Microbial community dispersal from wheat grains to sourdoughs: A contribution of participatory research.

Understanding microbial dispersal is critical to understand the dynamics and evolution of microbial communities. However, microbial dispersal is difficult to study because of uncertainty about their vectors of migration. This applies to both microbial communities in natural and human-associated environments. Here, we studied microbial dispersal along the sourdoughs bread-making chain using a participatory research approach. Sourdough is a naturally fermented mixture of flour and water. It hosts a community of bacteria and yeasts whose origins are only partially known. We analysed the potential of wheat grains and flour to serve as an inoculum for sourdough microbial communities using 16S rDNA and ITS1 metabarcoding. First, in an experiment involving farmers, a miller and bakers, we followed the microbiota from grains to newly initiated and propagated sourdoughs. Second, we compared the microbiota of 46 sourdough samples collected everywhere in France, and of the flour used for their back-slopping. The core microbiota detected on the seeds, in the flour and in the sourdough was composed mainly of microbes known to be associated with plants and not living in sourdoughs. No sourdough yeast species were detected on grains and flours. Sourdough lactic acid bacteria were rarely found in flour. When they were, they did not have the same amplicon sequence variant (ASV) as found in the corresponding sourdough. However, the low sequencing depth for bacteria in flour did not allow us to draw definitive conclusion. Thus, our results showed that sourdough yeasts did not come from flour, and suggest that neither do sourdough LAB.

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.

Sourdough microbial community dynamics: An analysis during French organic bread-making processes.

Natural sourdoughs are commonly used in bread-making processes, especially for organic bread. Despite its role in bread flavor and dough rise, the stability of the sourdough microbial community during and between bread-making processes is debated. We investigated the dynamics of lactic acid bacteria (LAB) and yeast communities in traditional organic sourdoughs of five French bakeries during the bread-making process and several months apart using classical and molecular microbiology techniques. Sourdoughs were sampled at four steps of the bread-making process with repetition. The analysis of microbial density over 68 sourdough/dough samples revealed that both LAB and yeast counts changed along the bread-making process and between bread-making runs. The species composition was less variable. A total of six LAB and nine yeast species was identified from 520 and 1675 isolates, respectively. The dominant LAB species was Lactobacillus sanfranciscensis, found for all bakeries and each bread-making run. The dominant yeast species changed only once between bread-making processes but differed between bakeries. They mostly belonged to the Kazachstania clade. Overall, this study highlights the change of population density within the bread-making process and between bread-making runs and the relative stability of the sourdough species community during bread-making process.

Bacterial spoilers of food: behavior, fitness and functional properties.

Most food products are highly perishable as they constitute a rich nutrient source for microbial development. Among the microorganisms contaminating food, some present metabolic activities leading to spoilage. In addition to hygienic rules to reduce contamination, various treatments are applied during production and storage to avoid the growth of unwanted microbes. The nature and appearance of spoilage therefore depend on the physiological state of spoilers and on their ability to resist the processing/storage conditions and flourish on the food matrix. Spoilage also relies on the interactions between the microorganisms composing the ecosystems encountered in food. The recent rapid increase in publicly available bacterial genome sequences, as well as the access to high-throughput methods, should lead to a better understanding of spoiler behavior and to the possibility of decreasing food spoilage. This review lists the main bacterial species identified as food spoilers, their ability to develop during storage and/or processing, and the functions potentially involved in spoilage. We have also compiled an inventory of the available genome sequences of species encompassing spoilage strains. Combining in silico analysis of genome sequences with experimental data is proposed in order to understand and thus control the bacterial spoilage of food better.

Bacteriocinogenic potential and safety evaluation of non-starter Enterococcus faecium strains isolated from home made white brine cheese.

Four LAB strains, isolated from Bulgarian home made white brine cheese, were selected for their effective inhibition against Listeria monocytogenes. According to their biochemical and physiological characteristics, the strains were classified as members of Enterococcus genus, and then identified as Enterococcus faecium by 16S rDNA sequencing. Their bacteriocin production and inhibitory spectrum were evaluated together with the occurrence of several bacteriocin genes (entA, entB, entP, entL50B). Their virulence potential and safety was assessed both using PCR targeted to the genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for antibiotic resistance, gelatinase, lipase, DNAse and α- and ß-haemolysis. The E. faecium strains harboured at least one enterocin gene while the occurrence of virulence, antibiotic resistance and biogenic amines genes was limited. Considering their strong antimicrobial activity against L. monocytogenes strains, the four E. faecium strains exhibited promising potential as bio-preservatives cultures for fermented food productions.

