No evidence for a relationship between farm or transformation process locations and antibiotic resistance patterns of Pseudomonas population associated with rainbow trout (Oncorhynchus mykiss).

AIMS: Study the relationship between antibiotic resistance patterns of Pseudomonas isolated from farmed rainbow trout fillets and farm or transformation process locations. METHODS AND RESULTS: Pseudomonas strains were isolated from rainbow trout sampled in two differently located farms and filleted in laboratory or in a processing factory. One hundred and twenty-five isolates were confirmed as belonging to Pseudomonas using CFC selective media, Gram staining, oxidase test and quantitative polymerase chain reaction methods. Fifty-one isolates from separate fish fillets were further identified using MALDI-TOF mass spectrometry, and the minimal inhibitory concentrations (MIC) of 11 antibiotics were also determined by microdilution method. Most of the isolates belonged to the Pseudomonas fluorescens group (94.1%), and no relationship was established between antibiotic resistance patterns and sampling locations (farms or filleting areas). Multiple resistance isolates with high MIC values (from 64 µg ml-1 to more than 1024 µg ml-1 ) were identified. CONCLUSIONS: Antibiotic resistance patterns found in Pseudomonas isolates were not influenced by farms or transformation process locations. Seven isolates were found highly resistant to four different antibiotic classes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study does not provide evidence of a relationship between farm or transformation process locations on antibiotic resistance patterns of Pseudomonas population.

Quantification of Viable Brochothrix thermosphacta in Cold-Smoked Salmon Using PMA/PMAxx-qPCR.

The aim of this study was to develop a rapid and accurate PMA-qPCR method to quantify viable Brochothrix thermosphacta in cold-smoked salmon. B. thermosphacta is one of the main food spoilage bacteria. Among seafood products, cold-smoked salmon is particularly impacted by B. thermosphacta spoilage. Specific and sensitive tools that detect and quantify this bacterium in food products are very useful. The culture method commonly used to quantify B. thermosphacta is time-consuming and can underestimate cells in a viable but not immediately culturable state. We designed a new PCR primer set from the single-copy rpoC gene. QPCR efficiency and specificity were compared with two other published primer sets targeting the rpoC and rpoB genes. The viability dyes PMA or PMAxx were combined with qPCR and compared with these primer sets on viable and dead B. thermosphacta cells in BHI broth and smoked salmon tissue homogenate (SSTH). The three primer sets displayed similar specificity and efficiency. The efficiency of new designed rpoC qPCR on viable B. thermosphacta cells in SSTH was 103.50%, with a linear determination coefficient (r2) of 0.998 and a limit of detection of 4.04 log CFU/g. Using the three primer sets on viable cells, no significant difference was observed between cells treated or untreated with PMA or PMAxx. When dead cells were used, both viability dyes suppressed DNA amplification. Nevertheless, our results did not highlight any difference between PMAxx and PMA in their efficiency to discriminate viable from unviable B. thermosphacta cells in cold-smoked salmon. Thus, this study presents a rapid, specific and efficient rpoC-PMA-qPCR method validated in cold-smoked salmon to quantify viable B. thermosphacta in foods.

Symbiont dynamics during the blood meal of Ixodes ricinus nymphs differ according to their sex.

Ticks harbour rich and diverse microbiota and, among the microorganisms associated with them, endosymbionts are the subject of a growing interest due to their crucial role in the biology of their arthropod host. Midichloria mitochondrii is the main endosymbiont of the European tick Ixodes ricinus and is found in abundance in all I. ricinus females, while at a much lower density in males, where it is even absent in 56 % of the individuals. This endosymbiont is also known to increase in numbers after the blood meal of larvae, nymphs or females. Because of this difference in the prevalence of M. mitochondrii between the two sexes, surveying the density of these bacteria in nymphs that will become either females or males could help to understand the behaviour of Midichloria in its arthropod host. To this aim, we have set up an experimental design by building 3 groups of unfed nymphs based on their scutum and hypostome lengths. After engorgement, weighing and moulting of a subset of the nymphs, a significant difference in sex-ratio among the 3 groups was observed. In parallel, Midichloria load in individual nymphs was quantified by qPCR both before and after engorgement. No difference in either body mass or Midichloria load was observed at the unfed stage, but following engorgement, both features were significantly different between each size group. Our results demonstrate that symbiont dynamics during nymphal engorgement is different between the two sexes, resulting in a significantly higher Midichloria load in nymphs that will become females. The consequences of those findings on our understanding of the interplay between the endosymbiont and its arthropod host are discussed.

Characterization of Bacterial Communities of Cold-Smoked Salmon during Storage.

