Water and food are major environmental transmission routes for Cryptosporidium, but our ability to identify the spectrum of oocyst contributions in current performance-based methods is limited. Determining risks in water and foodstuffs, and the importance of zoonotic transmission, requires the use of molecular methods, which add value to performance-based morphologic methods. Multi-locus approaches increase the accuracy of identification, as many signatures detected in water originate from species/genotypes that are not infectious to humans. Method optimisation is necessary for detecting small numbers of oocysts in environmental samples consistently, and further work is required to (i) optimise IMS recovery efficiency, (ii) quality assure performance-based methods, (iii) maximise DNA extraction and purification, (iv) adopt standardised and validated loci and primers, (v) determine the species and subspecies range in samples containing mixtures, and standardising storage and transport matrices for validating genetic loci, primer sets and DNA sequences.

Escherichia coli O157:H7, a Shiga-producing E. coli is a major pathogenic E. coli strain which since the early 1980s has become a crucial food and water-borne pathogen. Several management strategies can be applied to control the spread of infection; however early diagnosis represents the optimum preventive strategy to minimize the infection. Therefore, it is crucial to detect this pathogen in a fast and efficient manner in order to reduce the morbidity and mortality. Currently used gold standard tests rely on culture and pre-enrichment of E. coli O157:H7 from the contaminated source; they are time consuming and laborious. Molecular methods such as polymerase chain reaction are sensitive; however, they require expensive instrumentation. Therefore, there is a requirement for Accurate, Sensitive, Specific, User friendly, Rapid, Equipment free and Deliverable (ASSURED) detection methods for use in the laboratory and in the field. Emerging technologies such as isothermal amplification methods, biosensors, surface enhanced Raman Spectroscopy, paper-based diagnostics and smartphone-based digital methods are recognized as new approaches in the field of E. coli O157:H7 diagnostics and are discussed in this review. Mobile PCR and CRISPR-Cas diagnostic platforms have been identified as new tools in E. coli O157:H7 POC diagnostics with the potential for implementation by industry. This review describes advances and progress in the field of E. coli O157:H7 diagnosis in the context of food and water industry. The focus is on emerging high throughput point-of-care (POC) E. coli O157:H7 diagnostics and the requirement for the transformation to service routine diagnostics in the food and water industry.

Diarrheagenic E. coli (DEC) has been implicated in foodborne outbreaks worldwide and have been associated with childhood stunting in the absence of diarrhoea. Infection is extraordinarily common, but the routes of transmission have not been determined. Therefore, determining the most prevalent pathotypes in food and environmental sources may help provide better guidance to various stakeholders in ensuring food safety and public health and advancing understanding of the epidemiology of enteric disease. We characterized 205 E. coli strains previously isolated from producer distributor bulk milk (PDBM)(118), irrigation water (48), irrigated lettuce (29) and street vendor coleslaw (10) in South Africa. Enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC) were sought. We used PCR and partial gene sequencing for all 205 strains while 46 out of 205 that showed poor resolution were subsequently characterized using cell adherence (HeLa cells).PCR and partial gene sequencing of aatA and/or aaiC genes confirmed EAEC (2%, 5 out of 205) as the only pathotype. Phylogenetic analysis of sequenced EAEC strains with E. coli strains in GenBank showing ≥80% nucleotide sequence similarity based on possession of aaiC and aatA generated distinct clusters of strains separated predominantly based on their source of isolation (food source or human stool) suggesting a potential role of virulence genes in source tracking. EAEC 24%, 11 out of 46 strains (PDBM = 15%, irrigation water = 7%, irrigated lettuce = 2%) was similarly the predominant pathotype followed by strains showing invasiveness to HeLa cells, 4%, 2 out of 46 (PDBM = 2%, irrigated lettuce = 2%), among stains characterized using cell adherence.Therefore, EAEC may be the leading cause of DEC associated food and water-borne enteric infection in South Africa. Additionally, solely using molecular based methods targeting virulence gene determinants may underestimate prevalence, especially among heterogeneous pathogens such as EAEC.