Evaluation of the spoilage potential of bacteria isolated from spoiled cooked whole tropical shrimp (Penaeus vannamei) stored under modified atmosphere packaging.

The spoilage potential of isolates belonging to five bacterial groups/species (Shewanella baltica, Carnobacterium maltaromaticum, Aeromonas salmonicida, Vibrio sp., "other Gamma-Proteobacteria" [containing one strain of Pseudoalteromonas sp. and one strain of Psychrobacter sp.]) isolated from spoiled cooked and whole tropical shrimp stored under modified atmosphere packaging (MAP) was evaluated by inoculation into ionized cooked and peeled tropical shrimp followed by storage for 32 days at 8 °C. Microbial growth and sensory changes were monitored during the storage period. The major spoilage bacterial isolate groups were C. maltaromaticum and S. baltica. In order to characterize their spoilage potential further and to study the effect of their interactions, each of these two specific spoilage organisms (SSO) and one mixed-culture, C. maltaromaticum/S. baltica, were tested using a combination of complementary methods: molecular (PCR-TTGE), sensory, chemical, and conventional microbiological analyses. It was concluded that, in the mixed-culture-inoculated samples, both species groups imposed their spoilage characteristics.

Development of a rapid real-time PCR method as a tool to quantify viable Photobacterium phosphoreum bacteria in salmon (Salmo salar) steaks.

A specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of the Photobacterium phosphoreum species group in raw modified-atmosphere-packed salmon. Primers were designed to amplify a 350-bp fragment of the gyrase subunit B gene (gyrB) of P. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these primers were used for real-time PCR in pure culture, a good correlation (R(2) of 0.99) was obtained between this method and conventional enumeration on marine agar (MA). Quantification was linear over 5 log units as confirmed by using inoculated salmon samples. On naturally contaminated fresh salmon, the new real-time PCR method performed successfully with a quantification limit of 3 log CFU/g. A correlation coefficient (R(2)) of 0.963 was obtained between the PCR method and classic enumeration on MA, followed by identification of colonies (290 isolates identified by real-time PCR or by 16S rRNA gene sequencing). A good correlation with an R(2) of 0.940 was found between the new PCR method and an available specific conductance method for P. phosphoreum. This study presents a rapid tool for producing reliable quantitative data on viable P. phosphoreum bacteria in fresh salmon in 6 h. This new culture-independent method will be valuable for future fish inspection, the assessment of raw material quality in fish processing plants, and studies on the ecology of this important specific spoilage microorganism.

Genome sequence of Lactobacillus salivarius SMXD51, a potential probiotic strain isolated from chicken cecum, showing anti-campylobacter activity.

We report the draft genome sequence of Lactobacillus salivarius SMXD51, isolated from the cecum of healthy chickens showing an activity against Campylobacter--the food-borne pathogen that is the most common cause of gastroenteritis in the European Union (EU)--and potentially interesting features for a probiotic strain, explaining our interest in it.

Characterisation of the spoilage microbiota in raw salmon (Salmo salar) steaks stored under vacuum or modified atmosphere packaging combining conventional methods and PCR-TTGE.

In order to characterise the spoilage related to microbiota of raw salmon, a combination of culture-dependent and -independent methods, including PCR-TTGE, was used to analyse 3 raw salmon batches stored for 3 days at chilled temperature in modified atmosphere packaging (MAP) (50% CO2/50% N2) or under vacuum. Sensory evaluation, microbiological enumeration and chemical analysis were performed after 3, 7 and 10 days of storage. At the onset of spoilage, 65 bacterial isolates were picked from the plates. Thus, 13 different genera or species were identified by phenotypic and molecular tests: Serratia spp., Photobacterium phosphoreum, Yersinia intermedia, Hafnia alvei, Buttiauxella gaviniae, Pseudomonas sp., Carnobacterium maltaromaticum, Carnobacterium divergens, Lactococcus piscium, Lactobacillus fuchuensis, Vagococcus carniphilus, Leuconostoc gasicomitatum and Brochothrix thermosphacta. The PCR-TTGE profiles and band identification enabled a shift of the dominant populations during the storage to be visualised for all the batches, probably due to the temperature change and the packaging. At the beginning of storage, Pseudomonas sp. dominated the raw salmon microbiota while in the following days (7 and 10), P. phosphoreum and L. piscium were identified as the main bacterial groups. This study enhances the knowledge of MAP and vacuum-packed raw salmon spoilage microbiota.

