Isolation and typing methods for the epidemiologic investigation of thermotolerant campylobacters

Thermotolerant campylobacters, C. jejuni, C. coli, C. lari and C. upsaliensis, are spiral bacteria involved in human enteric disease. The prevalence of these emerging pathogens, mainly C. jejuni and to a lesser extent C. coli, as etiologic agents of enteric disease in industrialized countries has increased over the last decade. The isolation and culture of these microorganisms is tedious and timeconsuming mainly due to their complex nutritional and environmental requirements. This review discusses the techniques and methods developed for the selective isolation of thermotolerant campylobacters from food, environmental and clinical samples. Additionally, both traditional and newer molecular biology techniques applied to this group of thermophilic organisms for typing and taxonomic purposes are summarized

Preservative Effect of Aqueous and Ethanolic Extracts of the Macroalga Bifurcaria bifurcata on the Quality of Chilled Hake (Merluccius merluccius)

This work addressed the preservative behaviour of different icing media containing extracts from the alga Bifurcaria bifurcata. A comparative study of the antimicrobial and antioxidant effects of aqueous and ethanolic extracts of this macroalga was carried out. Whole hake (Merluccius merluccius) pieces were stored in ice containing either kind of extract and analysed for quality changes throughout a 13-day storage period. A progressive loss of microbial and biochemical quality was detected in all batches as chilling time increased. A significant inhibitory effect (p < 0.05) on microbial activity could be observed as a result of including the aqueous (lowering of psychrotrophic and lipolytic counts and pH value) and ethanolic (lowering of psychrotrophic and lipolytic counts) extracts. Additionally, both kinds of extract led to a substantial inhibition (p < 0.05) in the lipid hydrolysis rate (formation of free fatty acids), greater in the case of the batch containing ethanolic extract. Concerning lipid oxidation, a similar inhibitory effect (p < 0.05) on the formation of secondary compounds (thiobarbituric acid substances) was noticed in fish specimens corresponding to both alga extracts; however, more (p < 0.05) peroxide formation was detected in fish corresponding to the ethanolic extract batch. A preservative effect can be concluded for both kinds of extract; this effect agrees with previous studies reporting the presence of hydrophilic and lipophilic bioactive compounds in B. bifurcataThis research was funded by CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS (CSIC, Spain), GRANT NUMBER 2013-70E001S

Species Identification of Food Spoilage and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting

18 pages, 1 table, 2 figuresFood quality and safety is an increasingly important public health issue. Nowadays, the topics “food quality” and “food safety” are very close and two important issues in the food sector, due to the globalization of the food supply and the increased complexity of the food chain. The consumers need to purchase safe products that do not involve any kind of risk for health. On one hand, the aim of the “food safety” is to avoid health hazards for the consumer: microbiological hazards, pesticide residues, misuse of food additives and contaminants, such as chemicals, biological toxins and adulteration. On the other hand, “food quality” includes all attributes that influence the value of a product for the consumer; this includes negative attributes such as spoilage, contamination with filth, discoloration, off-odors and positive attributes such as the origin, color, flavor, texture and processing method of the food (FAO, 2003)This work was funded by project 10PXIB261045PR from Xunta de Galicia and by project AGL2010-19646 from the Spanish Ministry of Science and Technology. The work of K. Böhme and I.C. Fernández-No is supported by a “Maria Barbeito” and “Lucas Labrada” research contract from Xunta de GaliciaN

The effect of gelatine packaging film containing a Spirulina platensis protein concentrate on atlantic mackerel shelf life

The use of packaging films containing natural preservative compounds attracts great attention for the quality improvement of seafood. Microalga spirulina (Spirulina platensis) represents a potential source of high added-value and preservative biocompounds. The goal of this study was to enhance the quality of refrigerated Atlantic mackerel (Scomber scombrus) by including a protein concentrate (PC) of spirulina in a gelatine-based film. Quality changes in fish muscle were monitored by microbial and chemical analyses throughout an 11-day refrigerated storage (4 °C). As a result of the presence of spirulina PC in the film, an antimicrobial effect (p 0.05) was concluded for Enterobacteriaceae, proteolytics and lipolytics counts. Furthermore, a lower (p < 0.05) formation of trimethylamine and free fatty acids was detected. Lipid oxidation, measured by fluorescent compounds formation, also exhibited lower average values in fish corresponding to the batch containing spirulina concentrate. The preservative effects observed can be explained on the basis of the presence of antimicrobial and antioxidant compounds in the microalga concentrate. It is proposed that the current packaging system may constitute a novel and promising strategy to enhance the quality of commercial refrigerated fatty fish.Fil: Stejskal, Nadia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Miranda, José M.. Universidad de Santiago de Compostela; EspañaFil: Martucci, Josefa Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Ruseckaite, Roxana Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Ciencia y Tecnología de Materiales. Universidad Nacional de Mar del Plata. Facultad de Ingeniería. Instituto de Investigaciones en Ciencia y Tecnología de Materiales; ArgentinaFil: Aubourg, Santiago P.. Consejo Superior de Investigaciones Científicas; EspañaFil: Barros Velázquez, Jorge. Universidad de Santiago de Compostela; Españ