Cold-smoked salmon is a widely consumed ready-to-eat seafood product that is a fragile commodity with a long shelf-life. The microbial ecology of cold-smoked salmon during its shelf-life is well known. However, to our knowledge, no study on the microbial ecology of cold-smoked salmon using next-generation sequencing has yet been undertaken. In this study, cold-smoked salmon microbiotas were investigated using a polyphasic approach composed of cultivable methods, V3-V4 16S rRNA gene metabarcoding and chemical analyses. Forty-five cold-smoked salmon products processed in three different factories were analyzed. The metabarcoding approach highlighted 12 dominant genera previously reported as fish spoilers: Firmicutes Staphylococcus, Carnobacterium, Lactobacillus, ß-Proteobacteria Photobacterium, Vibrio, Aliivibrio, Salinivibrio, Enterobacteriaceae Serratia,Pantoea, γ-Proteobacteria Psychrobacter, Shewanella and Pseudomonas. Specific operational taxonomic units were identified during the 28-day storage study period. Operational taxonomic units specific to the processing environment were also identified. Although the 45 cold-smoked salmon products shared a core microbiota, a processing plant signature was found. This suggest that the bacterial communities of cold-smoked salmon products are impacted by the processing environment, and this environment could have a negative effect on product quality. The use of a polyphasic approach for seafood products and food processing environments could provide better insights into residential bacteria dynamics and their impact on food safety and quality.

Impact of DNA extraction and sampling methods on bacterial communities monitored by 16S rDNA metabarcoding in cold-smoked salmon and processing plant surfaces.

Amplicon sequencing approaches have been widely used in food bacterial ecology. However, choices regarding the methodology can bias results. In this study, bacterial communities associated with cold-smoked salmon products and their processing plant surfaces were monitored via sequencing of the V3-V4 region of the 16S rRNA gene. The impact of DNA extraction protocols, sampling methods (swabbing or sponging) and surface materials on bacterial communities were investigated. α and ß diversity analyses revealed that DNA extraction methods mainly influence the observed cold-smoked salmon microbiota composition. Moreover, different DNA extraction methods revealed significant differences in observed community richness and evenness. ß-Proteobacteria: Photobacterium, Serratia and Firmicutes: Brochothrix, Carnobacterium and Staphylococcus were identified as the dominant genera. Surface microbiota richness, diversity and composition were mainly affected by cleaning and disinfection procedures but not by DNA extraction methods. Surface community richness and evenness appeared higher when sampled by sponging compared to swabbing. ß-diversity analyses highlighted that surface topology, cleaning and disinfection and sampling devices seemed to affect the bacterial community composition. The dominant surface bacteria identified were mainly Flavobacteriaceae, ß-Proteobacteria and γ-Proteobacteria described as fish spoilers such as Acinetobacter, Pseudomonas and Shewanella. DNA extraction and sampling methods can have an impact on sequencing results and the ecological analysis of bacterial community structures. This study confirmed the importance of methodology standardization and the need for analytical validation before 16S rDNA metabarcoding surveys.

Antibiotic Resistance Genes and Bacterial Communities of Farmed Rainbow Trout Fillets (Oncorhynchus mykiss).

The rise of antibiotic resistance is not only a challenge for human and animal health treatments, but is also posing the risk of spreading among bacterial populations in foodstuffs. Farmed fish-related foodstuffs, the food of animal origin most consumed worldwide, are suspected to be a reservoir of antibiotic resistance genes and resistant bacterial hazards. However, scant research has been devoted to the possible sources of diversity in fresh fillet bacterial ecosystems (farm environment including rivers and practices, and factory environment). In this study bacterial communities and the antibiotic resistance genes of fresh rainbow trout fillet were described using amplicon sequencing of the V3-V4 region of the 16S rRNA gene and high-throughput qPCR assay. The antibiotic residues were quantified using liquid chromatography/mass spectrometry methods. A total of 56 fillets (composed of muscle and skin tissue) from fish raised on two farms on the same river were collected and processed under either factory or laboratory sterile filleting conditions. We observed a core-bacterial community profile on the fresh rainbow trout fillets, but the processing conditions of the fillets has a great influence on their mean bacterial load (3.38 ± 1.01 log CFU/g vs 2.29 ± 0.72 log CFU/g) and on the inter-individual diversity of the bacterial community. The bacterial communities were dominated by Gamma- and Alpha-proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The most prevalent genera were Pseudomonas, Escherichia-Shigella, Chryseobacterium, and Carnobacterium. Of the 73 antibiotic residues searched, only oxytetracycline residues were detected in 13/56 fillets, all below the European Union maximum residue limit (6.40-40.20 µg/kg). Of the 248 antibiotic resistance genes searched, 11 were found to be present in at least 20% of the fish population (tetracycline resistance genes tetM and tetV, ß-lactam resistance genes bla DHA and bla ACC, macrolide resistance gene mphA, vancomycin resistance genes vanTG and vanWG and multidrug-resistance genes mdtE, mexF, vgaB and msrA) at relatively low abundances calculated proportionally to the 16S rRNA gene.