A polymerase chain reaction (PCR) assay with two nested pairs of primers selected from conserved sequences within a 2.3 kb randomly cloned DNA fragment from the Salmonella typhimurium chromosome was developed. The nested PCR assay correctly identified 128 of a total of 129 Salmonella strains belonging to subspecies I, II, IIIb and IV. One strain of Salm. arizona (ssp. IIIa) tested negative. No PCR products were obtained from any of the 31 non-Salmonella strains examined. The sensitivity of the assay was 2 cfu, as determined by analysis of proteinase K-treated boiled lysates of Salm. typhimurium. The performance of the assay was evaluated for environmental water, sewage and food samples spiked with Salm. typhimurium. Water and sewage samples were filtered and filters were enriched overnight in a non-selective medium. Prior to PCR, the broth cultures were subjected to a rapid and simple preparation procedure consisting of centrifugation, proteinase K treatment and boiling. This assay enabled detection of 10 cfu 100 ml(-1) water with background levels of up to 8700 heterotrophic organisms ml(-1) and 10000 cfu of coliform organisms 100 ml(-1) water. Spiked food samples were analysed with and without overnight enrichment in a non-selective medium using the same assay as above. Nested PCR performed on enriched broths enabled detection of < 10 cfu g(-1) food. Variable results were obtained for food samples examined without prior enrichment and most results were negative. This rapid and simple assay provides a sensitive and specific means of screening drinking water or environmental water samples, as well as food samples, for the presence of Salmonella spp.

This study evaluated the effects of changing the composition of the pre-enrichment medium buffered peptone water (BPW) on the growth of stressed and unstressed Gram-negative foodborne pathogens in a one-broth enrichment strategy. BPW supplemented with an available iron source and sodium pyruvate, along with low levels of 8-hydroxyquinoline and sodium deoxycholate (BPW-S) improved the recovery of desiccated Cronobacter spp. from powdered infant formula. Growth of Salmonella and STEC was comparable in all BPW variants tested for different food matrices. In products with high levels of Gram-negative background flora (e.g. sprouts), the target organisms could not be reliably detected by PCR in any of the BPW variants tested unless the initial level exceeded 103cfu/10g of sprouts.Based on these results we suggest BPW-S for a one-broth enrichment strategy of stressed Gram-negative foodborne pathogens from dry products. However, a one-broth enrichment strategy based on BPW variants tested in this evaluation is not recommended for produce with a high level of Gram-negative background flora due to very high detection limits.

A two-stage study compared the BAX system PCR method with the reference culture method used by the Brazilian Ministry of Agriculture and Food Supply for the detection of Salmonella in food, water, and environmental samples. In stage 1, fish matrix samples (n = 258) were spiked at several levels with Salmonella and a combination of Salmonella and non-Salmonella competitive organisms. Replicates were analyzed by the BAX system PCR method and the reference method with comparable results (sensitivity >or=97.5%, specificity >or=83.3%) from both methods at the limit of detection. In stage 2, a total of 1,988 samples with 70 product types were analyzed with both methods. Five laboratories were involved in this study, and the samples used were from routine analyses. The BAX system PCR method was shown to be comparable to the reference method, with a limit of detection of 1.0 to 2.0 CFU/25 g of sample. Analysis of the results obtained in stage 2 and in the combination of stages 1 and 2 for the BAX system showed the following performance: sensitivity >or=99.0%, specificity >or=97.2%, false-negative rate <or=1.1%, and false-positive rate <or= 2.8%. Therefore, the BAX system appears to be equivalent to the reference method, with >or=97.3% agreement.

Salmonella is one of the most prevalent causing agents of food- and water-borne illnesses, posing an ongoing public health threat. These food-poisoning bacteria contaminate the resources at different stages such as production, aggregation, processing, distribution, as well as marketing. According to the high incidence of salmonellosis, effective strategies for early-stage detection are required at the highest priority. Since traditional culture-dependent methods and polymerase chain reaction are labor-intensive and time-taking, identification of early and accurate detection of Salmonella in food and water samples can prevent significant health economic burden and lessen the costs. The immense potentiality of biosensors in diagnosis, such as simplicity in operation, the ability of multiplex analysis, high sensitivity, and specificity, have driven research in the evolution of nanotechnology, innovating newer biosensors. Carbon nanomaterials enhance the detection sensitivity of biosensors while obtaining low levels of detection limits due to their possibility to immobilize huge amounts of bioreceptor units at insignificant volume. Moreover, conjugation and functionalization of carbon nanomaterials with metallic nanoparticles or organic molecules enables surface functional groups. According to these remarkable properties, carbon nanomaterials are widely exploited in the development of novel biosensors. To be specific, carbon nanomaterials such as carbon nanotubes, graphene and fullerenes function as transducers in the analyte recognition process or surface immobilizers for biomolecules. Herein the potential application of carbon nanomaterials in the development of novel Salmonella biosensors platforms is reviewed comprehensively. In addition, the current problems and critical analyses of the future perspectives of Salmonella biosensors are discussed.

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coil cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as less than or equal to 3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms.