Agricultural and management practices and bacterial contamination in greenhouse versus open field lettuce production.

The aim of this study was to gain insight into potential differences in risk factors for microbial contamination in greenhouse versus open field lettuce production. Information was collected on sources, testing, and monitoring and if applicable, treatment of irrigation and harvest rinsing water. These data were combined with results of analysis on the levels of Escherichia coli as a fecal indicator organism and the presence of enteric bacterial pathogens on both lettuce crops and environmental samples. Enterohemorragic Escherichia coli (EHEC) PCR signals (vt1 or vt2 positive and eae positive), Campylobacter spp., and Salmonella spp. isolates were more often obtained from irrigation water sampled from open field farms (21/45, 46.7%) versus from greenhouse production (9/75, 12.0%). The open field production was shown to be more prone to fecal contamination as the number of lettuce samples and irrigation water with elevated E. coli was significantly higher. Farmers comply with generic guidelines on good agricultural practices available at the national level, but monitoring of microbial quality, and if applicable appropriateness of water treatment, or water used for irrigation or at harvest is restricted. These results indicate the need for further elaboration of specific guidelines and control measures for leafy greens with regard to microbial hazards.

Relationships among hygiene indicators and enteric pathogens in irrigation water, soil and lettuce and the impact of climatic conditions on contamination in the lettuce primary production.

Eight Belgian lettuce farms located in the West Flanders were sampled to establish the relationships between levels of indicator bacteria, detection of enteric zoonotic pathogens and the temperature and precipitation during primary production. Pathogenic bacteria (PCR EHEC positives, Salmonella spp. or Campylobacter spp.) and indicator bacteria (total psychrotrophic aerobic plate count (TPAC), total coliforms, Escherichia coli, enterococci) were determined over a period of one and a half year from seedling leaves, peat-soil of the seedling, lettuce crops, field soil and irrigation water. Neither Salmonella isolates nor PCR EHEC signals were detected from lettuce although one out of 92 field soil samples contained Salmonella spp. and five soil samples provided PCR positives for EHEC virulence factors (vt1 or vt2 and eae gene). A low prevalence of Campylobacter (8/88) was noted in lettuce. It was shown that irrigation water is a major risk factor with regard to the bacterial contamination of the fresh produce as the water samples showed on a regular basis E. coli presence (59.2% of samples≥1CFU/100ml) and occasionally detection of pathogens (25%, n=30/120), in particular Campylobacter spp. The highest correlations between indicator bacteria, pathogens, temperature and the amount of precipitation were observed for the water samples in contrast to the soil or lettuce samples where no correlations were observed. The high correlations between E. coli, total coliforms and enterococci in the water implicated redundancy between analyses. Presence of elevated levels of E. coli increased the probability for the presence of pathogens (Campylobacter spp., EHEC and Salmonella spp.), but had a low to moderate predictive value on the actual presence of pathogens. The presence of pathogens and indicator bacteria in the water samples showed a seasonal effect as they tend to be more present during the months with higher temperature.

Moderate prevalence of antimicrobial resistance in Escherichia coli isolates from lettuce, irrigation water, and soil.

Fresh produce is known to carry nonpathogenic epiphytic microorganisms. During agricultural production and harvesting, leafy greens can become contaminated with antibiotic-resistant pathogens or commensals from animal and human sources. As lettuce does not undergo any inactivation or preservation treatment during processing, consumers may be exposed directly to all of the (resistant) bacteria present. In this study, we investigated whether lettuce or its production environment (irrigation water, soil) is able to act as a vector or reservoir of antimicrobial-resistant Escherichia coli. Over a 1-year period, eight lettuce farms were visited multiple times and 738 samples, including lettuce seedlings (leaves and soil), soil, irrigation water, and lettuce leaves were collected. From these samples, 473 isolates of Escherichia coli were obtained and tested for resistance to 14 antimicrobials. Fifty-four isolates (11.4%) were resistant to one or more antimicrobials. The highest resistance rate was observed for ampicillin (7%), followed by cephalothin, amoxicillin-clavulanic acid, tetracycline, trimethoprim, and streptomycin, with resistance rates between 4.4 and 3.6%. No resistance to amikacin, ciprofloxacin, gentamicin, or kanamycin was observed. One isolate was resistant to cefotaxime. Among the multiresistant isolates (n = 37), ampicillin and cephalothin showed the highest resistance rates, at 76 and 52%, respectively. E. coli isolates from lettuce showed higher resistance rates than E. coli isolates obtained from soil or irrigation water samples. When the presence of resistance in E. coli isolates from lettuce production sites and their resistance patterns were compared with the profiles of animal-derived E. coli strains, they were found to be the most comparable with what is found in the cattle reservoir. This may suggest that cattle are a potential reservoir of antimicrobial-resistant E. coli strains in plant primary production.