Faster monitoring of the invasive alien species (IAS) Dreissena polymorpha in river basins through isothermal amplification

Zebra mussel (Dreissena polymorpha) is considered as one of the 100 most harmful IAS in the world. Traditional detection methods have limitations, and PCR based environmental DNA detection has provided interesting results for early warning. However, in the last years, the development of isothermal amplification methods has received increasing attention. Among them, loop-mediated isothermal amplification (LAMP) has several advantages, including its higher tolerance to the presence of inhibitors and the possibility of naked-eye detection, which enables and simplifies its potential use in decentralized settings. In the current study, a real-time LAMP (qLAMP) method for the detection of Dreissena polymorpha was developed and tested with samples from the Guadalquivir River basin, together with two real-time PCR (qPCR) methods using different detection chemistries, targeting a specific region of the mitochondrial gene cytochrome C oxidase subunit I. All three developed approaches were evaluated regarding specificity, sensitivity and time required for detection. Regarding sensitivity, both qPCR approaches were more sensitive than qLAMP by one order of magnitude, however the qLAMP method proved to be as specific and much faster being performed in just 9 min versus 23 and 29 min for the qPCR methods based on hydrolysis probe and intercalating dye respectivelyThis work was supported by a partnership agreement project between the Confederación Hidrográfica del Guadalquivir and INL for the development of a system for early detection of zebra mussels through analysis of environmental DNA, and by project Nanotechnology Based Functional Solutions (NORTE-01-0145-FEDER-000019), supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund. S. A. acknowledges the Portuguese funding institution FCT – Fundação para Ciência e Tecnologia for Ph.D. scholarship SFRH/BD/140396/2018S

Evolution of Resistance in Poultry Intestinal Escherichia coli During Three Commonly Used Antimicrobial Therapeutic Treatments in Poultry

The resistance rates of intestinal Escherichia coli populations from poultry were determined during treatment and withdrawal period with 3 antimicrobial agents commonly used as therapeutics in poultry medicine. A total of 108 chickens were considered: 18 were treated orally with enrofloxacin, 18 with doxycycline, and 18 with sulfonamides, whereas another 18 chickens were maintained as controls for each antimicrobial group. Fecal samples were taken during the treatment and after the withdrawal period, and E. coli were isolated through Fluorocult media plating. A total of 648 E. coli strains (216 per antimicrobial tested) were isolated and identified though biochemical methods. Minimal inhibitory concentrations to the antimicrobials used were also determined using a broth microdilution method. The resistance rates of intestinal E. coli to all of the antimicrobials tested significantly increased during the course of the therapeutic treatment. In addition, significant differences (P = 0.0136) in resistance rates persisted between the intestinal E. coli of the enrofloxacin-treated and control batches until the end of the withdrawal period, but this difference was not observed for the cases of doxycycline or sulfonamides treatments. Antimicrobial use in poultry medicine seems to select for antimicrobial-resistant strains of pathogenic bacterial species such as E. coli. In some cases, the higher frequencies of resistant strains may persist in the avian intestinal tract until the end of the withdrawal period, when it is legal to use these animals for human consumptionThe authors wish to thank the Xunta de Galicia for granting research project (PGIDIT05TAL003E)S

Caracterización de Staphylococcus aureus aislado de productos lácteos por MALDI-TOF MS

Comunicaciones a congreso

Proteomic Characterization of Antibiotic Resistance, and Production of Antimicrobial and Virulence Factors in Streptococcus Species Associated with Bovine Mastitis. Could Enzybiotics Represent Novel Therapeutic Agents Against These Pathogens?

