Performance of bacterial and mitochondrial qPCR source tracking methods: A European multi-center study.

With the advent of molecular biology diagnostics, different quantitative PCR assays have been developed for use in Source Tracking (ST), with none of them showing 100% specificity and sensitivity. Most studies have been conducted at a regional level and mainly in fecal slurry rather than in animal wastewater. The use of a single molecular assay has most often proven to fall short in discriminating with precision the sources of fecal contamination. This work is a multicenter European ST study to compare bacterial and mitochondrial molecular assays and was set to evaluate the efficiency of nine previously described qPCR assays targeting human-, cow/ruminant-, pig-, and poultry-associated fecal contamination. The study was conducted in five European countries with seven fecal indicators and nine ST assays being evaluated in a total of 77 samples. Animal fecal slurry samples and human and non-human wastewater samples were analyzed. Fecal indicators measured by culture and qPCR were generally ubiquitous in the samples. The ST qPCR markers performed at high levels in terms of quantitative sensitivity and specificity demonstrating large geographical application. Sensitivity varied between 73% (PLBif) and 100% for the majority of the tested markers. On the other hand, specificity ranged from 53% (CWMit) and 97% (BacR). Animal-associated ST qPCR markers were generally detected in concentrations greater than those found for the respective human-associated qPCR markers, with mean concentration for the Bacteroides qPCR markers varying between 8.74 and 7.22 log10 GC/10 mL for the pig and human markers, respectively. Bacteroides spp. and mitochondrial DNA qPCR markers generally presented higher Spearman's rank coefficient in the pooled fecal samples tested, particularly the human fecal markers with a coefficient of 0.79. The evaluation of the performance of Bacteroides spp., mitochondrial DNA and Bifidobacterium spp. ST qPCR markers support advanced pollution monitoring of impaired aquatic environments, aiming to elaborate strategies for target-oriented water quality management.

Survival of Escherichia coli and Listeria innocua on Lettuce after Irrigation with Contaminated Water in a Temperate Climate.

Microbial disease outbreaks related to fresh produce consumption, including leafy green vegetables, have increased in recent years. Where contamination occurs, pathogen persistence may represent a risk for consumers' health. This study analysed the survival of E. coli and L. innocua on lettuce plants watered with contaminated irrigation water via a single irrigation event and within stored irrigation water. Separate lettuce plants (Lactuca sativa var. capitata) were irrigated with water spiked with Log10 7 cfu/mL of each of the two strains and survival assessed via direct enumeration, enrichment and qPCR. In parallel, individual 20 L water microcosms were spiked with Log10 7 cfu/mL of the individual strains and sampled at similar time points. Both strains were observed to survive on lettuce plants up to 28 days after inoculation. Direct quantification by culture methods showed a Log10 4 decrease in the concentration of E. coli 14 days after inoculation, and a Log10 3 decrease in the concentration of L. innocua 10 days after inoculation. E. coli was detected in water samples up to 7 days after inoculation and L. innocua was detected up to 28 days by direct enumeration. Both strains were recovered from enriched samples up to 28 days after inoculation. These results demonstrate that E. coli and L. innocua strains are able to persist on lettuce after a single contamination event up until the plants reach a harvestable state. Furthermore, the persistence of E. coli and L. innocua in water for up to 28 days after inoculation illustrates the potential for multiple plant contamination events from stored irrigation water, emphasising the importance of ensuring that irrigation water is of a high quality.

Application of plasma activated water for decontamination of alfalfa and mung bean seeds.

Microbial contamination of fresh produce is a major public health concern, with the number of associated disease outbreaks increasing in recent years. The consumption of sprouted beans and seeds is of particular concern, as these foodstuffs are generally consumed raw, and are produced in conditions favourable for the growth of zoonotic pathogens, if present in seeds prior to sprouting or in irrigation water. This work aimed to evaluate the activity of plasma activated water (PAW) as a disinfecting agent for alfalfa (Medicago sativa) and mung bean (Vigna radiata) seeds, during seed soaking. Each seed type was inoculated with Escherichia coli O157, E. coli O104, Listeria monocytogenes or Salmonella Montevideo, and treated with PAW for different times. A combination of PAW and ultrasound treatment was also evaluated. The germination and growth rate of both seeds were assessed after PAW treatments. PAW was demonstrated to have disinfecting ability on sprouted seeds, with reductions of up to Log10 1.67 cfu/g in alfalfa seeds inoculated with E. coli O104, and a reduction of Log10 1.76 cfu/g for mung bean seeds inoculated with E. coli O157 observed. The germination and growth rate of alfalfa and mung bean sprouts were not affected by the PAW treatments. The combination of a PAW treatment and ultrasound resulted in increased antimicrobial activity, with a reduction of Log10 3.48 cfu/g of S. Montevideo in mung bean seeds observed. These results demonstrate the potential for PAW to be used for the inactivation of pathogenic microorganisms which may be present on sprouted seeds and beans, thereby providing greater assurance of produce safety.

Impact of beef extract used for sample concentration on the detection of Escherichia coli DNA in water samples via qPCR.

There is increasing interest in methodologies for the simultaneous concentration and detection of multiple targets in individual samples. The aim of this study was to investigate the potential presence of E. coli DNA in beef extract powder used as part of a procedure to concentrate water samples for the simultaneous detection of bacteria, viruses and protozoa. DNA from E. coli was detected in five out of six beef extract lots tested, demonstrating the limitations of its inclusion when being used in assays that will be used for the detection of E. coli in water samples. Further work is required to clarify if this phenomenon also occurs for other microorganisms of interest in water.

Microbial Contamination of Fresh Produce: What, Where, and How?

Promotion of healthier lifestyles has led to an increase in consumption of fresh produce. Such foodstuffs may expose consumers to increased risk of foodborne disease, as often they are not subjected to processing steps to ensure effective removal or inactivation of pathogenic microorganisms before consumption. Consequently, reports of ready-to-eat fruit and vegetable related disease outbreak occurrences have increased substantially in recent years, and information regarding these events is often not readily available. Identifying the nature and source of microbial contamination of these foodstuffs is critical for developing appropriate mitigation measures to be implemented by food producers. This review aimed to identify the foodstuffs most susceptible to microbial contamination and the microorganisms responsible for disease outbreaks from information available in peer-reviewed scientific publications. A total of 571 outbreaks were identified from 1980 to 2016, accounting for 72,855 infections and 173 deaths. Contaminated leafy green vegetables were responsible for 51.7% of reported outbreaks. Contaminated soft fruits caused 27.8% of infections. Pathogenic strains of Escherichia coli and Salmonella, norovirus, and hepatitis A accounted for the majority of cases. Large outbreaks resulted in particular biases such as the observation that contaminated sprouted plants caused 31.8% of deaths. Where known, contamination mainly occurred via contaminated seeds, water, and contaminated food handlers. There is a critical need for standardized datasets regarding all aspects of disease outbreaks, including how foodstuffs are contaminated with pathogenic microorganisms. Providing food business operators with this knowledge will allow them to implement better strategies to improve safety and quality of fresh produce.