Comparison of Stomaching versus Rinsing for Recovering Bacterial Communities from Rainbow Trout (Oncorhynchus mykiss) Fillets.

ABSTRACT: The use of high-throughput methods allows a better characterization of food-related bacterial communities. However, such methods require large amounts of high-quality bacterial DNA, which may be a challenge when dealing with a complex matrix that has a low concentration of bacteria, such as fresh fish fillets. Therefore, the choice of method used to recover bacteria from a food matrix in a cost-effective way is critical, yet little information is available on the performance of commonly used methods. We assessed the recovery capacity of two such methods: stomaching and mechanical rinsing. The efficiency of the methods was evaluated through quantitative recovery and compatibility with end-point quantitative PCR (qPCR). Fresh rainbow trout (Oncorhynchus mykiss) fillets were inoculated with a bacterial marker, Brochothrix thermosphacta, at different concentrations (7.52 to 1.52 log CFU/g). The fillets were processed by one of the two methods, and the recovery of the marker in the suspensions was assessed by plate counting and qPCR targeting B. thermosphacta-rpoC. The same analyses were performed on six noninoculated fresh fillets. Stomaching and mechanical rinsing allowed efficient and repeatable recovery of the bacterial communities from the 42 inoculated fillets. No significant differences in recovery ratios were observed between the marker enumerated in the inoculation suspensions and in the corresponding recovery suspensions after rinsing and stomaching. However, the stomaching method allowed too many particles to pass through the filters bag, making necessary a limiting supplementary filtration step. As a consequence, only the rinsing recovery method allowed proper PCR quantification of the inoculated B. thermosphacta. The mean recovered bacterial level of the fillets was approximately 3 log CFU/g. It seems more relevant and cost-effective to recover the endogenous bacterial microbiota of a fish fillet structure using the rinsing method rather than the stomaching method.

Influence of lactate and acetate removal on the microbiota of French fresh pork sausages.

The microbiota of fresh French pork sausages were characterised in five batches of comminuted pork meat that were equally divided into two formulations either containing the acid-based preservatives lactate and acetate, or no preservatives. Conventional microbiological analysis and high-throughput 16S rDNA amplicon sequencing methods were performed on meat batches packed under modified atmosphere (70% oxygen and 30% carbon dioxide) during chilled storage. In addition, meat pH and colour, and gas composition of the packages were monitored until the end of the shelf-life. During storage, the population of mesophilic and lactic acid bacteria increased from 4 log CFU/g to 8 log CFU/g after 15 days of chilled storage, both with and without preservatives. Despite similar changes of the physical and chemical parameters, such as pH and package gas composition, spoilage was delayed in the meat containing the preservatives, suggesting that lactate and acetate are effective against spoilage. Metagenetic analysis showed that at the end of the shelf-life, the species distribution differed between both the formulations and the batches. Lactic acid bacteria were shown to dominate both with and without preservatives; however, samples containing no preservatives were characterised by the presence of an increased population of Brochothrix spp. and Pseudomonas spp. whereas, Leuconostoc mesenteroides/pseudomesenteroides and Lactobacillus curvatus/graminis were more abundant in the meat with preservatives.

Detection of Wolbachia in the tick Ixodes ricinus is due to the presence of the hymenoptera endoparasitoid Ixodiphagus hookeri.

The identification of micro-organisms carried by ticks is an important issue for human and animal health. In addition to their role as pathogen vectors, ticks are also the hosts for symbiotic bacteria whose impact on tick biology is poorly known. Among these, the bacterium Wolbachia pipientis has already been reported associated with Ixodes ricinus and other tick species. However, the origins of Wolbachia in ticks and their consequences on tick biology (known to be very diverse in invertebrates, ranging from nutritional symbionts in nematodes to reproductive manipulators in insects) are unknown. Here we report that the endoparasitoid wasp Ixodiphagus hookeri (Hymenoptera, Chalcidoidea, Encyrtidae)--strictly associated with ticks for their development--infested at almost 100% prevalence by a W. pipientis strain belonging to a Wolbachia supergroup that has already been reported as associated with other hymenopteran parasitoids. In a natural population of I. ricinus that suffers high parasitism rates due to I. hookeri, we used specific PCR primers for both hymenopteran and W. pipientis gene fragments to show that all unfed tick nymphs parasitized by I. hookeri also harbored Wolbachia, while unparasitized ticks were Wolbachia-free. We demonstrated experimentally that unfed nymphs obtained from larvae exposed to I. hookeri while gorging on their vertebrate host also harbor Wolbachia. We hypothesize that previous studies that have reported W. pipientis in ticks are due to the cryptic presence of the endoparasitoid wasp I. hookeri. This association has remained hidden until now because parasitoids within ticks cannot be detected until engorgement of the nymphs brings the wasp eggs out of diapause. Finally, we discuss the consequences of this finding for our understanding of the tick microbiome, and their possible role in horizontal gene transfer among pathogenic and symbiotic bacteria.

