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Ready-to-eat (RTE) leafy greens are popular products that unfortunately have been associated with numerous foodborne illness outbreaks. Since the influence of consumer practices is essential for their quality and safety, the objective of this study was to analyze the microbiota of RTE products throughout shelf life during simulated household conditions. Products from different companies were analyzed in terms of plate counts, and resealed and unopened packages were compared. High bacterial loads were found, up to a total plate count of 9.6 log

A novel and facile method is developed for rapid estimation of lactic acid bacterial concentration in fermented milk. Growth of bacteria in a liquid changes physicochemical property of the medium and its behavior at solid-liquid interface. Wettability determines characteristic of solid-liquid interface. Nano-rod, helical tetragonal and L-shaped morphologies were designed and fabricated. Hydrophobicity and zeta potential were measured for dried surfaces of 5 dairy bacterial strains. Relationship between microbial population and changes in solid-liquid interface was studied by wettability and surface free energy measurements. Due to hydrophobic surface property of conventional dairy strains, they strongly affect solid-liquid and liquid-vapor surface tensions when dispersed in a liquid, which are dependent on the bacterial concentration. Response surface methodology results showed that type and concentration of bacteria, droplet volume and solid–surface morphology affect wettability significantly. Higher hydrophobicity resulted in higher ∆θ (absolute value of the difference between the pure physiological saline and the bacterial suspension contact angles) dependence on the bacterial concentration. Probiotic bacteria concentration in fermented milk was estimated using the proposed method. A direct relationship was obtained between milk contact angle and bacterial concentration. Results show that this physical method can be applied for rapid estimation of bacterial concentration.

Pre-chilling leads to a temperature decline of the pre-rigor muscle of poultry carcasses, and a reduction of the initial bacterial load may occur. Both ultrasound (US) and slightly acidic electrolyzed water (SAEW) have been used alone in the meat industry for the manufacture of emulsions, pasteurization, and prevention of bacteria growth. However, the impact of the combination of these technologies during the pre-chilling of chicken carcasses has not been evaluated. In this study, breast chicken cylinders (CBCs) were pre-chilled for 10 min at 10 °C using SAEW and different US frequencies (25 and 130 kHz). The microbiological characteristics, lipid and protein oxidation, shear force, and anaerobic glycolysis were evaluated. The US + SAEW combination led to an effective reduction (P 0.05) of enterobacteria, mesophilic bacteria, lactic acid bacteria, and psychrotrophic bacteria, while the lipid and protein oxidation, shear force, anaerobic glycolysis, and muscle structure were not affected (P 0.05). Therefore, the combination of these technologies may be promising in the pre-chilling stage of chicken carcasses.

Bacteriophages have been envisioned as tools to control a variety of foodborne pathogenic bacteria. Salmonella is a foodborne pathogen that is a threat to public health around the world. Contaminated tomatoes have been associated with several Salmonella outbreaks. Hence, the objective of this work was to identify and characterize different lytic bacteriophages against Salmonella Newport, as one of top ten Salmonella serovars associated with human salmonellosis in North America, and then apply these phages to enhance the safety of cherry tomatoes. Four lytic phages against Salmonella Newport were selected based on their ability to lyse a majority of the 26 screened Salmonella serovars. The selected phages belong to Myoviridae (vB_SnwM_CGG4-1, vB_SnwM_CGG4-2) and Siphoviridae (vB_SnwM_CGG3-1, vB_SnwM_CGG3-2) families. They were found to be stable at different temperatures and pH, have latent periods ranging from 53 to 65 min and burst sizes from 92 to 177. In addition, the two Myoviridae phages have a lower frequency of developing bacteriophage insensitive mutants when compared with the Siphoviridae phages. No significant change in virulence gene expression was observed in the developed bacteriophage insensitive mutants when compared to the parental phage sensitive strain. Furthermore, the vB_SnwM_CGG4-1 genome revealed no homology to virulence or lysogenic genes. A phage cocktail was used to control the growth of S. Newport in broth medium and on contaminated cherry tomato. Complete inhibition of bacterial growth in broth medium was observed at 25 °C for 24 h. In addition, a 4.5 log10 unit reduction in the bacterial count was observed when applying the phage cocktail onto contaminated tomatoes stored at 22 °C for 3 days. These findings suggest that the isolated phages can be used for biocontrol of S. Newport to improve the safety of ready-to-eat (RTE) produce.

Thawed hake (Merluccius capensis and M. paradoxus) and plaice (Pleuronectes platessa) fillets were used as a model to evaluate the effect of storage temperature (0 or 10 degrees C) and biological variability (fish species, lot to lot) on bacterial growth kinetics and microbial successions. Both culture dependent methods (plate counts on nonselective and selective media) and culture independent methods (qPCR and 16S rRNA gene metabarcoding) were used. Bacterial counts exceeded 10(7) cfu/g within 2-3 days at 10 degrees C and 7-8 days at 0 degrees C. Plate counts on three media (Plate Count Agar +0.5% NaCl, Iron Agar Lyngby and Pseudomonas Selective medium) and 16S rRNA gene counts estimated by qPCR were highly correlated. Growth was modelled using the D-model and specific growth rate ranged between 0.97 and 1.24 d(-1) and 3.54 and 5.90 d(-1) at 0 and 10 degrees C, respectively. The initial composition of the microbiota showed lot-to-lot variation, but significant differences between the two fish species were detected. Alpha diversity significantly decreased during storage. When bacterial counts exceeded 10(7) cfu/g, the microbiota was dominated by members of the genera Pseudomonas, Psychrobacter, Acinetobacter, Serratia, Flavobacterium, Acinetobacter, Carnobacterium, Brochothrix and Vagococcus. However, Photobacterium and Shewanella, two genera frequently associated with fish spoilage, were either absent or minor components of the microbiota. As expected, storage temperature significantly affected the abundance of several species. The inference of microbial association networks with three different approaches (an ensemble approach using the CoNet app, Sparse Correlations for Compositional data, and SParse InversE Covariance Estimation for Ecological Association Inference) allowed the detection of both a core microbiota, which was present throughout storage, and a number of taxa, which became dominant at the end of spoilage and were characterized by a disproportionate amount of negative interactions.

The U.S. FDA Food Safety Modernization Act Preventive Controls for Human Food Rule underlines the importance of an effective environmental monitoring (EM) program. EM is used to determine harborage sites of microorganisms on processing equipment, assess effectiveness of sanitation programs, and prevent transmission of foodborne pathogens. This study characterizes commercially-available polyurethane foam (PUF) and cellulose (CELL) EM tools for their efficacy in the release of foodborne pathogens from their sponge matrices. Specifically, the objectives of this study were to 1) compare the ability of EM tools to release microorganisms into a recovery eluent, 2) characterize EM tool performance at decreasing inoculum concentrations, and 3) assess the impact of various operators during the processing of EM samples. Two bacteria (Listeria monocytogenes, Salmonella Typhimurium) and one human norovirus surrogate (Tulane virus [TV]) were compared at decreasing inoculum levels utilizing two elution techniques (mechanical stomacher, manually by operator), and across six operators. Data indicated that EM tool material composition impacted the release of microorganisms (p = 0.0001), where the PUF EM tool released TV more readily than the CELL EM tool. Conversely, the decreasing inoculum levels did not statistically differ in the release of microorganisms from the EM tool matrices. In addition, no significant difference was found between the machine stomacher and manual elution by human operator or between operators. Overall, the study provides a detailed characterization of two commercially-available EM tools, and the differences identified in this study can be used to improve the effectiveness of EM programs.