The biosynthetic gene cluster for sophorolipids: a biotechnological interesting biosurfactant produced by Starmerella bombicola.

Sophorolipids are promising biological derived surfactants or detergents which find application in household cleaning, personal care and cosmetics. They are produced by specific yeast species and among those, Starmerella bombicola (former Candida bombicola) is the most widely used and studied one. Despite the commercial interest in sophorolipids, the biosynthetic pathway of these secondary metabolites remained hitherto partially unsolved. In this manuscript we present the sophorolipid gene cluster consisting of five genes directly involved in sophorolipid synthesis: a cytochrome P450 monooxygenase, two glucosyltransferases, an acetyltransferase and a transporter. It was demonstrated that disabling the first step of the pathway - cytochrome P450 monooxygenase mediated terminal or subterminal hydroxylation of a common fatty acid - results in complete abolishment of sophorolipid production. This phenotype could be complemented by supplying the yeast with hydroxylated fatty acids. On the other hand, knocking out the transporter gene yields mutants still able to secrete sophorolipids, though only at levels of 10% as compared with the wild type, suggesting alternative routes for secretion. Finally, it was proved that hampering sophorolipid production does not affect cell growth or cell viability in laboratory conditions, as can be expected for secondary metabolites.

Physicochemical quality and chemical safety of chlorine as a reconditioning agent and wash water disinfectant for fresh-cut lettuce washing.

Chlorine was assessed as a reconditioning agent and wash water disinfectant in the fresh-cut produce industry. Artificial fresh-cut lettuce wash water, made from butterhead lettuce, was used for the experiments. In the reconditioning experiments, chlorine was added to artificial wash water inoculated with Escherichia coli O157 (6 log CFU/ml). Regression models were constructed based on the inactivation data and validated in actual wash water from leafy vegetable processing companies. The model that incorporated chlorine dose and chemical oxygen demand (COD) of the wash water accurately predicted inactivation. Listeria monocytogenes was more resistant to chlorine reconditioning in artificial wash water than Salmonella spp. and Escherichia coli O157. During the washing process with inoculated lettuce (4 log CFU/g), in the absence of chlorine, there was a rapid microbial buildup in the water that accumulated to 5.4 ± 0.4 log CFU/100 ml after 1 h. When maintaining a residual concentration of 1 mg/liter free chlorine, wash water contamination was maintained below 2.7, 2.5, and 2.5 log CFU/100 ml for tap water and artificial process water with COD values of 500 and 1,000 mg O2/liter, respectively. A model was developed to predict water contamination during the dynamic washing process. Only minor amounts of total trihalomethanes were formed in the water during reconditioning. Total trihalomethanes accumulated to larger amounts in the water during the wash water disinfection experiments and reached 124.5 ± 13.4 µg/liter after 1 h of execution of the washing process in water with a COD of 1,000 mg O2/liter. However, no total trihalomethanes were found on the fresh-cut lettuce after rinsing.

Insight into the prevalence and distribution of microbial contamination to evaluate water management in the fresh produce processing industry.

This study provided insight into the degree of microbial contamination in the processing chain of prepacked (bagged) lettuce in two Belgian fresh-cut produce processing companies. The pathogens Salmonella and Listeria monocytogenes were not detected. Total psychrotrophic aerobic bacterial counts (TPACs) in water samples, fresh produce, and environmental samples suggested that the TPAC is not a good indicator of overall quality and best manufacturing practices during production and processing. Because of the high TPACs in the harvested lettuce crops, the process water becomes quickly contaminated, and subsequent TPACs do not change much throughout the production process of a batch. The hygiene indicator Escherichia coli was used to assess the water management practices in these two companies in relation to food safety. Practices such as insufficient cleaning and disinfection of washing baths, irregular refilling of the produce wash baths with water of good microbial quality, and the use of high product/water ratios resulted in a rapid increase in E. coli in the processing water, with potential transfer to the end product (fresh-cut lettuce). The washing step in the production of fresh-cut lettuce was identified as a potential pathway for dispersion of microorganisms and introduction of E. coli to the end product via cross-contamination. An intervention step to reduce microbial contamination is needed, particularly when no sanitizers are used as is the case in some European Union countries. Thus, from a food safety point of view proper water management (and its validation) is a critical point in the fresh-cut produce processing industry.