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.

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.