High Disinfectant Tolerance in Pseudomonas spp. Biofilm Aids the Survival of Listeria monocytogenes.

Pseudomonas spp. are the most commonly found bacteria in food-processing environments due to properties such as a high growth rate at low temperatures, a high tolerance of antimicrobial agents, and biofilm formation. In this study, a set of Pseudomonas isolates originating from cleaned and disinfected surfaces in a salmon processing facility were screened for biofilm formation at 12 °C. A high variation in biofilm formation between the isolates was observed. Selected isolates, in both planktonic and biofilm states, were tested for resistance/tolerance to a commonly used disinfectant (peracetic acid-based) and antibiotic florfenicol. Most isolates showed a much higher tolerance in the biofilm state than in the planktonic state. In a multi-species biofilm experiment with five Pseudomonas strains with and without a Listeria monocytogenes strain, the Pseudomonas biofilm appeared to aid the survival of L. monocytogenes cells after disinfection, underscoring the importance of controlling the bacterial load in food-processing environments.

Bacterial community development and diversity during the first year of production in a new salmon processing plant.

The bacterial diversity and load on equipment in food processing facilities is constantly influenced by raw material, water, air, and staff. Despite regular cleaning and disinfection, some bacteria may persist and thereby potentially compromise food quality and safety. Little is known about how bacterial communities in a new food processing facility gradually establish themselves. Here, the development of bacterial communities in a newly opened salmon processing plant was studied from the first day and during the first year of operation. To focus on the persisting bacterial communities, surface sampling was done on strategical sampling points after cleaning and disinfection. To study the diversity dynamics, isolates from selected sampling and time points were classified by Oxford Nanopore Technology-based rep-PCR amplicon sequencing (ON-rep-seq) supplemented by 16S rRNA gene or rpoD gene sequencing (for Pseudomonas). An overall increase in bacterial numbers was only observed for food-contact surfaces in the slaughter department, but not in filleting department, on non-food contact surfaces or on the fish. Changes in temporal and spatial diversity and community composition were observed and our approach revealed highly point-specific bacterial communities.

Antibiotic Resistance Properties among Pseudomonas spp. Associated with Salmon Processing Environments.

Continuous monitoring of antimicrobial resistance in bacteria along the food chain is crucial for the assessment of human health risks. Uncritical use of antibiotics in farming over years can be one of the main reasons for increased antibiotic resistance in bacteria. In this study, we aimed to classify 222 presumptive Pseudomonas isolates originating from a salmon processing environment, and to examine the phenotypic and genotypic antibiotic resistance profiles of these isolates. Of all the analyzed isolates 68% belonged to Pseudomonas, and the most abundant species were Pseudomonas fluorescens, Pseudomonas azotoformans, Pseudomonas gessardii, Pseudomonas libanesis, Pseudomonas lundensis, Pseudomonas cedrina and Pseudomonas extremaustralis based on sequencing of the rpoD gene. As many as 27% of Pseudomonas isolates could not be classified to species level. Phenotypic susceptibility analysis by disc diffusion method revealed a high level of resistance towards the antibiotics ampicillin, amoxicillin, cefotaxime, ceftriaxone, imipenem, and the fish farming relevant antibiotics florfenicol and oxolinic acid among the Pseudomonas isolates. Whole genome sequencing and subsequent analysis of AMR determinants by ResFinder and CARD revealed that no isolates harbored any acquired resistance determinants, but all isolates carried variants of genes known from P. aeruginosa to be involved in multidrug efflux pump systems.

ON-rep-seq as a rapid and cost-effective alternative to whole-genome sequencing for species-level identification and strain-level discrimination of Listeria monocytogenes contamination in a salmon processing plant.