Brucellosis is one of the most prevalent zoonotic diseases with worldwide distribution. Although the incidence of brucellosis varies widely in different regions, it is a major concern for public health around the world. This study aimed to determine the prevalence and quantity of Brucella spp. in sheep and goat raw milk, as well as artisanal cheeses produced in the North-west of Iran. To evaluate the intrinsic parameters that may affect the survival of Brucella spp., some of the cheese properties (e.g., pH, salt, moisture, and storage time before selling) were also assessed. A total of 572 samples consisting in 214 sheep raw milk, 92 goat raw milk, and 266 local artisanal cheese samples were collected. The artisanal cheeses were manufactured from a mixture of raw sheep and goat milk. According to the results, using quantitative real-time PCR (qPCR), 17.29%, 15.22%, and 22.93% of the sheep raw milk, goat raw milk, and artisanal cheese samples were found positive for Brucella spp., respectively. In comparison with culture assay, qPCR was 3.5 to 5 times (p

Listeria monocytogenes has caused outbreaks of foodborne illness from apples in the USA, and is also a major issue for regulatory compliance worldwide. Due to apple's significance as an important export product from New Zealand, we aimed to determine the effect of long-term, low-temperature sea-freight from New Zealand to the USA (July) and Europe (March–April), two key New Zealand markets, on the survival and/or growth of L. monocytogenes on fresh apples. Temperature and humidity values were recorded during a shipment to each market (USA and Europe), then the observed variations around the 0.5 °C target temperature were simulated in laboratory trials using open (‘Scired’) and closed (‘Royal Gala’ for the USA and ‘Cripps Pink’ for Europe) calyx cultivars of apples inoculated with a cocktail of 107–108 cells of seven strains of L. monocytogenes. Samples were analysed for L. monocytogenes quantification at various intervals during the simulation and on each occasion, an extra set was analysed after a subsequent 8 days at 20 °C. When both the sea-freight simulations concluded, L. monocytogenes showed 5 log reductions on the equatorial surface of skin of apples, but only about 2.5 log reduction for USA and about 3.3 log reduction for Europe in the calyx. Cultivar type had no significant effect on the survival of L. monocytogenes for both sea-freight simulations, either in the calyx or on the skin (P > 0.05). Most of the reduction in the culturable cells on the skin occurred during the initial 2 weeks of the long-term storage simulations. There was also no significant difference in the reduction of L. monocytogenes at 0.5 or 20 °C. No correlation was observed between firmness or total soluble solids and survival of L. monocytogenes. Because the inoculated bacterial log reduction was lower in the calyx than on the skin, it is speculated that the risk of causing illness is higher if contaminated apple cores are eaten. The result suggested that the international sea-freight transportation does not result in the growth of L. monocytogenes irrespective of time and temperature. The results of this study provide useful insights into the survival of L. monocytogenes on different apple cultivars that can be used to develop effective risk mitigation strategies for fresh apples during long-term, low-temperature international sea-freight transportation.

Freezing broiler carcasses, industrially or at home, not only delays spoilage, but also is expected to increase food safety by hampering growth of food pathogens. However, detailed knowledge on microbial changes after a short or longer freezing period of fresh broiler meat in home freezing setting is lacking and no comparison between different freezing periods has been published yet. The present study combined classical isolation techniques and identification by MALDI-TOF MS with 16S rRNA amplicon sequencing to assess bacterial contamination on broiler carcasses that were either bought fresh and then frozen for short periods (total n = 20) in home freezing, or industrial frozen one (total n = 4) at retail. Changes in total aerobic bacteria (TAB) were also studied on 78 freshly bought broiler carcasses that were then stored frozen for up to 6 months in domestic freezers. Salmonella and Campylobacter were examined to assess the effect of freezing on controlling common foodborne pathogens. The contamination level of mesophilic and psychrotrophic TAB was numerically equal on carcasses at retail, either fresh or frozen at different time points. After short and long freezing period, a decrease in counts of mesophilic TAB was observed, while changes in counts of psychrotrophic TAB were rarely observed. No correlation between home freezing period and TAB load, either mesophilic (R = -0.006, p = 0.949) or psychrotrophic (R = 0.080, p = 0.389), was observed. No Salmonella and Campylobacter was detected on industrial frozen carcasses but on fresh carcasses at retail, either pre-freezing or after freezing. The bacterial communities were influenced by freezing, in which some genera showed significantly changes in relative abundance after freezing. In conclusion, from a food safety point of view, freezing of meat products does not serve as safety hurdle, and freezing should only be considered as a method for extending shelf life compared with fresh chicken meat. Applying hygienic slaughter procedures to keep the initial contamination as low as possible, and the maintenance of the cold chain during further processing are the key factors in food safety.

Food producing animals are considered a reservoir for Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC) producing Enterobacteriaceae. Therefore, meat is discussed to be a potential source for the transmission of these resistant bacteria to humans. There is only limited information about the quantitative load of ESBL-/AmpC-producing Enterobacteriaceae in different sample matrices during slaughter and their distribution in the slaughterhouse environment. Therefore, the aim of this study was to determine the prevalence as well as quantitative load of ESBL-/AmpC-producing Enterobacteriaceae in caecum, skin and filet samples of different broiler chicken flocks during slaughter in Germany. In addition, environmental samples were taken during slaughter of the respective flocks. To gain insights into possible transmission routes of ESBL-/AmpC-producing Enterobacteriaceae, the corresponding phylogroup and beta-lactamase genes were determined for selected isolates. ESBL-/AmpC-producing Enterobacteriaceae were detected during slaughter of all seven investigated flocks. On average, 47% (83/175) of caecum, 55% (96/175) of skin, 28% (49/175) of filet and 28% (25/89) of environmental samples harboured ESBL-/AmpC-producing Enterobacteriaceae. Prevalence varied widely between the flocks as well as between the different sample matrices. In about half of the caecum (23/40) and skin (19/40) samples as well as 85% (17/20) of the filet samples, the number of putative ESBL-/AmpC-producing Enterobacteriaceae (cefotaxime resistant Enterobacteriaceae) was below quantification limit. The median of cefotaxime resistant Enterobacteriaceae was 2.5 × 103 cfu/g in caecum, 1.5 × 103 cfu/g in skin and 1.5 × 102 cfu/g in filet samples. The median of cefotaxime resistant Enterobacteriaceae was, depending on the sample matrix, 1–4 log units below the median of total Enterobacteriaceae. Using real-time PCR, in 82% (629/767) of the cefotaxime resistant Enterobacteriaceae at least one of the investigated beta-lactamase genes blaCTX-M, blaSHV, blaTEM, blaAmpC-CIT was detected. The respective resistance genes of 322 isolates were further sequenced. The predominant bla-gene was blaCMY-2 (48%), followed by blaSHV-12 (23%). A contamination from the broiler chicken to the slaughterhouse environment and vice versa seems probable as isolates of the same species and phylogroup, encoding the same resistance genes were detected in all matrices during slaughter of the respective flock as well as in the slaughterhouse environment.

Biofilms on the surface of dairy manufacturing plants are potential reservoirs of microbial contamination. These microbial aggregates may harbour pathogenic and spoilage organisms which contaminate dairy products. The biofilm forming capacity of many spore forming isolates of dairy origin has not been given much attention. The present study explored the biofilm forming potential of 148 isolates, comprising mesophilic and thermophilic bacteria, with particular emphasis on Bacillus licheniformis on polystyrene and stainless steel (SS) surfaces. We concluded that only four species are of significance for biofilm development on the surface of SS in the presence of skimmed milk, namely, B. licheniformis, Geobacillus stearothermophilus, Geobacillus thermoleovorans group and Anoxybacillus flavithermus. The maximum number of cells recovered from the biofilms developed on SS coupons in the presence of skimmed milk for these four species was as follows: 4.8, 5.2, 4.5 and 5.3logCFU/cm2, respectively. Number of cells recovered from biofilms on 1cm2 SS coupons increased in the presence of tryptic soy broth (TSB) for all mesophiles including B. licheniformis, while decreased for G. stearothermophilus, G. thermoleovorans group and A. flavithermus. The crystal violet staining assay on polystyrene proved to be inadequate to predict cell counts on SS for the bacteria tested in our trial in the presence of either TSB or skimmed milk. The results support the idea that biofilm formation is an important part of bacterial survival strategy as only the most prevalent isolates from milk powders formed good biofilms on SS in the presence of skimmed milk. Biofilm formation also proved to be a strain-dependent characteristic and interestingly significant variation in biofilm formation was observed within the same RAPD groups of B. licheniformis which supports the previously reported genetic and phenotypic heterogeneity within the same RAPD based groups. The work reported in this manuscript will broaden our knowledge on biofilm formation of a large number of dairy isolates and emphasize strain and substrate dependence.