Natural transmission of Zoonotic Babesia spp. by Ixodes ricinus ticks.

To determine characteristics of natural transmission of Babesia sp. EU1 and B. divergens by adult Ixodes ricinus ticks, we examined tick salivary gland contents. We found that I. ricinus is a competent vector for EU1 and that their sporozoites directly invade erythrocytes. We conclude that EU1 is naturally transmitted by I. ricinus.

Complete sequence of the floR-carrying multiresistance plasmid pAB5S9 from freshwater Aeromonas bestiarum.

OBJECTIVES: A multiresistant Aeromonas bestiarum strain, shown to be persistent and spreading in a freshwater stream, was investigated for the presence, location and organization of antimicrobial resistance genes. METHODS: The plasmid pAB5S9 was transferred by electroporation into Escherichia coli TG1. The resistance phenotype mediated by pAB5S9 was determined. Moreover, the plasmid was sequenced completely and analysed for its structure and organization of reading frames. RESULTS: Plasmid pAB5S9 mediated resistances to phenicols, sulphonamides, streptomycin and tetracycline. The analysis of the 24.7 kb sequence revealed the presence of 20 predicted coding sequences (CDSs), which included the floR, sul2 and strA-strB resistance genes and a tetR-tet(Y) determinant. Approximately 7.5 kb of pAB5S9 showed 100% nucleotide sequence identity to three non-contiguous segments of the SXT element of Vibrio cholerae. Regions identical to SXT comprised the floR gene, flanked upstream by a complete and downstream by a truncated ISCR2 element, and the region of the sul2 and strA-strB genes. Other CDSs of pAB5S9 related to plasmid replication and partitioning, metabolic and gene regulation functions as well as conjugative transfer showed homology to sequences from diverse bacterial species, indicating a mosaic structure. CONCLUSIONS: This study provides the first report of a floR-carrying plasmid in the genus Aeromonas and the first description of a tetR-tet(Y) determinant. The analysis of the multiresistant A. bestiarum strain indicates that strains of this species, some of which are opportunistic pathogens for fish, might also act as a resistance gene reservoir in the freshwater environment.

Survey of antibiotic resistance in an integrated marine aquaculture system under oxolinic acid treatment.

The consequences of antibiotic use in aquatic integrated systems, which are based on trophic interactions between different cultured organisms and physical continuity through water, need to be examined. In this study, fish reared in a prototype marine integrated system were given an oxolinic acid treatment, during and after which the level of resistance to this quinolone antibiotic was monitored among vibrio populations from the digestive tracts of treated fish, co-cultured bivalves and sediments that were isolated on thiosulfate-citrate-bile-sucrose. Oxolinic acid minimum inhibitory concentration distributions obtained from replica plating of thiosulfate-citrate-bile-sucrose plates indicated that a selection towards oxolinic acid resistance had occurred in the intestines of fish under treatment. In contrast, and despite oxolinic acid concentrations higher than minimum inhibitory concentrations of susceptible bacteria, no clear evolution of resistance levels was detected either in bivalves or in sediments.

Mechanisms of quinolone resistance and clonal relationship among Aeromonas salmonicida strains isolated from reared fish with furunculosis.

The mechanisms of resistance to quinolone and epidemiological relationships among A. salmonicida strains isolated from diseased fish in French marine farms from 1998 to 2000 were investigated. The quinolone resistance-determining regions of the gyrA and parC genes of 12 clinical A. salmonicida isolates with different levels of quinolone susceptibility were sequenced. MICs were determined in the presence of the efflux pump inhibitor (EPI) Phe-Arg beta-naphthylamide and E(max) values (MIC without EPI/MIC in the presence of EPI) were calculated. Isolates fell into two classes: (i) those that had a wild-type gyrA gene with oxolinic acid MIC Asn with oxolinic acid MIC >/= 2, flumequine MIC >/= 4 and ciprofloxacin MIC >/= 0.125 micro g ml(-1). No mutations were found in parC. High E(max) values obtained for flumequine and oxolinic acid (up to 16 and 8, respectively, for the most resistant isolates of the two classes) indicated an important contribution of efflux to the resistance phenotype. Flumequine accumulation experiments confirmed that high E(max) values were associated with a much lower level of accumulation. PCR/RFLP assays conducted on 34 additional isolates showed the presence of a mutation at codon 87 of gyrA in nearly all the quinolone-resistant isolates. This finding, together with PFGE typing results, strongly suggests a common clonal origin of these quinolone-resistant isolates.