Identification, source tracking, and surveillance of food pathogens are crucial factors for the food-producing industry. Over the last decade, the techniques used for this have moved from conventional enrichment methods, through species-specific detection by PCR to sequencing-based methods, whole-genome sequencing (WGS) being the ultimate method. However, using WGS requires the right infrastructure, high computational power, and bioinformatics expertise. Therefore, there is a need for faster, more cost-effective, and more user-friendly methods. A newly developed method, ON-rep-seq, combines the classical rep-PCR method with nanopore sequencing, resulting in a highly discriminating set of sequences that can be used for species identification and also strain discrimination. This study is essentially a real industry case from a salmon processing plant. Twenty Listeria monocytogenes isolates were analyzed both by ON-rep-seq and WGS to identify and differentiate putative L. monocytogenes from a routine sampling of processing equipment and products, and finally, compare the strain-level discriminatory power of ON-rep-seq to different analyzing levels delivered from the WGS data. The analyses revealed that among the isolates tested there were three different strains. The isolates of the most frequently detected strain (n = 15) were all detected in the problematic area in the processing plant. The strain level discrimination done by ON-rep-seq was in full accordance with the interpretation of WGS data. Our findings also demonstrate that ON-rep-seq may serve as a primary screening method alternative to WGS for identification and strain-level differentiation for surveillance of potential pathogens in a food-producing environment.

Occurrence, diversity and temperature-dependent growth kinetics of Aeromonas spp. in lettuce.

Bagged, pre-cut and prewashed lettuce products are marketed as ready to eat. This concept poses a food safety concern, due to lack of efficient hurdles to eliminate possible microbial contaminants from the fresh produce and/or the processing itself. Aeromonas spp. are potential foodborne pathogens that are frequently isolated from lettuce. High counts of, e.g., A. hydrophila have been found in retail ready-to-eat (RTE) vegetable salads. The aim of this study was to assess the general microbiological quality, the occurrence and diversity of potential human pathogenic mesophilic Aeromonas spp. of retail RTE lettuce products. Additionally, temperature-dependent growth kinetic parameters of Aerobic Plate Counts (APC) and Aeromonas spp. in one selected RTE lettuce product, rocket lettuce, were quantified by performing storage experiments at 4 °C, 8 °C and 12 °C. The Aeromonas isolates were further characterized regarding pathogenic traits and phylogenetic relationship. The overall hygienic quality of the lettuce products was unsatisfactory, as 60% of the products had an APC level higher than 7.0 log CFU/g. Presumptive Aeromonas spp. were detected in 52% of the samples, levels ranging from approximately 2.0-6.0 log CFU/g. Significantly lower counts of APC and Aeromonas spp. were found in uncut and unwashed products. Presumptive Aeromonas spp. were able to proliferate in rocket lettuce stored at 4 °C (µmax = 0.39 ± 0.06/d and µmax = 0.43 ± 0.05/d for lettuce from producers A and B, respectively), and µmax was approximately 2× higher at 8 °C and 3× higher at 12 °C. Eighty-four percent of the collected isolates were identified as A. media, based on partial gyrB sequencing. Additionally A. salmonicida and A. bestiarum were detected. The pathogenic potential in this material was high, most of the isolates harbored at least one of the toxin genes, act, ast, alt.

Assessment of microbiological quality of retail fresh sushi from selected sources in Norway.

Retail fresh sushi is gaining popularity in Europe. This study was conducted to investigate the microbiological quality of selected samples of fresh sushi with a shelf life of 2 to 3 days offered as complete meals in Norwegian supermarkets. Analysis of aerobic plate counts in 58 sushi samples from three producers revealed large variations in microbiological quality, and 48% of the analyzed sushi boxes were rated as unsatisfactory (> 6.0 log CFU/g). Mesophilic Aeromonas spp. was detected in 71% of the samples. In a follow-up study, we collected products and raw materials directly from the production facility of one producer and observed a significant decrease (P < 0.01) in aerobic plate counts compared with the initial sampling. The observed difference between products purchased in stores compared with those collected directly from the factory suggests that poor temperature control during distribution and display in stores leads to reduced microbiological quality. Microbiological analysis of the sushi ingredients revealed that potentially pathogenic bacteria such as mesophilic Aeromonas spp. or bacteria belonging to the Enterobacteriaceae can be introduced into sushi through both raw vegetables and fish. The results highlight the importance of high quality ingredients and proper temperature control to ensure stable quality and safety of these food products.