Raw milk may contain pathogenic microorganism that can seriously affect the health of consumers. In Southwest Ethiopia, raw cow milk is consumed more than the processed products, but its microbiological quality and its predictors are not studied well. The aim of this study was to determine the microbial quality of raw cow milk and its predictors along the dairy value chain in Southwest Ethiopia. A total of 150 milk and 300 environmental samples were collected randomly from dairy farms, milk distribution centers, and retailer outlets for microbiological analysis using standard protocols. One milk handler from each milk production or distribution stage was also interviewed to assess the knowledge, attitude, and practices regarding milk handling. Descriptive statistics and multiple linear regression models were used to summarize the data and to identify predictors of milk microbial quality, respectively. As the milk transported from dairy farm to milk retailer outlet, the mean total bacterial count has increased from 5.0 ± 0.3 to 7.2 ± 0.1 log CFU/ml respectively. The mean coliform count of the milk sample was 4.4 ± 0.4 log CFU/ml at the dairy farm and 7.0 ± 0.2 log CFU/ml at milk selling points, indicating the deterioration of milk quality along the dairy value chain. All of the analyzed water samples were positive for fecal coliform bacteria. The highest coliform bacteria were reported from milk storage equipment found at milk retailer outlet with the count of 4.8 ± 0.5 log CFU/ml. Educational status and attitude of milk handlers and the quality of water used to wash milk equipment and hands of milk handlers were the major factors affecting the microbial quality of raw cow milk. The findings of this study revealed that the microbial quality of raw milk in the study area was poor. Hence, improving the attitude and educational status of milk handlers, and the quality of water is an important step to enhance milk quality and consequently to prevent milk borne diseases.

The study examined the relative fate of the top six non-O157 shiga-toxin producing Escherichia coli (STEC) and E. coli O157:H7 during the manufacture of dry fermented sausages (DFS). Three separate batches of sausages containing a five-strain cocktail for each serogroup and uninoculated control were manufactured and subjected to identical fermentation, maturation and dry curing conditions. Changes in physicochemical properties and inoculated STEC numbers were enumerated during the DFS production stages and log reduction and log reduction rates were calculated. Inoculation of very high concentrations (8logCFUg-1) of STEC in the sausage batter did not significantly (P>0.05) affect the changes in the pH, aw, moisture, protein, fat content compared to the uninoculated DFS. There was a significant (P 0.05) from each other. These results indicate that the lethality of DFS production processes observed against E. coli O157:H7 would result in a similar inactivation of the top six non-O157 STEC.

The objective of this study was to investigate the effect of novel antimicrobial packaging materials containing poly-[2-(tertbutylamino) methylstyrene] (poly(TBAMS)) on the growth of typical spoilage and pathogenic bacteria present on meat. The antimicrobial activity of materials containing different poly(TBAMS) concentrations was determined by comparing the bacterial counts on reference and sample materials at different temperatures and times and in the presence of meat components. Storage tests with poultry fillets and veal cutlets were conducted with samples vacuum packaged in the reference foil and foil containing 10% poly(TBAMS). After specific time intervals, typical spoilage microorganisms, total viable count (TVC), sensory changes and pH value were analysed. The results of the different poly(TBAMS) containing packaging materials showed an increase of the antimicrobial activity with an increasing amount of poly(TBAMS) in the base polymer. A high antimicrobial activity against inoculum of spoilage and pathogenic organisms typical for meat products was detected of a multilayer foil containing 10% poly(TBAMS) in the inner layer after 24 h at 7 °C. Gram positive-bacteria were more sensitive to poly(TBAMS) foil than gram-negative bacteria. In storage tests however, over the entire storage, a significant effect of this poly(TBAMS) foil on microbial growth on chicken breast fillets and veal cutlets could not be identified. Poly(TBAMS) packaging materials showed very good antimicrobial properties against a wide range of bacteria. However, for a significant inhibition of microbial growth on fresh meat, a higher amount of poly(TBAMS) was necessary to prolong the shelf life of meat.

Campylobacter and Salmonella are leading causes of foodborne illnesses worldwide, vastly harboured by raw meat as their common food reservoir. Both microbes are prevalent in meat processing environments in the form of biofilms that contribute to cross-contamination and foodborne infection. This study applied raw meat juice (chicken juice and pork juice) as a minimally processed food model to study its effects on bacterial biofilm formation. Meat juice was collected during the freeze-thaw process of raw meat and sterilized by filtration. In 96-well polystyrene plates and glass chambers, supplementation of over 25% meat juice (v/v) in laboratory media led to an increase in biofilm formation of Campylobacter and Salmonella. During the initial attachment stage of biofilm development, more bacterial cells were present on surfaces treated with meat juice residues compared to control surfaces. Meat juice particulates on abiotic surfaces facilitated biofilm formation of Campylobacter and Salmonella under both static and flow conditions, with the latter being assessed using a microfluidic platform. Further, the deficiency in biofilm formation of selected Campylobacter and Salmonella mutant strains was restored in the presence of meat juice particulates. These results suggested that meat juice residues on the abiotic surfaces might act as a surface conditioner to support initial attachment and biofilm formation of Campylobacter and Salmonella. This study sheds light on a possible survival mechanism of Campylobacter and Salmonella in meat processing environments, and indicates that thorough cleaning of meat residues during meat production and handling is critical to reduce the bacterial load of Campylobacter and Salmonella.

The processing environment of salmon processing plants represents a potential major source of bacteria causing spoilage of fresh salmon. In this study, we have identified major contamination routes of important spoilage associated species within the genera Pseudomonas, Shewanella and Photobacterium in pre-rigor processing of salmon. Bacterial counts and culture-independent 16S rRNA gene analysis on salmon fillet from seven processing plants showed higher levels of Pseudomonas spp. and Shewanella spp. in industrially processed fillets compared to salmon processed under strict hygienic conditions. Higher levels of Pseudomonas spp. and Shewanella spp. were found on fillets produced early on the production day compared to later processed fillets. The levels of Photobacterium spp. were not dependent on the processing method or time of processing. In follow-up studies of two plants, bacterial isolates (n=2101) from the in-plant processing environments (sanitized equipment/machines and seawater) and from salmon collected at different sites in the production were identified by partial 16S rRNA gene sequencing. Pseudomonas spp. dominated in equipment/machines after sanitation with 72 and 91% of samples from the two plants being Pseudomonas-positive. The phylogenetic analyses, based on partial 16S rRNA gene sequencing, showed 48 unique sequence profiles of Pseudomonas of which two were dominant. Only six profiles were found on both machines and in fillets in both plants. Shewanella spp. were found on machines after sanitation in the slaughter department while Photobacterium spp. were not detected after sanitation in any parts of the plants. Shewanella spp. and Photobacterium spp. were found on salmon in the slaughter departments. Shewanella was frequently present in seawater tanks used for bleeding/short term storage.In conclusion, this study provides new knowledge on the processing environment as a source of contamination of salmon fillets with Pseudomonas spp. and Shewanella spp., while Photobacterium spp. most likely originate from the live fish and seawater. The study show that strict hygiene during processing is a prerequisite for optimal shelf life of salmon fillets and that about 90% reductions in the initial levels of bacteria on salmon fillets can be obtained using optimal hygienic conditions.

