Biofilm Formation and Morphotypes of Salmonella enterica subsp.arizonae Differs from Those of Other Salmonella enterica Subspecies in Isolates from Poultry Houses.

Salmonella serovars are responsible for foodborne diseases around the world. The ability to form biofilms allows microorganisms to survive in the environment. In this study, 73 Salmonella strains, belonging to four different subspecies, were isolated from poultry houses and foodstuffs and tested. Biofilm formation was measured at four different temperatures and two nutrient concentrations. Morphotypes and cellulose production were evaluated at three different temperatures. The presence of several genes related to biofilm production was also examined. All strains and subspecies of Salmonella had the ability to form biofilms, and 46.57% of strains produced biofilms under all conditions tested. Biofilm formation was strain dependent and varied according to the conditions. This is the first study to analyze biofilm formation in a wide number of Salmonella enterica subsp. arizonae strains, and no direct relationship between the high prevalence of Salmonella enterica subsp. arizonae strains and their ability to form biofilm was established. Morphotypes and cellulose production varied as the temperature changed, with 20°C being the optimum temperature for expression of the red, dry, and rough morphotype and cellulose. Salmonella enterica subsp. arizonae, whose morphotype is poorly studied, only showed a smooth and white morphotype and lacked the csgD and gcpA genes that are implicated in biofilm production. Thus, Salmonella biofilm formation under different environmental conditions is a public health problem because it can survive and advance through the food chain to reach the consumer.

Prevalence, molecular characterization and antimicrobial resistance of Salmonella serovars isolated from northwestern Spanish broiler flocks (2011-2015).

The present study investigated the prevalence, antimicrobial resistance to twenty antibiotics, and class 1 integron and virulence genes of Salmonella isolated from poultry houses of broilers in northwestern Spain between 2011 and 2015. Strains were classified to the serotype level using the Kauffman-White typing scheme and subtyping with enterobacterial repetitive intergenic consensus PCR. The prevalence of Salmonella spp. was 1.02%. Sixteen different serotypes were found, with S. typhimurium and S. arizonae 48:z4, z23:- being the most prevalent. A total of 59.70% of strains were resistant to at least one, and 19.70% were resistant to multiple drugs. All Salmonella spp. were susceptible to cefotaxime, ciprofloxacin, gentamicin, kanamycin, levofloxacin, neomycin, and trimethoprim. The highest level of resistance was to sulfamethoxazole (40.29%), doxycycline (17.91%), and nalidixic acid (17.91%). None of the isolates carried class 1 integron and only isolates of S. enterica subspecies enterica were positive for all virulence factors tested, whereas S. arizonae lacked genes related to replication and invasion in nonphagocytic cells. This study demonstrates that the prevalence and antimicrobial resistance of Salmonella spp. in poultry houses of broilers of northwestern Spain is low compared with those found in other studies and in other steps of the food chain.

Characterization of yeasts isolated from artisanal short-ripened cows' cheeses produced in Galicia (NW Spain).

A total of 143 presumptive yeast isolates were obtained from the predominant microflora of 21 short-ripened starter-free raw cow's milk cheeses made in Galicia (NW Spain), and the following 68 isolates were identified by both genotyping and sequencing methods: Yarrowia lipolytica (21 isolates), Kluyveromyces lactis (18), Debaryomyces hansenii (11), Pichia guilliermondii (11), Pichia fermentans (4) and Saccharomyces cerevisiae (3). Of these, Y. lipolytica and K. lactis displayed the strongest extracellular proteolytic activity on skim milk agar, and none of the D. hansenii isolates showed any activity on this medium. Y. lipolytica also displayed the highest lipolytic activity on Tween 80 and on tributyrin. This species, which was characterized by production of butanoic acid, free fatty acid esters and sulfur compounds in pasteurized whole milk, was responsible for rancid and cheesy flavors. K. lactis mainly produced acetaldehyde, ethanol, branched chain aldehydes and alcohols, and acetic acid esters, which were responsible for alcoholic, fruity and acetic notes. The volatile profiles of D. hansenii were rather limited and characterized by high levels of methyl ketones. Most of the yeast isolates were described as tryptamine producers, although low concentrations of histamine were produced by five Y. lipolytica and two P. fermentans isolates. We conclude that selected Y. lipolytica strains could be used as adjunct cultures in the manufacture of Arzúa-Ulloa and Tetilla cheeses, and selected K. lactis strains could be used as co-starters in the manufacture of acid curd Cebreiro cheese, thus contributing to the sensory quality and typicality of the cheeses.

Identification of single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of foodborne Bacillus spp.

The main goal of this work was the identification of single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of foodborne Bacillus spp. that may be useful for typing purposes. These species include, among others, Bacillus cereus, an important pathogenic species involved in food poisoning, and Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus, which are causative agents of food spoilage described as responsible for foodborne disease outbreaks. With this purpose in mind, 52 Bacillus strains isolated from culture collections and fresh and processed food were considered. SNP type "Y" at sites 212 and 476 appeared in the majority of B. licheniformis studied strains. SNP type "R" at site 278 was detected in many strains of the B. subtilis/Bacillus amyloliquefaciens group, while polymorphism "Y" at site 173 was characteristic of the majority of strains of B. cereus/Bacillus thuringiensis group. The analysis of SNPs provided more intra-specific information than phylogenetic analysis in the cases of B. cereus and B. subtilis. Moreover, this study describes novel SNPs that should be considered when designing 16S rRNA-based primers and probes for multiplex-PCR, Real-Time PCR and microarray systems for foodborne Bacillus spp.