A case study from the interaction of strawberry and Botrytis cinerea highlights the benefits of comonitoring both partners at genomic and mRNA level.

Strawberry Fragaria x ananassa (cv. Korona) was inoculated with Botrytis cinerea by dipping berries in a conidial suspension. Colonization by the pathogen was monitored using real-time PCR, ELISA and ergosterol assays, the first showing the highest sensitivity. The expression of pathogen beta-tubulin and six polygalacturonases (Bcpg1-6) and three host defence genes (polygalacturonase-inhibiting protein (FaPGIP) and two class II chitinases) were monitored using real-time RT-PCR. The maximum transcript levels of the host defence genes occurred at 16 h postinoculation (hpi) at the presumed initial penetration stage. The unique transcript profile of Bcpg2 over the 96-h incubation time and its high transcript levels relative to those of the other Bcpgs at 8-24 hpi suggest that the gene has a specific role in the penetration stage. Bcpg1 was expressed constitutively at a relatively high level in actively growing mycelia throughout the experimental period. Comparison of the transcript profiles indicated that Bcpg1 and Bcpg3-6 were coordinately regulated.

Quantification of allele-specific expression of a gene encoding strawberry polygalacturonase-inhibiting protein (PGIP) using Pyrosequencing.

Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated the transcript abundance of six individual, highly homologous alleles of a polygalacturonase-inhibiting protein gene (FaPGIP) from octoploid strawberry (Fragaria x ananassa). We applied the highly quantitative Pyrosequencing method which, for the gene under study, detected allele frequency differences as small as 4.0 +/- 2.8%. Pyrosequencing of RT-PCR products showed that one FaPGIP allele was preferentially expressed in leaf tissue, while two other alleles were expressed in a fruit-specific way. For fruits that were inoculated with Botrytis cinerea a strong increase in overall FaPGIP gene expression was observed. This upregulation was accompanied by a significant change in FaPGIP allele frequencies when compared with non-treated fruits. Remarkably, in the five cultivars tested, the allele frequency in cDNA from the inoculated fruits was similar to that in genomic DNA, suggesting uniform upregulation of all FaPGIP alleles present as a result of pathogenesis-related stress. The results demonstrate that when Pyrosequencing of RT-PCR products is performed, novel allele-specific gene regulation can be detected and quantified.

A gene encoding a polygalacturonase-inhibiting protein (PGIP) shows developmental regulation and pathogen-induced expression in strawberry.

• Polygalacturonase-inhibiting proteins (PGIPs) have been demonstrated to play a role in host defence in several plants. • The PGIP now cloned from strawberry (Fragaria × ananassa) showed a high degree of homology to other fruit PGIPs. The gene expression of strawberry PGIP was monitored in healthy leaves, flowers and fruit at different maturity stages. PGIP transcript levels were also analysed following fruit inoculation with the fungal pathogen Botrytis cinerea in strawberry cultivars displaying variation in susceptibility. • Healthy mature berries showed the highest constitutive PGIP gene expression levels compared with leaves, flowers and immature fruit, indicating that the gene is developmentally regulated. Among the cultivars studied ('Elsanta', 'Korona', 'Polka', 'Senga sengana', 'Tenira'), 'Polka' had the highest constitutive expression level of PGIP. After inoculation with B. cinerea, all five cultivars displayed a significant induction of PGIP gene expression, but the differences between them were not statistically significant. • The high induction of the PGIP gene after inoculation with B. cinerea indicates that PGIP has a role in defence of strawberry.