Evaluation of ATP bioluminescence-based methods for hygienic assessment in fish industry.

AIMS: To evaluate ATP bioluminescence-based hygiene monitoring systems under conditions relevant to fish processing environments. METHODS AND RESULTS: The ATP bioluminescence of fish fractions that are potentially present after insufficient cleaning of fish processing environments was determined. Different fractions and interfering substances representing the stages from slaughtering to smoking were prepared and measured using two different commercial systems (SystemSURE Plus and Clean-Trace). ATP bioluminescence was quenched by acidic liquid smoke and by sodium chloride even at concentrations as low as 0·9% NaCl. Large variations were observed between different types of trout homogenates: the ATP bioluminescence from raw belly fat homogenate was 100-1000 times lower than for trout blood. There were about a 1000-fold lower ATP bioluminescence in raw compared to heat-treated fractions from trout, with the exception of blood. The bioluminescence from Listeria monocytogenes was very low. Results from fish processing plants supported the laboratory findings. CONCLUSIONS: The output from ATP-monitoring instruments depends on the nature of fish soil present, as well as the presence of sodium chloride and low pH. This may lead to considerable under- or overestimation of the level of organic soil. SIGNIFICANCE AND IMPACT OF THE STUDY: ATP bioluminescence instruments are widely used by the fish industry for monitoring hygiene. The monitoring method will only give valuable information about the hygiene if critical limits are set after a validation period, distinguishing between areas with different types of soil and between different hygiene zones.

A novel library-independent approach based on high-throughput cultivation in Bioscreen and fingerprinting by FTIR spectroscopy for microbial source tracking in food industry.

Microbiological source tracking (MST) for food industry is a rapid growing area of research and technology development. In this paper, a new library-independent approach for MST is presented. It is based on a high-throughput liquid microcultivation and FTIR spectroscopy. In this approach, FTIR spectra obtained from micro-organisms isolated along the production line and a product are compared to each other. We tested and evaluated the new source tracking approach by simulating a source tracking situation. In this simulation study, a selection of 20 spoilage mould strains from a total of six genera (Alternaria, Aspergillus, Mucor, Paecilomyces, Peyronellaea and Phoma) was used. The simulation of the source tracking situation showed that 80-100% of the sources could be correctly identified with respect to genus/species level. When performing source tracking simulations, the FTIR identification diverged for Phoma glomerata strain in the reference collection. When reidentifying the strain by sequencing, it turned out that the strain was a Peyronellaea arachidicola. The obtained results demonstrated that the proposed approach is a versatile tool for identifying sources of microbial contamination. Thus, it has a high potential for routine control in the food industry due to low costs and analysis time. SIGNIFICANCE AND IMPACT OF THE STUDY: The source tracking of fungal contamination in the food industry is an important aspect of food safety. Currently, all available methods are time consuming and require the use of a reference library that may limit the accuracy of the identification. In this study, we report for the first time, a library-independent FTIR spectroscopic approach for MST of fungal contamination along the food production line. It combines high-throughput microcultivation and FTIR spectroscopy and is specific on the genus and species level. Therefore, such an approach possesses great importance for food safety control in food industry.

Microbial dynamics in mixed culture biofilms of bacteria surviving sanitation of conveyor belts in salmon-processing plants.

AIMS: The microbiota surviving sanitation of salmon-processing conveyor belts was identified and its growth dynamics further investigated in a model mimicking processing surfaces in such plants. METHODS AND RESULTS: A diverse microbiota dominated by Gram-negative bacteria was isolated after regular sanitation in three salmon processing plants. A cocktail of 14 bacterial isolates representing all genera isolated from conveyor belts (Listeria, Pseudomonas, Stenotrophomonas, Brochothrix, Serratia, Acinetobacter, Rhodococcus and Chryseobacterium) formed stable biofilms on steel coupons (12°C, salmon broth) of about 10(9) CFU cm(-2) after 2 days. High-throughput sequencing showed that Listeria monocytogenes represented 0·1-0·01% of the biofilm population and that Pseudomonas spp dominated. Interestingly, both Brochothrix sp. and a Pseudomonas sp. dominated in the surrounding suspension. CONCLUSIONS: The microbiota surviving sanitation is dominated by Pseudomonas spp. The background microbiota in biofilms inhibit, but do not eliminate L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The results highlights that sanitation procedures have to been improved in the salmon-processing industry, as high numbers of a diverse microbiota survived practical sanitation. High-throughput sequencing enables strain level studies of population dynamics in biofilm.