Genetic discrimination of foodborne pathogenic and spoilage Bacillus spp. based on three housekeeping genes.

Bacillus genus includes foodborne pathogenic and spoilage-associated species, such as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis and Bacillus pumilus. Bacillus is also a heterogeneous genus that includes closely related species that are difficult to discriminate among, especially when well-conserved genes such as 16S rRNA and 23S rRNA are considered. The main goal of the present work was to study the usefulness of three housekeeping genes, the TU elongation factor (tuf), the DNA gyrase ß subunit (gyrB) and the RNA polymerase ß subunit (rpoB) genes, for use in differentiating among the most important foodborne Bacillus spp. sequences from 20 foodborne isolated Bacillus strains, and sequences belonging to different Bacillus spp. retrieved from the GenBank were analysed. In general terms, gyrB, rpoB and tuf gene regions for the strains considered in this study exhibited interspecific similarities of 57.8%, 67.23% and 77.66% respectively. Novel tufGPF and tufGPR universal primers targeted to the tuf gene were designed and proved to be useful for the amplification of all Bacillus spp considered. In conclusion, the tuf gene can be considered to be a good target for the differential characterisation of foodborne Bacillus species, especially for differentiating B. subtilis and B. cereus from other closely related species.

Detection of food spoilage and pathogenic bacteria based on ligation detection reaction coupled to flow-through hybridization on membranes.

Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific for Aeromonas spp., Pseudomonas spp., Shewanella spp., and Morganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.

Characterisation and profiling of Bacillus subtilis, Bacillus cereus and Bacillus licheniformis by MALDI-TOF mass fingerprinting.

The Bacillus genus includes species such as Bacillus cereus, Bacillus licheniformis and Bacillus subtilis, some of which may be pathogenic or causative agents in the spoilage of food products. The main goal of this work was to apply matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass fingerprinting to the classification of these Bacillus species. Genetic analyses were also compared to phyloproteomic analyses. A collection of 57 Bacillus strains isolated from fresh and processed food and from culture collections were studied and their mass spectra compiled. The resulting mass fingerprints were compared and characteristic peaks at the strain and species levels were assigned. The results showed that MALDI-TOF was a good complementary approach to 16S rRNA sequencing and even a more powerful tool in the accurate classification of Bacillus species, especially for differentiating B. subtilis and B. cereus from Bacillus amyloliquefaciens and Bacillus thuringiensis, respectively. MALDI-TOF was also found to provide valuable information at both intra- and interspecies levels in the Bacillus species studied.

Isolation and characterization of Streptococcus parauberis from vacuum-packaging refrigerated seafood products.

Streptococcus parauberis is known as an etiological agent of mastitis in cows and for producing streptococcosis in farmed fish, although its presence in foods has seldom been reported. In this work, two bacterial isolates were recovered from a spoiled vacuum-packaged refrigerated seafood product. Both isolates were identified by 16S rRNA gene sequencing, exhibiting 99% homology with respect to S. parauberis. Both isolates were also characterized by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Genetic analysis revealed the clonal homogeneity of the isolates and their grouping together with other S. parauberis strains in a different cluster with respect to Streptococcus uberis strains. Proteomic analysis by MALDI-TOF MS allowed for the identification of five mass peaks in the range of 2200-6000 m/z that resulted to be specific to the species S. parauberis and allowed its rapid and direct identification with respect to other pathogenic and spoilage bacteria potentially present in seafood and other food products. This study represents, to our knowledge, the first report of S. parauberis in seafood in general and in vacuum-packed food products in particular. Moreover, it provides a rapid method based on MALDI-TOF MS for the identification of S. parauberis.

Characterisation of histamine-producing bacteria from farmed blackspot seabream (Pagellus bogaraveo) and turbot (Psetta maxima).

Turbot (Psetta maxima) and blackspot seabream (Pagellus bogaraveo) represent two of the most important emerging farmed fish species in European countries. However, no information of the presence and development of histamine-producing bacteria on them has been reported so far. Accordingly, the aim of this study was to isolate and identify the main histamine-producing bacteria in farmed turbot and blackspot seabream. For this study, 24 isolates (12 from turbot and 12 from blackspot seabream) were preliminarily selected on Niven medium. Two of these isolates were confirmed as prolific histamine producers by HPLC. Thus, Pseudomonas fragi (isolated from turbot) and Pseudomonas syringae (isolated from blackspot seabream) were able to produce 272±69ppm and 173±45ppm of histamine in vitro, respectively, after incubation at 30°C/24h. While turbot fillets proved to be quite resistant to histamine formation at temperatures below 10°C, blackspot seabream fillets inoculated with P. syringae and the prolific histamine former Morganella morganii accumulated 696±84 and 760±59ppm histamine, respectively, under such conditions. Genetic identification based on 16S rRNA sequencing was performed in parallel with the investigation of characteristic mass spectral profiles of the isolates by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS analyses provided species-specific fingerprints, which allow rapid identification and classification of the isolates. Six genus-specific mass peaks in the range of 2218-4434 m/z were shared by both strains. Bacterial identification was achieved by the identification of six species-specific mass peaks in the ranges of 2534-7183 m/z and 2536-9113 m/z for P. fragi and P. syringae, respectively.

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR.

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R(2) values of 0.9969 and 0.9958 respectively. Linear correlations between the log(10) input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10(1) CFU/mL to 1.65 × 10(6) CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.