Quantification of viable Brochothrix thermosphacta in cooked shrimp and salmon by real-time PCR.

Brochothrix thermosphacta, a Gram-positive bacterium, is considered as the predominant spoilage microbiota of modified atmosphere packing (MAP) shrimp and fish. Traditional methods currently used to detect B. thermosphacta in foods are time-consuming and labour-intensive. The aim of this study was to develop a real-time PCR quantification method combined with a propidium monoazide (PMA) sample treatment step to monitor the population of B. thermosphacta in cooked shrimp and salmon. The specificity of the two primers MO405 and MO404 used to amplify a 70 bp fragment of the 16S rRNA gene was demonstrated by using purified DNA from 30 strains, among 21 bacterial species including 22 reference strains. Using these primers for real-time PCR and in pure culture, a good correlation was obtained between real-time PCR and the conventional plating method. Quantification was linear over 7-log units using artificially inoculated samples. The method performed successfully when tested on naturally contaminated cooked shrimp and fresh salmon, with a minimum threshold of 1.9×10² CFU/g for accurate quantification of B. thermosphacta. The correlation between the B. thermosphacta counts obtained by real-time PCR and plate counts on naturally contaminated shrimp and salmon was high (R²=0.895). Thus, this study presents a rapid tool for producing reliable quantitative data on B. thermosphacta in cooked shrimp and fresh salmon.

Purification and characterization of a new bacteriocin active against Campylobacter produced by Lactobacillus salivarius SMXD51.

Strain SMXD51, isolated from chicken ceca and identified as Lactobacillus salivarius, produced a component that inhibits the growth of Gram-positive and Gram-negative bacteria and especially Campylobacter jejuni. The active peptide from the cell-free supernatant of Lb. salivarius SMXD51 was purified in three steps: (i) precipitation with 80% saturated ammonium sulfate, (ii) elution on a reversed phase SPE UPTI-CLEAN cartridge using different concentrations of acetonitrile, (iii) final purification by reversed phase HPLC on a C(18) column. The mode of action of this peptide of 5383.2 Da was identified as bactericidal, and its amino acid composition was established. This new bacteriocin SMXD51 appears potentially very useful to reduce Campylobacter in poultry prior to processing.

Identification of lactobacilli residing in chicken ceca with antagonism against Campylobacter.

Bacteriocins produced by Lactobacillus salivarius have been recently recognized as a natural means to control Campylobacter and Salmonella in live poultry. This finding is of relevance since Campylobacter jejuni and Campylobacter coli are the predominant species isolated from poultry that are associated with human campylobacteriosis. In the present work, lactic acid bacteria (LAB) isolated from the cecum of twenty Tunisian chickens were identified and those isolates with antagonism against Campylobacter were further characterized. Following their preliminary confirmation as LAB, 150 strains were identified by combining morphological criteria, biochemical tests, and molecular methods, the latter inluding intergenic 16S- 23S PCR, specific lactobacilli PCR, and a biphasic approach. Most of the LAB isolated belonged to the genus Lactobacillus, among them Lb. sakei (33.3%), Lb. salivarius (19.4%), Lb. reuteri (8.6%), and Lb. curvatus (8.6%). The other LAB strains included those of the genus Weissella (16.7%), Enterococcus faecalis (5.3%), Leuconostoc mesenteroides (2.7%), Lactococcus graviae (2.7%), and Streptococcus sp. (2.7%). The Lactobacilli strains were tested for their antagonism against C. jejuni and C. coli. The activity of three of them, Lb. salivarius SMXD51, Lb. salivarius MMS122, and Lb. salivarius MMS151, against the aforementioned target strains could be ascribed to the production of bacteriocins.