Streptococcus spp. are major mastitis pathogens present in dairy products, which produce a variety of virulence factors that are involved in streptococcal pathogenicity. These include neuraminidase, pyrogenic exotoxin, and M protein, and in addition they might produce bacteriocins and antibiotic-resistance proteins. Unjustifiable misuse of antimicrobials has led to an increase in antibiotic-resistant bacteria present in foodstuffs. Identification of the mastitis-causing bacterial strain, as well as determining its antibiotic resistance and sensitivity is crucial for effective therapy. The present work focused on the LC–ESI–MS/MS (liquid chromatography–electrospray ionization tandem mass spectrometry) analysis of tryptic digestion peptides from mastitis-causing Streptococcus spp. isolated from milk. A total of 2706 non-redundant peptides belonging to 2510 proteins was identified and analyzed. Among them, 168 peptides were determined, representing proteins that act as virulence factors, toxins, anti-toxins, provide resistance to antibiotics that are associated with the production of lantibiotic-related compounds, or play a role in the resistance to toxic substances. Protein comparisons with the NCBI database allowed the identification of 134 peptides as specific to Streptococcus spp., while two peptides (EATGNQNISPNLTISNAQLNLEDKNK and DLWC*NM*IIAAK) were found to be species-specific to Streptococcus dysgalactiae. This proteomic repository might be useful for further studies and research work, as well as for the development of new therapeutics for the mastitis-causing Streptococcus strainsThis work was supported by the Xunta de Galicia; the European Union Social Fund (ESF); the Spanish Ministry of Economy and Competitiveness (Project AGL 2.013-48.244-R); and by the European Regional Development Fund (ERDF) (2007-2013)S