Sprouted seeds have been implicated in a number of serious outbreaks caused by Salmonella and Shiga toxinproducing Escherichia coli. Sprouts pose a very complex challenge to bacterial pathogen enrichment and detection since they naturally contain high levels of background microflora including members of theEnterobacteriaceae. As such, the currently used method cannot ensure reliable detection of STEC in sprouts. In this study, we compared different media for the enrichment of Enterobacteriaceae in their ability to promote the growth of stressed STEC at 37 °C and 42 °C. Mung bean sprouts were spiked with low levels of STEC and their growth was recorded over time. In addition, the microbiome of mung bean sprouts was analysed before and after enrichment. Our results indicate that the growth of dry-stressed STEC is comparable in all of the testedenrichment media except for mTSB + Novobiocin and not influenced by the incubation temperature. Low levels of STEC spiked into the sprouts resuspended in media only grew to levels of around 4 log cfu/ml during enrichment,which could reduce the probability of detection. Proteobacteria was the dominant phylum detected within the microbiome of non-enriched mung bean sprouts. During enrichment in EE-broth, Proteobacteria remained the most abundant phylum. In contrast, during enrichment in BPW the relative abundance of Proteobacteria decreased whereas Firmicutes increased when compared to the non-enriched mung bean sprout microbiome. The microbiome composition was not significantly influenced by the incubation temperature during enrichmentin both BPW and EE-broth. This is the first study to examine the microbiome on sprouted mung bean seeds during BPW and EE enrichment and relates the bacterial community composition changes to the enrichment of pathogens.

This study investigated the bacterial dynamics along the beef chain for clean and dirty cattle in the slaughter and processing lines, using classic quantitative methods and molecular analyses. In addition, the Norwegian national guidelines for Good Hygiene Practices in Norway were evaluated. In these guidelines, cattle presented for slaughter are categorised according to hide cleanliness, resulting in separate processing lines for meat from very dirty animals and reduced prices to farmers. The study was conducted in two commercial abattoirs in Norway. Two groups were compared; 40 visually clean cattle and 40 visually dirty cattle presented for slaughter, with 20 from each group at each abattoir. The same animals were sampled at five sampling sites: hides, carcass surfaces after dehiding, just before chilling, after chilling, and meat trimmings. Meat trimmings were sampled in only one abattoir. Three hundred and sixty samples were collected by swabbing 100 cm(2) of the brisket area at the first four sampling sites, and sampling 200 g of meat trimmings at the fifth site. The results showed that the hides of dirty cattle had more Enterobacteriaceae and higher Aerobic Plate Counts (APC) than visually clean cattle (P

Proliferation of microbial population on fresh poultry meat over time elicits spoilage when reaching unacceptable levels, during which process slime production, microorganism colony formation, negative organoleptic impact and meat structure change are observed. Spoilage organisms in raw meat, especially Gram-negative bacteria can be difficult to combat due to their cell wall composition. In this study, the natural antimicrobial agents s-poly-L-lysine (epsilon-PL) and isoeugenol were tested individually and in combinations for their activities against a selection of Gram-negative strains in vitro. All combinations resulted in additive interactions between epsilon-PL and isoeugenol towards the bacteria tested. The killing efficiency of different ratios of the two antimicrobial agents was further evaluated in vitro against Pseudomonas putida. Subsequently, the most efficient ratio was applied to a raw turkey meat model system which was incubated for 96 h at spoilage temperature. Half of the samples were challenged with P. putida, and the bacterial load and microbial community composition was followed over time.CFU counts revealed that the antimicrobial blend was able to lower the amount of viable Pseudomonas spp. by one log compared to untreated samples of challenged turkey meat, while the single compounds had no effect on the population. However, the compounds had no effect on Pseudomonas spp. CFU in unchallenged meat. Next-generation sequencing offered culture-independent insight into population diversity and changes in microbial composition of the meat during spoilage and in response to antimicrobial treatment. Spoilage of unchallenged turkey meat resulted in decreasing species diversity over time, regardless of whether the samples received antimicrobial treatment. The microbiota composition of untreated unchallenged meat progressed from a Pseudomonas spp. to a Pseudomonas spp., Photobacterium spp., and Brochothrix thermosphacta dominated food matrix on the expense of low abundance species. We observed a similar shift among the dominant species in meat treated with epsilon-PL or the antimicrobial blend, but the samples differed markedly in the composition of less abundant species. In contrast, the overall species diversity was constant during incubation of turkey meat challenged with P. putida although the microbiota composition did change over time. Untreated or epsilon-PL treated samples progressed from a Pseudomonas spp. to a Pseudomonas spp. and Enterobacteriaceae dominated food matrix, while treatment with the antimicrobial blend resulted in increased relative abundance of Hafnia spp., Enterococcaceae, and Photobacterium spp.We conclude that the blend delayed the onset of spoilage of challenged meat, and that all antimicrobial treatments of unchallenged or challenged meat affect the progression of the microbial community composition. Our study confirms that the antimicrobial effects observed in vitro can be extrapolated to a food matrix such as turkey meat. However, it also underlines the consequence of species-to-species variation in susceptibility to antimicrobials, namely that the microbial community change while the CFU remains the same. Addition of antimicrobials may thus prevent the growth of some microorganisms, allowing others to proliferate in their place. (C) 2015 Elsevier B.V. All rights reserved.

Biofilms formed on food contact surfaces are frequently exposed to disinfectants at different concentrations. This study was designed to evaluate how S. Enteritidis in single species and dual species biofilms with P. fluorescens respond to quaternary ammonium compounds (QAC) residues on food contact surfaces. The 48 h-biofilms of S. Enteritidis and P. fluorescens in single/dual species were continuously exposed to 20 ppm QAC for 5 days, followed by QAC challenge at 200 ppm and 100 ppm for attached and detached cells, respectively. Biofilm structures were observed by confocal laser scanning microscopy (CLSM) and extracellular polymeric substances (EPS)-related gene expression was also evaluated. Results showed that QAC stress led to one log lower cell counts of S. Enteritidis and P. fluorescens single species biofilms. More cellulose observed by CLSM images and increased transcript levels of cellulose-related genes (csgD, bcsA and ardA) of S. Enteritidis were induced by QAC stress. Nevertheless, high percentage of membrane damaged cells in QAC pre-exposed biofilms might contribute to the increased sensitivity of S. Enteritidis in both attached and detached cells. Previous QAC exposure did not influence S. Enteritidis viable cell counts in dual specie biofilms, in which S. Enteritidis showed strong resistance to QAC with2 log CFU/cm

Freezing vegetables requires pre-treatments to reduce microbial load and destroy enzymes that impair the frozen product quality. So far blanching has been the most effective pre-treatment, preferred by the food industry, despite its severity: heating up to temperatures close to 100 °C for 1-3 min causes sensory and texture changes in most horticultural products. Alternative blanching treatments, using UV-C radiation combined with milder thermal treatments or with thermosonication, may improve the quality of the final frozen vegetables. Zucchini (Cucurbita pepo L.), the vegetable under study, has an availability in fresh restricted to a season, needing therefore to be often frozen to be used throughout the year. In this study, its surface was first inoculated with two vegetable contaminants, Enterococcus faecalis and Deinococcus radiodurans cells, which are resistant, respectively, to high temperatures and to radiation and then submitted to several blanching treatments, single or combined, and the effect on these microorganisms reduction was evaluated. As single treatments, water blanching (the control treatment, as it is the blanching treatment traditionally used) was applied up to 180 s at temperatures ranging from 65 to 90 °C, and UV-irradiation applied in continuous. As combined pre-treatments, water blanching combined with UV-C (continuous or in pulses), and thermosonication (20 kHz at 50% of power) combined with UV-C pulses were also studied. The continuous UV-C radiation incident irradiance was 11 W/m2 up to 180 s, and the pulses at incident radiance of 67 W/m2, lasting 3.5 s each (35 pulses). Mathematical modeling of bacterial reduction data was carried out using the Bigelow, the Weibull and Weibull modified models, and estimation of their respective kinetic parameters proved that the latter models presented a better fit below 75 °C. The best results proved to be the combination of water blanching at temperatures as low as 85 °C during

Pantoea agglomerans strain CPA-2 is an effective biocontrol agent (BCA) against the major postharvest pathogens present on pome and citrus fruits. Dehydration, such as freeze-drying, spray-drying and fluidized bed drying is one of the best ways to formulate BCAs. In this work, the survival of CPA-2 cells after formulation was determined by dilution plating and molecular methods as qPCR alone and combined with a sample pretreatment with a propidium monoazide dye (PMA-qPCR) and they were used to calculate treatment concentrations in efficacy trials on postharvest oranges. Furthermore, no significant differences in CPA-2 survival were observed as determined by dilution plating and PMA-qPCR after both the freeze drying and fluidized bed drying processes; however, an interesting significant difference was observed in the spray dried product comparing all quantitative methods. A difference of 0.48 and 2.17 log10 CFU or cells g/dw was observed among PMA-qPCR with qPCR and dilution plating, respectively. According to our study, dilution plating was shown to be an unreliable tool for monitoring the survival of CPA-2 after spray drying. In contrast, the combination of PMA and qPCR enabled a quick and unequivocal methodology to enumerate viable and VBNC CPA-2 cells under stress-dried conditions. Efficacy trials showed that, after 3 days, spray drying formulation rehydrated with 10% non-fat skimmed milk (NFSM) was as effective as fresh cells to control Penicillium digitatum in oranges.