The effects of different hygiene procedures in reducing bacterial contamination in a model domestic kitchen.

AIMS: Few studies have compared the effectiveness of hygienic cleaning under simulated use conditions. This study compares commonly used and novel cleaning methods for food contact and hand contact surfaces in kitchens. METHODS AND RESULTS: We report results from two surveys on Norwegian consumers' cleaning procedures. Laboratory models involving cutting boards, tap handles and mobile phones contaminated with Escherichia coli and Staphylococcus aureus were used to compare the hygiene efficacy of commonly used cleaning methods together with new technologies (sprays, single-use wipes, and chlorine-based disinfectants). Commonly used cleaning methods produced a mean log10 reduction (LR) in contamination of 1.5-2.5. The efficacy could be improved by drying or including a disinfection step (mean LR 3.1-4.6). Cleaning of mobile phones was common and was improved by including humidity (1.5-1.9 mean LR). CONCLUSIONS: In many situations, traditional methods used by consumers may be sufficient to hygienically clean surfaces. However, in some situations, such as where there are infected or immune-compromised individuals, or where high risk foods are being handled, hygiene practices resulting in higher LR should be recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that data from models simulating use conditions are required to estimate the effectiveness of detergent-based removal practices and how these can be enhanced by inactivation processes such as drying and disinfection to ensure that contamination from food-borne pathogens is reduced to acceptable levels to prevent infection transmission.

The persistence of Salmonella following desiccation under feed processing environmental conditions: a subject of relevance.

UNLABELLED: Although Salmonella persistence has been predominantly linked to biofilm formation, the physiological state of Salmonella should also be considered as a possible pathway for persistence and survival in the feed industry. Hence, the purpose of this study was to assess the extent of viability of Salmonella cells through long-term desiccation periods under conditions typically found in feed processing environments, and whether these same cells could resuscitate and cause salmonellosis in vivo. We showed that upon desiccation, Salmonella Agona, a representative feed industry isolate and Salmonella Typhimurium ATCC 14028, a laboratory strain, were induced into a nonculturable state at 35 and 85% relative humidity conditions, at defined temperatures of 30 and 12°C, respectively. Although the reduction in culturable cells was more than 6 log10 , metabolic activity was found in more than 1% of the population. Desiccation-induced nonculturable Salm. Typhimurium could not be revived and were nonvirulent in a mouse model following infection through oral gavage. These results suggest that the specific conditions for reviving nonculturable Salmonella after long periods of desiccation are yet to be fully identified. The need for mapping key factors involved in the persistence of Salmonella would help better detect it and improve feed safety measures. SIGNIFICANCE AND IMPACT OF THE STUDY: While Salmonella has been shown to persist for years in feed processing environments, it is still unknown how temperature and humidity affect the persistence of Salmonella cells over time in terms of their metabolic states and cultivability. Here, we show that long-term exposure to feed processing environmental conditions induces Salmonella into a nonculturable state even though about 1% of the population remains metabolically active. This has significant implications when monitoring Salmonella from the environment which could yield false-negative results using conventional pre-enrichment detection methods.

Microbial background flora in small-scale cheese production facilities does not inhibit growth and surface attachment of Listeria monocytogenes.

