissue-response lesions affect fruit quality and some Penicillium spp. sporulated from the lesions, causing the inoculated species to complete their life cycle. New citrus-Penicillium spp. interactions were observed and the importance of monitoring inoculum loads of pathogens and nonhost pathogens were highlighted.

Pyrosequencing analysis of roof-harvested rainwater and river water used for domestic purposes in Luthengele village in the Eastern Cape Province of South Africa.

Pyrosequencing targeting the V1-V3 hypervariable of the 16S rDNA was used to investigate the bacterial diversity in river and roof-harvested rainwater (RHRW) used for potable purposes by rural households in Luthengele village in the Eastern Cape Province of South Africa. The phylum Proteobacteria dominated the data set (80.5 % of all reads), while 4.2 % of the reads could not be classified to any of the known phyla at a probability of 0.8 or higher (unclassified bacteria). At class level, the classes; Betaproteobacteria (50.4 % of all reads), Alphaproteobacteria (16.2 %), Verrucomicrobiae (6.6 %), Planctomycetacia (5.7 %), and Sphingobacteria (3 %) dominated the data set in all the samples. Although the class Verrucomicrobiae constituted 6.6 % of all sequences, 88.6 % of the sequences were from the river sample where the class represented 43.7 % of the observed sequences in the sample. The bacteria community structure clearly showed significant similarities between RHRW and differences with the river water control sample, suggesting different levels of contamination and environmental factors affecting the various water sources. Moreover, signatures of potential pathogens including Legionella, Acinetobacter, Pseudomonas, Clostridia, Chromobacterium, Yersinia, and Serratia were detected, and the proportions of Legionella were relatively higher suggesting a potential health risk to households using RHRW. This work provides guidance for prioritizing subsequent culturable and quantitative analysis to ensure that potentially significant pathogens are not left out of risk estimations.

Antibiotic resistance in Escherichia coli isolates from roof-harvested rainwater tanks and urban pigeon faeces as the likely source of contamination.

The objective of this study was to investigate the risks associated with the use of roof-harvested rainwater (RHRW) and the implication of pigeons as the most likely source of contamination by testing for antibiotic resistance profiles of Escherichia coli. A total of 239 E. coli were isolated from thirty fresh pigeon faecal samples (130 isolates), 11 RHRW tanks from three sites in Pretoria (78) and two in Johannesburg (31). E. coli isolates were tested against a panel of 12 antibiotics which included ampicillin, amoxicillin, amikacin, cefoxitin, ceftriaxone, chloramphenicol, ciprofloxacin, cotrimoxazole, enrofloxacin, gentamicin, nalidixic acid and tetracycline. In all samples, resistance to ampicillin (22.7.9%), gentamicin (23.6%), amikacin (24%), tetracycline (17.4) and amoxicillin (16.9%) were the most frequently encountered form of resistance. However, a relatively higher proportion of isolates from pigeon faeces (67.3%) were antibiotic resistant than those from RHRW (53.3%). The highest number of phenotypes was observed for single antibiotics, and no single antibiotic resistance was observed for chloramphenicol, ceftriaxone, gentamicin, cefoxitin, cotrimoxazole, although they were detected in multiple antibiotic resistance (MAR) phenotypes. The highest multiple antibiotic resistance (MAR) phenotypes were observed for a combination of four antibiotics, on isolates from JHB (18.8%), pigeon faeces (15.2%) and Pretoria (5.1%). The most abundant resistance phenotype to four antibiotics, Ak-Gm-Cip-T was dominated by isolates from pigeon faeces (6.8%) with Pretoria and Johannesburg isolates having low proportions of 1.3 and 3.1%, respectively. Future studies should target isolates from various environmental settings in which rainwater harvesting is practiced and the characterisation of the antibiotic resistance determinant genes among the isolates.

Pathogenic Penicillium spp. on Apple and Pear.

Numerous Penicillium spp. have been associated with postharvest fruit spoilage. This study investigates pathogenicity and aggressiveness of selected Penicillium spp. previously isolated from South African and European Union fruit export chains. Penicillium expansum was the most aggressive and P. crustosum the second most aggressive on all apple cultivars ('Royal Gala', 'Granny Smith', 'Golden Delicious', 'Topred', and 'Cripps Pink') and two pear cultivars ('Packham's Triumph' and 'Forelle') tested. P. digitatum was the most aggressive on 'Beurre Bosc', 'Beurre Hardy', and 'Sempre' ('Rosemarie') pear cultivars and the third most aggressive on Granny Smith and Cripps Pink apple cultivars. To our knowledge, this is the first report where P. digitatum has been described as aggressive on certain pome fruit cultivars. These pear cultivars are also the most commonly associated with decay on the export markets, resulting in considerable end-market losses. P. brevicompactum was detected as pathogenic on pear but was not further evaluated in the study. P. solitum covered a broader cultivar range, expressed higher disease incidence, and was more aggressive (larger lesions) on pear cultivars than on apple cultivars. This study provides new information on host specificity and the importance of pathogenic Penicillium spp. isolated from various environments in the shipping and marketing channels.

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.

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.