We here developed a novel loop-mediated isothermal amplification (LAMP) method to detect Streptococcus suis in raw pork meat. This method, designated LAMPSS, targeted the recombination/repair protein (recN) gene of S. suis and detected all serotypes of S. suis, except those taxonomically removed from authentic S. suis, i.e., serotypes 20, 22, 26, 32, 33, and 34. The specificity of LAMPSS was confirmed and its detection limit was 5.4cfu/reaction. Among the 966 raw pork meat samples examined, including sliced pork, minced pork, and the liver, tongue, heart, and small intestine, 255 samples tested positive with LAMPSS. The rate of contamination was higher in the organs than in pork. No significant difference was observed in the total bacterial count between LAMPSS-positive and -negative samples. The number of shops that provided LAMPSS-positive pork was slightly higher in those that sold swine organs and pork than in those that sold only pork, suggesting that cross contamination occurred from the organs to pork. Among the 255 which tested positive for LAMPSS, only 47 samples tested positive for the previously described LAMP specific for S. suis serotype 2. Two isolates of S. suis serotype 2, belonging to sequence type 28, which is potentially hazardous to humans, as well as those of some other serotypes were obtained from 19 out of 47 samples by combining LAMP with a replica plating method. These results suggest that LAMPSS will be a useful tool for the surveillance of raw pork meat in the retail market.

Little work has been reported on the use of commercial antimicrobials against foodborne pathogens on duck meat. We investigated the effectiveness of trisodium phosphate (TSP) and sodium hypochlorite (SH) as antimicrobial treatments against Campylobacter and Salmonella on duck meat under simulated commercial water chilling conditions. The results were compared to the same treatments on well-studied chicken meat. A six strain Campylobacter or Salmonella cocktail was inoculated (5 ml) at two dilution levels (10(4) and 10(8) cfu/ml) onto 25 g duck or chicken meat with skin and allowed to attach for 10 min. The meat was exposed to three concentrations of pH adjusted TSP (8, 10 and 12% (w/v), pH 11.5) or SH (40, 50 and 60 ppm, pH 5.5) in 30 ml water under simulated spin chiller conditions (4 °C, agitation) for 10 min. In a parallel experiment the meat was placed in the antimicrobial treatments before inoculation and bacterial cocktails were added to the meat after the antimicrobial solution was removed while all other parameters were maintained. Untreated controls and controls using water were included in all experiments. Bacterial numbers were determined on Campylobacter blood-free selective agar and Mueller Hinton agar or xylose deoxycholate agar and tryptone soya agar using the thin agar layer method for Campylobacter and Salmonella, respectively. All TSP concentrations significantly (p

This study quantified cefotaxime-resistant E. coli (CREC) on nine different carcass areas of 104 freshly slaughtered pig carcasses. In 49% [95% confidence interval (95% CI): 29-69%] of the carcasses CREC could be isolated and enumerated (using Tryptone Bile Agar with X-Glucuronide supplemented with 1 mg/L cefotaxime). Proportions of positive samples varied between carcass areas from 1% [95% CI: 0-10%] (loin) to 23% [95% CI: 10-44%] (head). Maximum concentrations on positive samples ranged between -0.6 log10 CFU/cm2 (loin, elbow before evisceration) and 1.7 log10 CFU/cm2 (head). The head was significantly more frequently contaminated than the loin (p = 0.027) and ham (3% [95% CI: 1-15%]). The foreleg was significantly more frequently contaminated (20% [95% CI: 13-30%]) than the ham. Combination disk diffusion assays revealed that 81% of the CREC isolates were extended-spectrum beta-lactamases (ESBL) producers, 13% were AmpC cephalosporinases (AmpC) producers and 2% ESBL and AmpC co-producers. Genotyping denoted blaCTX-M-gr1 (63%) and blaTEM (40%) as most present antibiotic resistance genes. Multiple gene combinations in one isolate and multiple combinations of genotypes and phenotypes among isolates of one sample were observed. These quantitative data can be used for intervention strategies to lower human exposure to CREC.

The European and International Standard method for the detection of Salmonella spp. in samples from the primary production stage, EN ISO 6579:2002/Amd.1:2007, was validated by an interlaboratory study in the frame of Mandate M/381, ordered by the European Commission and accepted by the European Standardisation Organisation (CEN). In addition to this study, results from two interlaboratory studies organised earlier by the European Union Reference Laboratory (EURL) for Salmonella were used for determination of the performance characteristics. Parallel to the performance evaluation for the Mandate, the revision of EN ISO 6579:2002 started. Part of this revision was the incorporation of the standardised method for detection of Salmonella in samples from the primary production stage (EN ISO 6579:2002/Amd.1:2007) and its performance characteristics in the new part 1 of EN ISO 6579. The 2002 version of EN ISO 6579 already contained performance characteristics for the detection of Salmonella in food samples, but LOD50 values (contamination level at which 50% of the samples are found positive) were not yet included. To be in line with the performance characteristics determined for detection of Salmonella spp. in samples from the primary production stage, LOD50 values for detection of Salmonella in food samples were calculated from the raw data of the validation studies performed in 2000. In this paper, the performance characteristics of EN ISO 6579-1:2017 are determined based not only on the results of the interlaboratory study carried out in 2013 under the Mandate, but also on several other interlaboratory studies. These performance characteristics consist of specificity, sensitivity and LOD50.

Microbiological analyses of lettuce, radish, carrot and beetroot were conducted to determine the effect of production system (organic and conventional) on the microbial quality of vegetables in Poland. During 2010–2014 growing seasons, 600 organic and 372 conventional samples were collected from certified farms. The vegetables were analyzed for aerobic mesophilic bacteria, yeasts and molds, Enterobacteriaceae, coliforms and Escherichia coli according to Polish standards. The farmer's survey was conducted to collect information on farm management practices. The index (from 0 – no risk to 4 – high risk) of potential contamination of the produce by human pathogens, related to fertilization system was developed. The mesophilic bacteria, yeasts and molds, coliforms and Enterobacteriaceae numbers for the radish and carrot were similar for organic and conventional cultivation systems (mesophilic bacteria 7.0 log10 cfu g−1 and 6.6 log10 cfu g−1; yeasts and molds 5.1 and 4.8; coliforms 1.3 and 1.5; Enterobacteriaceae 2.1 and 2.3 for radish and carrot respectively). Organic lettuce harbored significantly more bacteria than conventional (mesophilic 6.7 log10 cfu g−1 and 6.4 log10 cfu g−1, coliforms 1.8 and 1.4; Enterobacteriaceae 2.5 and 1.9 for organic and conventional respectively). Organic beetroot contained higher number of yeasts and molds (5.1 log10 cfu g−1) and Enterobacteriaceae (2.9 log10 cfu g−1) than conventional (4.9 and 2.5 log10 cfu g−1). The vegetables from organic farms showed significantly higher load of E. coli (on average 0.42 log10 cfu g−1) than conventionally cultivated vegetables (in average 0.05 log10 cfu g−1). The index 0–4 of potential risk of produce contamination by human pathogens was created according to fertilization practices in both farm types. Its value increased with enhanced contribution of manures and other animal wastes. In organic production the main fertilization practice was application of animal manures, composted and not composted. A popular practice was also top dressing of growing plants with fermented plant extracts, sometimes enriched with dungwater. In conventional farming system mineral fertilization was the main source of the vegetable nutrition. Therefore, organic produce indicated higher index of contamination risk (2–4) than conventional vegetables (1–2). High indexes were positively associated with higher number of E. coli. It was found that fertilization system practiced in organic farms may deteriorate sanitary quality of the produce.