Desired section: Sensory and nutritive qualities of food

Short title: Extended shelf life of horse mackerel in slurry ice Horse mackerel shelf-life in slurry ice Slurry ice, a biphasic system consisting of small spherical ice crystals surrounded by seawater, was evaluated in parallel to flake ice for the storage of horse mackerel (Trachurus trachurus). Storage in slurry ice implied a significant enhancement of the shelf life (5d for flake ice to 15d for slurry ice), better control of pH value, and lower counts of total aerobes, proteolytic and lipolytic bacteria, these reaching average differences between batches of 2, 1.43 and 1.98 log units, respectively, after 8 d of Slurry ice can be defined as a biphasic system consisting of small spherical ice crystals suspended in iced water at a temperature slightly above the initial freezing point of fish (0ºC to -2ºC). Among the main advantages of slurry ice, two should be highlighted: its faster chilling rate -due to its higher heat-exchange capacity-, and the limited physical damage that causes to fish food products -due to the spherical geometry of its microscopic ice crystals-. Other advantages of slurry ice derive from its complete coverage of the fish surface, which affords a better protection of the fish surface with respect to oxidation and dehydration events. Slurry ice can also be pumped, this guaranteeing a more hygienic handling of the fish products, and may be combined with other agents, such as ozone, to achieve an antiseptic surface effect, or melanosis inhibitors, to prevent browning reactions in shellfish (Huidobro and others 2002). Chapman (1990) reported a better maintenance of quality of finfish stored on-board in slurry ice as compared with other chilling methods, a result similar to that found for the on-board storage of albacore tuna by other authors (Price and others 1991). Harada (1991) also underlined the advantages of slurry ice as a pre-cooling method for fish. The scientific literature recently accounts for the use of slurry ice systems for the storage of Australian prawns (Chinivasagam and others 1998), and shrimp (Huidobro and others 2002). Other authors have also reported that slurry ice represents a good slaughter method to sacrifice and store farmed seabream (Huidobro and others 2001). Horse mackerel is a medium-fat species abundant in Northeast Atlantic In this work we have applied an advanced slurry ice system to the storage of horse mackerel (Trachurus trachurus) during 22 d, and compared with a control batch stored in parallel in conventional flake ice. With a view to investigating the shelf life of horse mackerel, here the effects of storage of horse mackerel in slurry ice on sensory and microbiological quality were investigated during 22 d. In addition, the isolation and identification of major bacteria involved in the proteolytic and lipolytic breakdown of horse mackerel muscle was also undertaken. Slurry ice and flake ice systems A slurry ice prototype (FLO-ICE, Kinarca S.A.U., Vigo, Spain) was used in the present work. The composition of the slurry ice binary mixture was 40% ice and 60% water, prepared from filtered seawater (salinity: 3.3%). The temperature of the slurry ice mixture was -1.5ºC. Flake ice was prepared with an Icematic F100 Compact device (CASTELMAC SPA, Castelfranco, Italy). Fish material, processing and sampling Specimens of horse mackerel (Trachurus trachurus) were caught during the day at a local fishing bank close to Northwestern Spain and kept on ice until they arrived at our laboratory. The fish specimens were neither headed nor gutted. The length of the specimens was in the range of 16-21 cm; the weight was in the range of 230-270 g. The fish specimens were placed in either slurry or flake ice at a fish:ice proportion of 1:1, and stored for up to 22 d in a refrigerated room at 2ºC. When required, the flake ice and the slurry ice mixture were renewed. For each chilling treatment, three different batches were used and studied separately along the whole experimental period. Samples were taken from each batch on days 0, 2, 5, 8, 12, 15, 19 and 22. Once the intact specimens had been subjected to sensory analyses, the white muscle was separated and used for microbiological and chemical analyses; all analyses were performed in triplicate. Sensory analyses 6 Sensory analyses were conducted by a taste panel consisting of five experienced judges, according to the guidelines presented in Microbiological analyses Samples of 25 g of fish muscle were dissected aseptically from chilled horse mackerel specimens, mixed with 225 ml of 0.1% peptone water, and homogenised in a stomacher (Seward Medical, London, UK) as previously described others 1998, 1999). For assays at abusive temperatures, whole fish fillets were placed inside sterile bags and kept at 30ºC for 3 d before the fish extracts were prepared. In all cases, serial dilutions from the microbial extracts were prepared in 0.1% peptone water. Total aerobes were investigated in plate count agar (PCA, Oxoid Ltd., London, UK) after incubation at 31ºC for 72 h. Anaerobes were investigated in the same way, except that an anaerobic atmosphere kit (Oxoid) was placed together with the plates inside the anaerobiosis jar. Lactose-fermenting Enterobacteriaceae (coliforms) were investigated in Violet Red Bile Agar (VRBA medium, Merck, Darmstadt, Germany) after incubation at 30ºC±1ºC for 24±2 h, as recommended by the manufacturer (Merck Microbiology Manual, 2002). Microorganisms exhibiting a proteolytic phenotype were investigated in casein-agar medium (30ºC/48 h) (Phaff and others 1994), as previously described (Ben-Gigirey and others 2000). Bacterial colonies exhibiting a lipolytic phenotype were detected in tributyrine-agar medium, as described elsewhere (Ben-Gigirey and others 2000). Chemical analyses The evolution of pH values along the storage time was carried out using a 6-mm diameter insertion electrode (Crison, Barcelona, Spain). Total volatile base-nitrogen (TVB-N) values were measured according to Aubourg and others (1997). On it, fish muscle (10 g) is extracted with perchloric acid (6%) and made up to 50 ml, the TVB-N content being determined -after steam-distillation of the acid extracts rendered alkaline to pH 13 with NaOH (20%)-by titration of the distillate with 10 mM hydrochloric acid. The results are expressed as mg TVB-N/100 g muscle. Trimethylamine-nitrogen (TMA-N) values were obtained by the Tozawa and others (1971) method, which involves the preparation of a 5% trichloracetic acid extract of fish muscle (10 g/25 ml) and reaction with picric acid. Data are expressed as mg TMA-N/100 g muscle. RESULTS AND DISCUSSION Sensory analyses According to the results of the sensory analyses Quantitative microbiological analyses a Freshness categories are as expressed in Thus, the average numbers of coliforms throughout storage were very low (&lt; 1 log CFU/g), no statistically significant (p&lt;0.05) difference being observed between both batches, and neither with respect to the initial counts at day 0. Similar low numbers of anaerobes and coliforms had been obtained by Figueroa and others (1990) in jack mackerel. It should also be remarked that the counts of total aerobes, proteolytic and lipolytic bacteria, correlated well with the differences observed in the sensory evaluation, a result that also agreed with previous reports for shrimp (Huidobro and others 2002) Chemical analyses Statistically significant (p&lt;0.05) differences were observed for the pH value in horse mackerel stored in slurry ice and flake ice With respect to the P. penneri strains, two of them -P8 and P9-produced cisteine aryl amidase, showed a weak production of extracellular lipases and proteases and almost equivalent phenotypic profiles. Isolates L5, L7 and L8 of P. penneri did not produce cisteine aryl amidase and exhibited moderate proteolytic/moderate lipolytic activity. Finally, isolate L1 did not produce cisteine aryl amidase and exhibited very 24 strong proteolytic/weak lipolytic activity. Thus, at least three different P. penneri strains -P8, L5 and L1-were isolated from horse mackerel under abusive temperature conditions. Interestingly, all P. penneri strains isolated from horse mackerel also biosynthethized and secreted alkaline phosphatase, acid phosphatase, leucine aryl amidase, naphtol-phosphohydrolase and α-glycosidase