Characterization of a bacteriocin produced by Lactobacillus sakei R1333 isolated from smoked salmon.

Strain R1333, isolated from commercially available smoked salmon, was identified as Lactobacillus sakei based on biochemical tests, sugar fermentation reactions (API 50 CHL), PCR with species-specific primers and sequencing of the 16S rRNA gene. Strain R1333 produces a 3811 kDa class IIa bacteriocin, active against Streptococcus caprinus, Streptococcus macedonicus, Streptococcus spp., L. sakei, Lactococcus lactis subsp. lactis, Listeria innocua, Listeria ivanovii subsp. ivanovii and Listeria monocytogenes. The mode of activity against L. innocua 2030C and L. ivanovii subsp. ivanovii ATCC 19119 was bactericidal, resulting in cell lysis and enzyme- and DNA-leakage. The highest level of activity (1600 AU/mL) was recorded when cells were grown at 30°C in MRS broth (initial pH 6.5). Only 800 AU/mL was recorded when strain R1333 was grown in MRS without Tween 80. Lower levels of bacteriocin production were recorded when strain R1333 was grown in MRS at 20°C. Peptide R1333 adsorbs at low levels (200 AU/mL) to producer cells. Purification of bacteriocin R1333 was performed by 60% ammonium sulfate precipitation, followed by separation on a SepPak C(18) column and reverse-phase HPLC on a Nucleosil C(18) column with a linear gradient from 0.1% TFA to 90% acetonitryl. A molecular mass of 3811 kDa was determined by mass spectrometry. Based on mass spectrometry and sequencing of the PCR amplified fragment targeting the sakG gene, L. sakei R1333 is a potential producer of sakacin G. This is the first report of the identification of sakacin G produced by L. sakei isolated from smoked salmon.

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.

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.

Characterisation of an antiviral pediocin-like bacteriocin produced by Enterococcus faecium.

The bacteriocin-producing strain Enterococcus faecium ST5Ha was isolated from smoked salmon and identified by biomolecular techniques. Ent. faecium ST5Ha produces a pediocin-like bacteriocin with activity against several lactic acid bacteria, Listeria spp. and some other human and food pathogens, and remarkably against HSV-1 virus. Bacteriocin ST5Ha was produced at high levels in MRS broth at 30 degrees C and 37 degrees C, reaching a maximum production of 1.0 x 10(9) AU/ml, checked against Listeria ivanovii ATCC19119 as target strain and surrogate of pathogenic strain Listeria monocytogenes. The molecular weight of bacteriocin ST5Ha was estimated to be 4.5 kDa according to tricine-SDS-PAGE data. Ent. faecium ST5Ha harbors a 1.044 kb chromosomal DNA fragment fitting in size to that of pediocin PA-1/AcH. In addition, the sequencing of bacteriocin ST5Ha gene indicated 99% of DNA homology to pediocin PA-1/AcH. The combined application of low levels (below MIC) of ciprofloxacin and bacteriocin ST5Ha resulted in a synergetic effect in the inhibition of target strain L. ivanovii ATCC19119. Bacteriocin ST5Ha displayed antiviral activity against HSV-1, an important human pathogen, with a selectivity index of 173. To the best of our knowledge, this is the first report on Ent. faecium as a potential producer of pediocin-like bacteriocin with antiviral activity.

Polyphasic taxonomic studies of lactic acid bacteria associated with Tunisian fermented meat based on the heterogeneity of the 16S-23S rRNA gene intergenic spacer region.

The objective of this work was to investigate the structure and diversity of lactic acid bacteria (LAB) communities in traditionally fermented meat collected from different areas of Tunisia. A polyphasic study, which involves phenotypic tests and ribosomal DNA-based techniques, was used to identify Gram-positive and catalase-negative isolates. PCR amplification of the 16S-23S rDNA ISR of 102 isolates and other reference LAB strains gave (1) one type of rrn operon (M-ISR) for lactococci, (2) two types of rrn operon (S-ISR and M-ISR) for enterococci, (3) two types of rrn operon (S-ISR and L-ISR) for Lactobacilli, and (4) three PCR amplicons (S-ISR, M-ISR, and L-ISR) obtained for Pediococcus spp. and Weissella genus. The clustering and comparison of ISR-RFLP profiles given by the isolates with those given by reference LAB strains, allowed their identification as Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis, Enterococcus sanguinicola, Enterococcus hawaiiensis, Lactobacillus sakei, Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus alimentarius, Pediococcus pentosaceus, and Weissella confusa. Combined 16S-23S rDNA ISR and RFLP patterns can be considered as a good potential target for a rapid and reliable differentiation between isolates of LAB and provided further information on the organization of their rrn operons.