Propidium monoazide (PMA) coupled with qPCR has been successfully used for specific quantification of viable bacteria cells in diverse matrices food. The present study aimed to develop PMA-qPCR assay for quantification of Lactobacillus paracasei viable cells in probiotic yoghurt. L. paracasei grown in culture medium was submitted to heat treatment at 60°C for different periods of time and probiotic yoghurt containing L. paracasei were prepared and stored at 4°C for 30days. The viable cells were quantified using qPCR and PMA-qPCR assays targeting tuf gene and also by plate counting. Standard curves were prepared and mean efficiency obtained was 94% and 96% (R2>0.98) to L. paracasei in culture medium and probiotic yoghurt stored one day, respectively. The limit of detection (LOD) for both samples was 104 genome copies, corresponding to 32.1pg of DNA. For viable cells quantification, standard curves Cq versus log CFU were plotted using mean CFU by plate counting of L. paracasei grown in culture medium and probiotic yoghurt. Results obtained for L. paracasei heat-treated cells were concordant by PMA-qPCR and plate count, CFU decreased as the heat treatment time increased, while qPCR count remained constant. L. paracasei enumerations obtained by qPCR for probiotic yoghurt stored one day and 30days were higher than enumerations by PMA-qPCR for the same samples. The plate count values were similar to CFU values obtained by PMA-qPCR. These results showed that PMA-qPCR is a powerful approach compared with culture-dependent methods for quantification of L. paracasei viable cells in yoghurt. PMA-qPCR allowed reliable obtained results much faster than plate counting.

During the last decade Campylobacter has been the most commonly reported gastrointestinal bacterial infection in humans in the European Union. The use of a sensitive detection method based on enrichment of Campylobacter spp. is often needed when examining foods. However, as background flora developed resistance to third generation β-lactams used in selective culture media, the ISO method was adapted. It now consists of three different procedures (A, B, and C) depending on the expected concentration and condition of Campylobacter and the background microflora. As the diagnostic sensitivity of the detection test varies between laboratories, this justifies the validation of the method in an interlaboratory study. The matrices selected for testing in the collaborative trials were frozen spinach (procedure A, Bolton enrichment broth), minced meat (procedure A, Bolton enrichment broth), raw milk (procedure B, Preston enrichment broth), chicken skin (procedure B, Preston enrichment broth), and broiler caecal material (procedure C, direct plating on mCCD agar). Each matrix was artificially inoculated with a different Campylobacter strain at a low and high contamination level, and with sterile diluent for 'blanks'. Seventeen laboratories participated in the interlaboratory study. The sensitivity and specificity of the methods for the five selected matrices were determined, as well as the level of detection (LOD50). Calculated LOD50 values ranged from 0.84 cfu/test portion in frozen spinach and 2.2 cfu/test portion in minced meat to 14 cfu/test portion in chicken skin and 57 cfu/test portion in raw milk, all based on test portions of 10 g. The test portion size for broiler caecal material was a 10 μl-loop, yielding a LOD50 of 6.1 cfu/test portion. The validation data were incorporated in the newly published ISO standard EN ISO 10272-1:2017 - Microbiology of the food chain - Horizontal method for detection and enumeration of Campylobacter - Part 1: Detection method.

The methods for the detection and enumeration of Enterobacteriaceae, described in EN ISO 21528, parts 1 and 2, were validated by order of the European Commission under the mandate M/381. Fourteen laboratories from seven European countries participated in the collaborative trials, organized by The Netherlands Food and Consumer Product Safety Authority (Wageningen/Utrecht, The Netherlands). Five different matrices from different food categories were selected to be tested in the collaborative trials, in order to validate the method horizontally, according to ISO 16140. The matrices included meat, tiramisu, infant formula, liquid egg, smoked salmon (detection method only) and animal feed (enumeration method only). The raw meat and liquid egg were naturally contaminated and the other matrices were artificially contaminated with a cocktail of four different Enterobacteriaceae strains. The samples used in the trial were tested for homogeneity and stability before distribution. The method for detection of Enterobacteriaceae showed a specificity and sensitivity above 95% for all matrices. The method for the enumeration had a repeatability limit r of 0.37 (expressed as a difference between log10-transformed test results) and a reproducibility limit R of 0.87 (expressed as a difference between log10-transformed test results). The validation data were incorporated in the newly published ISO standards EN ISO 21528:2017- Microbiology of the food chain - Horizontal methods for the detection and enumeration of Enterobacteriaceae - Part 1: Detection method, and Part 2: Colony-count technique.

The reference method for the detection and enumeration of L . monocytogenes in food (Standards EN ISO 11290-1&2) have been validated by inter-laboratory studies in the frame of the Mandate M381 from European Commission to CEN. In this paper, the inter-laboratory studies led in 2013 on 5 matrices (cold-smoked salmon, milk powdered infant food formula, vegetables, environment, and cheese) to validate Standard EN ISO 11290-2 are reported. According to the results obtained, the method of the revised Standard EN ISO 11290-2 can be considered as a good method for the enumeration of L. monocytogenes in foods and food processing environment, in particular for the matrices included in the study. Values of repeatability and reproducibility standard deviations can be considered satisfactory for this type of method with a confirmation stage, since most of them were below 0.3 log 10 , also at low levels, close to the regulatory limit of 100 CFU/g.

According to Regulation (EC) No 2017/1495, amending Regulation (EC) No 2073/2005, a slaughterhouse process hygiene criterion based on a limit of 103 CFU/g of Campylobacter for no more than 20 (for the years 2018 and 2019), 15 (for the years 2020–2024) and 10 (starting from 2025) of 50 neck skin samples of broiler carcasses could be an effective measure to reduce the incidence of human campylobacteriosis. In order to stimulate the poultry industry to improve the control of Campylobacter along the slaughter-line, the quantification of indicator bacteria such as Escherichia coli or Enterobacteriaceae could be a useful strategy. The aims of this study were: a) to investigate the possible relationship between Campylobacter and indicator bacteria counts at two different points of the broiler slaughter-line and b) to evaluate the probability that carcasses have Campylobacter counts above 103 CFU/g in relation to indicator bacteria counts. E. coli, Enterobacteriaceae and Campylobacter were simultaneously enumerated on neck skin samples of broiler carcasses sampled at the post-evisceration (n = 75) and at the post-chilling points (n = 75) of three Italian poultry slaughterhouses. In general, the log counts of all the investigated microorganisms were significantly lower at the post-chilling point, and the indicator bacteria (E. coli and Enterobacteriaceae) counts were significantly higher than Campylobacter counts at both sampling points. A multilevel linear mixed model, relating the Campylobacter log10 counts with the E. coli and Enterobacteriaceae log10 counts, showed that the Campylobacter log10 counts increased significantly for every additional E. coli log10 count, and this was more evident at the post-chilling than at the post-evisceration sampling point. With regards to the Enterobacteriaceae, the increase was similar at the two sampling points. An additional model, developed to assess the probability that carcasses would have Campylobacter counts above 3 log10 CFU/g, showed that this probability increased significantly if the level of E. coli count also increased. Carcasses classified as Campylobacter > 3 log10 CFU/g according to the observed results of E. coli ≥ 4 log10 CFU/g did have high Campylobacter counts. Conversely, it was not possible to conclude anything for carcasses with E. coli

Bifidobacterium bifidum is a bacterial species exclusively found in the human intestinal tract. This species is becoming increasingly popular as a probiotic organism added to lyophilized products. In this study, porcine mucin was used as the sole carbon source for the selective enumeration of B. bifidum in probiotic food additives. Thirty-six bifidobacterial strains were cultivated in broth with mucin. Only 13 strains of B. bifidum utilized the mucin to produce acids. B. bifidum was selectively enumerated in eight probiotic food supplements using agar (MM agar) containing mupirocin (100 mg/L) and mucin (20 g/L) as the sole carbon source. MM agar was fully selective if the B. bifidum species was presented together with Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium longum subsp. longum species and with lactic acid bacteria (lactobacilli, streptococci). Isolated strains of B. bifidum were identified using biochemical, PCR, MALDI-TOF procedures and 16S rRNA gene sequencing. The novel selective medium was also suitable for the isolation of B. bifidum strains from human fecal samples.