The background microbiota of 5 Norwegian small-scale cheese production sites was examined and the effect of the isolated strains on the growth and survival of Listeria monocytogenes was investigated. Samples were taken from the air, food contact surfaces (storage surfaces, cheese molds, and brine) and noncontact surfaces (floor, drains, and doors) and all isolates were identified by sequencing and morphology (mold). A total of 1,314 isolates were identified and found to belong to 55 bacterial genera, 1 species of yeast, and 6 species of mold. Lactococcus spp. (all of which were Lactococcus lactis), Staphylococcus spp., Microbacterium spp., and Psychrobacter sp. were isolated from all 5 sites and Rhodococcus spp. and Chryseobacterium spp. from 4 sites. Thirty-two genera were only found in 1 out of 5 facilities each. Great variations were observed in the microbial background flora both between the 5 producers, and also within the various production sites. The greatest diversity of bacteria was found in drains and on rubber seals of doors. The flora on cheese storage shelves and in salt brines was less varied. A total of 62 bacterial isolates and 1 yeast isolate were tested for antilisterial activity in an overlay assay and a spot-on-lawn assay, but none showed significant inhibitory effects. Listeria monocytogenes was also co-cultured on ceramic tiles with bacteria dominating in the cheese production plants: Lactococcus lactis, Pseudomonas putida, Staphylococcus equorum, Rhodococcus spp., or Psychrobacter spp. None of the tested isolates altered the survival of L. monocytogenes on ceramic tiles. The conclusion of the study was that no common background flora exists in cheese production environments. None of the tested isolates inhibited the growth of L. monocytogenes. Hence, this study does not support the hypothesis that the natural background flora in cheese production environments inhibits the growth or survival of L. monocytogenes.

FTIR spectroscopic characterization of differently cultivated food related yeasts.

The application of Fourier Transform Infrared Spectroscopy for characterization of yeasts is growing rapidly. Since it is known that the phenotypic expression of yeast cells depends sensitively on the nutrients that are available in the growth medium, one standardized growth medium is usually used for identification and characterization purposes in order to obtain reproducible FTIR signals. Since our recently developed high-throughput micro-cultivation protocol has the capacity to use more than one standardized growth medium, we wanted to investigate if the parallel use of multiple growth media can improve identification results. For this purpose, five different cultivation media (YP, YPD, YMB, SAB and SD) were used. In total 91 food spoilage yeast strains of 12 different genera were cultivated in different cultivation media and subsequently characterized by FTIR spectroscopy. For spectral identifications, Radial Basis Function-Partial Least Squares (RBF-PLS) was used in combination with cross-model validation where an inner cross-validation loop was used to optimize the model, while in an outer loop an independent test set was kept aside to test the optimized model. Sensitivity and specificity were evaluated for each studied genus class. The results show that the YMB selective medium gave the best discrimination results for 9 of the 12 genera with sensitivity above 90%. Only three genera showed better identification results on other media (Clavispora and Metschnikowia on medium SD, Debaryomyces on medium YPD). We therefore suggest to use the media SD, YPD in combination with the YMB medium for the identification of food spoilage yeasts.

Characterization of food spoilage fungi by FTIR spectroscopy.

AIMS: The objective of the study was to evaluate a high-throughput liquid microcultivation protocol and FTIR spectroscopy for the differentiation of food spoilage filamentous fungi. METHODS AND RESULTS: For this study, fifty-nine food-related fungal strains were analysed. The cultivation of fungi was performed in liquid medium in the Bioscreen C microtitre plate system with a throughput of 200 samples per cultivation run. Mycelium was prepared for FTIR analysis by a simple procedure, including a washing and a homogenization step. Hierarchical cluster analysis was used to study affinity among the different species. Based on the hierarchical cluster analysis, a classification and validation scheme was developed by artificial neural network analysis. The classification network was tested by an independent test set. The results show that 93.9 and 94.0% of the spectra were correctly identified at the species and genus level, respectively. CONCLUSIONS: The use of high-throughput liquid microcultivation protocol combined with FTIR spectroscopy and artificial neural network analysis allows differentiation of food spoilage fungi on the phylum, genus and species level. SIGNIFICANCE AND IMPACT OF THE STUDY: The high-throughput liquid microcultivation protocol combined with FTIR spectroscopy can be used for the detection, classification and even identification of food-related filamentous fungi. Advantages of the method are high-throughput characteristics, high sensitivity, low costs and relatively short time of analysis.