Screening for anti-listerial bacteriocin-producing lactic acid bacteria from "Gueddid" a traditionally Tunisian fermented meat.

Forty eight lactic acid bacteria strains isolated from "Gueddid", a traditionally Tunisian fermented meat, were screened for bacteriocin production. Four strains named MMZ 04, 09, 13, and 17 showed antimicrobial activity and were identified as Enterococcus faecium by molecular methods based on the 16S-23S rDNA ISR, PCR-RFLP analysis of the 16S-23S rDNA ISR and species-specific primers. The four antimicrobial compounds were heat stable (121°C for 15min), active over a wide pH range (3-9) and the antimicrobial activity was lost after treatment with trypsin, α-chymotrypsin and proteinase K but not by lysozyme and lipase. The mode of action of enterocin MMZ17 was identified as bactericidal. The MMZ17 bacteriocin was partially purified by ammonium sulphate precipitation and C(18) Sep-Pack chromatography. The apparent molecular size of enterocin MMZ17 as indicated by activity detection after SDS-PAGE was lower than 6.5 KDa. According to these assays, enterocin MMZ17 can be classified as a small, heat-stable Listeria-active peptide, presumably belonging to class IIa bacteriocins.

Use of the potential probiotic strain Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers.

Campylobacteriosis is the most frequently reported zoonotic disease in humans in the EU since 2005. As chicken meat is the main source of contamination, reducing the level of Campylobacter in broiler chicken will lower the risk to consumers. The aim of this project was to evaluate the ability of Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers and to investigate the mechanisms that could be involved. Thirty broilers artificially contaminated with C. jejuni were treated by oral gavage with MRS broth or a bacterial suspension (107CFU) of Lb. salivarius SMXD51 (SMXD51) in MRS broth. At 14 and 35days of age, Campylobacter and Lb. salivarius loads were assessed in cecal contents. The impact of the treatment on the avian gut microbiota at day 35 was also evaluated. At day 14, the comparison between the control and treated groups showed a significant reduction (P<0.05) of 0.82 log. After 35days, a significant reduction (P<0.001) of 2.81 log in Campylobacter loads was observed and 73% of chickens treated with the culture exhibited Campylobacter loads below 7log10CFU/g. Taxonomic analysis revealed that SMXD51 treatment induced significant changes (P<0.05) in a limited number of bacterial genera of the avian gut microbiota and partially limited the impact of Campylobacter on Anaerotruncus sp. decrease and Subdoligranulum sp. increase. Thus, SMXD51 exhibits an anti-Campylobacter activity in vivo and can partially prevent the impact of Campylobacter on the avian gut microbiota.

Genetic diversity analysis of isolates belonging to the Photobacterium phosphoreum species group collected from salmon products using AFLP fingerprinting.

An accurate amplified fragment length polymorphism (AFLP) method, including three primer sets for the selective amplification step, was developed to display the phylogenetic position of Photobacterium isolates collected from salmon products. This method was efficient for discriminating the three species Photobacterium phosphoreum, Photobacterium iliopiscarium and Photobacterium kishitanii, until now indistinctly gathered in the P. phosphoreum species group known to be strongly responsible for seafood spoilage. The AFLP fingerprints enabled the isolates to be separated into two main clusters that, according to the type strains, were assigned to the two species P. phosphoreum and P. iliopiscarium. P. kishitanii was not found in the collection. The accuracy of the method was validated by using gyrB-gene sequencing and luxA-gene PCR amplification, which confirmed the species delineation. Most of the isolates of each species were clonally distinct and even those that were isolated from the same source showed some diversity. Moreover, this AFLP method may be an excellent tool for genotyping isolates in bacterial communities and for clarifying our knowledge of the role of the different members of the Photobacterium species group in seafood spoilage.