Vibrio parahaemolyticus is a major cause of foodborne illness and related with the consumption of raw contaminated seafood, especially oysters. To evaluate the effectiveness of various applications of a bacteriophage (phage), pVp-1, against a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain (CRS 09-17), we designed artificial contamination models that are most likely to be encountered during oyster processing. When live oysters were treated with bath immersion with pVp-1 after CRS 09-17 challenge, the growth of bacterial strain was significantly reduced. After 72 h of phage application with bath immersion, bacterial growth reduction was observed to be 8.9 × 10 6 CFU/ml (control group) to 1.4 × 10 CFU/ml (treatment group). When pVp-1 was surface-applied on the flesh of oysters after CRS 09-17 inoculation, bacterial growth was properly inhibited. After 12 h of phage application on the surface of oysters, bacterial growth inhibition was revealed to be 1.44 × 10 6 CFU/ml (control group) to 1.94 CFU/ml (treatment group). This is the first report, to the best of our knowledge, of oyster surface-application of a phage against a multiple-antibiotic-resistant V. parahaemolyticus pandemic strain, and our successful phage application to various situations emphasizes the potential use of the phage to avoid V. parahaemolyticus infection from aquaculture to consumption.

Current methods of monitoring the microbial ecology of food fermentation system are generally labor intensive and/or time consuming. This study developed two methods based on high-resolution melting curves (HRM) to monitor sourdough microbiota during fermentation and to investigate the effect of cereal substrate on microbial ecology. A strain cocktail of Lactobacillus fermentum FUA3165, Lactobacillus plantarum FUA3309, Lactobacillus paracasei FUA3166 and Lactobacillus reuteri FUA3168 was used to ferment red (Town and PAN8609) and white (commercial and Segaolane) sorghum sourdough, and wheat sourdough. The microbial composition of sourdoughs was determined by plate count and HRM-qPCR to differentiate at the species level. The resistance of each species to sorghum phenolic extract was measured. There was no difference in microbial composition among the four sorghum sourdoughs, with L. fermentum FUA3165 in all sourdoughs. The competiveness of the strains in sorghum sourdoughs corresponded to their resistance to sorghum phenolic extract. In a second experiment, five L. reuteri strains, the human-lineage strains FUA3400 and 3401 isolated from wheat sourdough, the rodent-lineage strain FUA5448 isolated from rye sourdough and the sorghum isolates FUA3168 and 3324, were used to ferment wheat, rye and sorghum sourdoughs. The microbial composition of sourdoughs was determined by plate counts and HRM-qPCR to different L. reuteri strains representing different host-adapted lineages. No difference among different substrates was observed; indicating cereal type had no selective effect on sourdough microbial ecology. In conclusion, HRM-qPCR assays were established as rapid and highly specific tool for monitoring of sourdough microbiota. The ability to distinguish highly similar microbes in samples containing only few genotypes makes HRM-qPCR suitable for quality control in other food fermentation systems. The presence of phenolic compounds in sorghum sourdough favored organisms with higher resistance.

Tonsils from 150 pigs slaughtered at 270 days or older were tested for Yersinia enterocolitica with different cultural methods. Samples were collected in three different abattoirs of Northern Italy between April and November 2012 and were analysed by direct plating on cefsulodin–irgasan–novobiocin (CIN) agar and by enrichment procedures following the ISO 10273:2003 reference method. Twenty-three (15.3%) samples were positive: 22 tonsils (14.7%) were positive for human pathogenic Y. enterocolitica bio-serotype 4/O:3 and one tonsil (0.7%) for Y. enterocolitica bio-serotype 1A/7,8-8,8,19. Seventeen samples out of 23 (73.9%) were positive by direct plating method. Among the enrichment procedures, the best recovery rate (8 positives out of 23; 34.8%) was obtained by the two-day enrichment in peptone–sorbitol–bile (PSB) broth followed by plating on CIN agar plates. The two-day enrichment in PSB followed by potassium hydroxide (KOH) treatment before plating onto CIN agar gave 7 positives out of 23 (30.4%), decreasing to 3 positives (13.0%) without KOH treatment. The worst results were obtained by prolonged (five days) enrichment in PSB, with or without KOH treatment, followed by plating on CIN agar: 4.3% (1 out of 23) and 0.0% recovery rates, respectively. The mean concentration was 1.9 × 104 CFU/g, with a minimum of 1.0 × 102 CFU/g and a maximum of 5.8 × 104 CFU/g, thus demonstrating that tonsils may play an important role in contamination of pluck sets, carcasses, and slaughterhouse environment. Prevalence of virulence genes among the Y. enterocolitica 4/O:3 isolates was as follows: 12/22 (54.5%) for yadA, 21/22 (95.5%) for ail, 21/22 (95.5%) for inv and 22/22 (100%) for ystA. All Y. enterocolitica 4/O:3 isolates were sensitive to amoxicillin/clavulanic acid, ciprofloxacin and ceftazidime and resistant to ampicillin and cephalotin. High proportions of 4/O:3 isolates (95%) were sensitive to cefotaxime, gentamicin, kanamicin and nalidixic acid. High levels of resistance were observed to sulphonamide compounds (91%), streptomycin (64%) and chloramphenicol (55%). Multi-resistant isolates were very common; resistance to three or more antimicrobials was observed in 91% (20/22) of 4/O:3 isolates. High level of resistance to chloramphenicol was possibly due to coresistance to tiamphenicol, which was detected in 100% of the isolates. XbaI-PFGE detected four clusters among the 22 Y. enterocolitica 4/O:3 isolates. The most represented accounted for 77% (17/22) of the isolates, the second most common was found in 14% (3/22) of the isolates and the two other profiles were observed in single isolates. The comparison with a selection of human isolates supported the role of the pig as reservoir of 4/O:3 Y. enterocolitica.

Microbial quality of frozen Nile crocodile ( Crocodylus niloticus ) meat from three farms in Zimbabwe was assessed based on 2051 samples collected for pre-export testing during 2006 to 2011. Data were perused by season and year in terms of aerobic plate (APC), coliform (CC), Escherichia coli (ECC) and Listeria monocytogenes (LMC) counts and the presence of Salmonella spp. The log 10 -transformed data were compared among the farms and seasons using the Kruskal–Wallis test. Microbial quality of the samples was graded based on the EC No. 2073.2005 criteria for beef. The mean APC and CC for the crocodile meat differed significantly ( P = 0.000) among the farms with the highest APC (3.2 ± 0.05 log 10 cfu/g) and the lowest (2.7 ± 0.05 log 10 cfu/g) recorded from farms A and C, respectively. There were no significant differences ( P > 0.05) in ECC and LMC among the farms, while Salmonella spp. were only isolated from one farm. Although the microbial quality of frozen crocodile meat from these farms was generally within acceptable limits, the isolation of E. coli and Salmonella spp. is of public health concern. Thus, implementing of measures to control the pasteurizing process and to minimize bacterial contamination of crocodile meat after pasteurization need to be carefully considered.

A major community outbreak of salmonellosis occurred in France in October 2010. Classical epidemiological investigations led to the identification of beef burgers as the cause of the outbreak and the presence of the emerging monophasic Salmonella Typhimurium 4,5,12:i:−. The objective of this study was to understand the events that led to this large outbreak, that is to say, what are the contributing factors associated with consumer exposure to Salmonella. To this end, intensive microbiological investigations on several beef burgers were conducted and a risk assessment model was built. The microbiological results confirm the presence of Salmonella in all analysed frozen burgers at high levels of contamination above 1000 MPN/g. These results in frozen burgers combined with a model of thermal destruction were used to estimate the dose ingested by the exposed persons. Most people that consumed cooked beef burgers were exposed from 1.6 to 3.1 log10 (MPN). The number of sick people predicted with a dose–response relationship for Salmonella is consistent with the observed number of salmonellosis cases. The very high initial contamination level in frozen beef burgers is the primary cause of this large outbreak rather than bad cooking practices. Intensive investigations, modelling of the initial contamination and quantitative exposure and risk assessments are complementary to epidemiological investigation. They can be valuable elements for the assessment of missing information or the identification of the primary causes of outbreaks.

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H′ L = 0.4 and H′ Y = 0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis , Lactobacillus plantarum , and Leuconostoc mesenteroides , while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis , Enterococcus faecalis , and Leuconostoc pseudomesenteroides . Among the 20 yeast species found, Debaryomyces hansenii , Candida zeylanoides , and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.

The objectives of this study were to evaluate the effect of different controlled atmospheres, containing 3% O2+10% CO2, 5% O2+10% CO2 and 8% O2+10% CO2, on changes in microbial population of 'Ambrunés' sweet cherries throughout storage during 30days and subsequent shelf-life, as well as to identify the main genera of yeast, mould, lactic acid bacteria, Staphylococcus spp., Pseudomonas spp., Enterobacteriaceae spp., and coliforms. The results indicated that controlled atmospheres with 5% O2+10% CO2 and 8% O2+10% CO2 were highly effective to control the growth of mesophilic aerobic bacteria, psychrotrophs, Pseudomonas spp., yeasts, and moulds after 15days of storage, showing, in the case of yeasts and moulds, counts that ranged between1 and 1.75logCFU/g. The genera of yeasts, moulds, lactic acid bacteria, Enterobacteriaceae spp., and coliforms identified were Aureobasidium spp., Penicillium spp., Leuconostoc spp., and Rahnella spp., respectively. In addition, the genera Staphylococcus spp. and Pseudomonas spp. were also identified. On the other hand, cherries of Stage 3 ripening presented the highest counts for all microbial groups.

Cheddar cheese is a complex ecosystem where both the bacterial population and the cheese making process contribute to flavor and texture development. The aim of this study was to use molecular methods to evaluate the impact of milk heat treatment and ripening temperature on starter lactococci and non-starter lactic acid bacteria (NSLAB) throughout ripening of Cheddar cheese. Eight Cheddar cheese batches were manufactured (four with thermized and four with pasteurized milk) and ripened at 4, 7 and 12°C to analyze the bacterial composition and rRNA transcriptional activity reflecting the ability of lactococci and lactobacilli to synthesize proteins. Abundance and rRNA transcription of lactococci and lactobacilli were quantified after DNA and RNA extraction by using quantitative PCR (qPCR) and reverse transcription-quantitative PCR (RT-qPCR) targeting the 16S rRNA gene, respectively. Results showed that lactococci remained dominant throughout ripening, although 16S rRNA genome and cDNA copies/g of cheese decreased by four and two log copy numbers, respectively. Abundance and rRNA transcription of Lactobacillus paracasei, Lactobacillus buchneri/parabuchneri, Lactobacillus rhamnosus, Lactobacillus brevis, and Lactobacillus coryniformis as well as total lactobacilli were also estimated using specific 16S rRNA primers. L. paracasei and L. buchneri/parabuchneri concomitantly grew in cheese made from thermized milk at 7 and 12°C, although L. paracasei displayed the most rRNA transcription among Lactobacillus species. This work showed that rRNA transcriptional activity of lactococci decreased throughout ripening and supports the usefulness of RNA analysis to assess which bacterial species have the ability to synthesize proteins during ripening, and could thereby contribute to cheese quality.

Blood-yolk-polymyxin B-trimethoprim agar (BYPTA) was developed by the addition of egg yolk, laked horse blood, sodium pyruvate, polymyxin B, and trimethoprim, and compared with mannitol-yolk-polymyxin B agar (MYPA) for the isolation and enumeration of Bacillus cereus (B. cereus) in pure culture and various food samples. In pure culture, there was no statistical difference (p0.05) between the recoverability and sensitivity of MYPA and BYPTA, whereas BYPTA exhibited higher specificity (p0.05). To evaluate BYPTA agar with food samples, B. cereus was experimentally spiked into six types of foods, triangle kimbab, sandwich, misugaru, Saengsik, red pepper powder, and soybean paste. No statistical difference was observed in recoverability (p0.05) between MYPA and BYPTA in all tested foods, whereas BYPTA exhibited higher selectivity than MYPA, especially in foods with high background microflora, such as Saengsik, red pepper powder, and soybean paste. The newly developed selective medium BYPTA could be a useful enumeration tool to assess the level of B. cereus in foods, particularly with high background microflora.

In the present study, eggs from commercial caged layer flocks at different stages of lay in Australia were collected. Enterobacteriaceae populations from eggshell surface and eggshell pore were enumerated and these populations characterized using API® Rapid 20E strips. The eggshell surface, eggshell pore and egg internal content samples were also processed for the isolation of Salmonella and these isolates were tested for the presence or absence of several virulence genes (prgH, sopB, spiC, orfL, invA, sifA, sitC, misL). Results indicated that there was no significant difference in total Enterobacteriaceae count on the eggs of the flock from early, mid or late lay flocks. Enterobacteriaceae isolates were of 11 different genera which included: Cedecea, Citrobacter, Enterobacter, Escherichia, Klebsiella, Kluyvera, Leclercia, Pantoea, Salmonella, Serratia and Yersinia. Out of all 153 identified Enterobacteriaceae isolates, the Escherichia genus was reported most frequently (60.78%). Results also indicated that overall there were 4.51% (14/310) Salmonella positive pooled samples. In this study, 14 Salmonella strains were isolated, serotyping confirmed that 12 out of them were Salmonella Infantis and the 2 others were Salmonella enterica subsp. enterica serovar 4,12:d: Polymerase chain reaction results indicated that all Salmonella Infantis isolates harboured invA, misL, orfL, prgH, sifA, sitC, sopB and spiC genes which suggests that Salmonella Infantis strains isolated from eggshell surface may have the capacity to invade and survive in macrophages.

The survival of eight undesired (spoilage/pathogenic) food related bacteria (Citrobacter freundii PSS60, Enterobacter spp. PSS11, Escherichia coli PSS2, Klebsiella oxytoca PSS82, Serratia grimesii PSS72, Pseudomonas putida PSS21, Stenotrophomonas maltophilia PSS52 and Listeria monocytogenes ATCC 19114T) was investigated in mineral nutrient solution (MNS) during the crop cycle of radishes (Raphanus sativus L.) cultivated in hydroponics in a greenhouse. MNSs were microbiologically analyzed weekly by plate count. The evolution of the pure cultures was also evaluated in sterile MNS in test tubes. The inoculated trials contained an initial total mesophilic count (TMC) ranging between 6.69 and 7.78 Log CFU/mL, while non-sterile and sterile control trials showed levels of 4.39 and 0.97 Log CFU/mL, respectively. In general, all inoculated trials showed similar levels of TMC in MNS during the experimentation, even though the levels of the inoculated bacteria decreased. The presence of the inoculums was ascertained by randomly amplified polymorphic DNA (RAPD) analysis applied on the isolates collected at 7-day intervals. At harvest, MNSs were also analyzed by denaturing gradient gel electrophoresis (DGGE). The last analysis, except P. putida PSS21 in the corresponding trial, did not detect the other bacteria, but confirmed that pseudomonads were present in un-inoculated MNSs. Despite the high counts detected (6.44 and 7.24 CFU/g), only C. freundii PSS60, Enterobacter spp. PSS11 and K. oxytoca PSS82 were detected in radishes in a living form, suggesting their internalization.