Factors affecting survival of Shigatoxin-producing Escherichia coli on abiotic surfaces.

Shigatoxin-producing Escherichia coli (STEC) causes severe infections, and has been the cause of a number of foodborne outbreaks. Knowledge on the survival of STEC is crucial in order to limit the risk of cross contamination and transfer of STEC to food during processing. In this study survival of STEC and non-STEC on surfaces under various humidities, temperatures and in the presence of different types of soil was investigated. A model system with controlled relative humidity and temperature was established by using saturated salt solutions. All the 12 STEC strains had a reduction in viable count during incubation at 70% RH at 12 degrees C. The reduction was 2-3.5 log and 4.5-5.5 log after 1 and 7 days of incubation, respectively. Surviving cells were observed after 19 days of incubation. The STEC strains were more resistant to desiccation than non-STEC strains. STEC survived better at 12 degrees C, compared to 20 degrees C. The survival of STEC was much lower than the survival of a Staphylococcus simulans strain tested, which showed less than 1 log reduction until day 7 at 70% RH at 12 degrees C, while several STEC strains had comparable survival to a Salmonella Agona strain. The survival of two STEC strains tested was highest at 98% RH. The lowest survival was observed at 85% RH, with better survival at drier conditions. Presence of proteins and glucose protected the cells at dry conditions. Two commercial disinfectants tested at in-use concentration had limited effect (0.8-2.5 log reduction) against STEC on stainless steel, especially for cells incubated at high relative humidity (98% RH). STEC surviving on surfaces in the food industry may impose a risk for cross contamination. Cleaning and use of suitable disinfectants will reduce the survival of STEC, but surfaces should be allowed to dry completely since humid conditions will promote the survival and growth of STEC.

A synthetic furanone potentiates the effect of disinfectants on Salmonella in biofilm.

AIMS: To study a possible effect of a synthetic brominated furanone on biofilm formation and biofilm resistance to disinfectants in Salmonella enterica. METHODS AND RESULTS: The effect of a synthetic furanone on biofilm formation of Salm. enterica serovar Agona and Salm. enterica serovar Typhimurium (11 strains of different origins) was evaluated in a microtiterplate assay. A significant reduction in biofilm build-up in microtiterplates by the furanone was observed for seven of the strains tested. Biofilms by two Salm. Agona feed factory strains and the effects on survival after exposures to disinfectants (hypochlorite and benzalkonium chloride) were assessed for both strains. Pretreatment with furanone significantly potentiated the effect of the two disinfectants for both strains. CONCLUSIONS: The effect of disinfectants on Salmonella in biofilm was significantly enhanced when the biofilm was grown in the presence of furanone. This was probably because of an effect on biofilm architecture, composition and in some cases also biofilm build-up. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study gives valuable new knowledge in the fight against Salmonella biofilm in the environment because of the potentiated effect of conventional disinfectants.

Physiological and Structural Differences Between Enterococcus faecalis JH2-2 and Mutant Strains Resistant to (P)-Divercin RV41.

The aim of this study was to show the differences that could exist at the physiological and structural levels between Enterococcus faecalis JH2-2 (wild type) and three mutant strains resistant to divercin RV41. These mutant strains were recently isolated and characterized for their intermediate resistance to recombinant DvnRV41; a subclass IIa bacteriocin produced by Escherichia coli. These mutant strains were named 35A1 (altered in gene coding phosphoesterase activity), 35H1 (altered in gene coding σ(54) factor) and 36H4 (altered in gene coding glycerophosphodiesterase). The growth and resistance of each strain were tested against lysozyme. The inhibitory substance did not show any cross-resistance but exhibited an additive effect ascribed to the combined action of lysozyme and (P)-DvnRV41. The use of Fourier transform infrared spectroscopy (FT-IR) allowed to unravelling differences at the structural levels between the aforementioned strains. Thus, mutants 35H1 and 36H4 showed clear differences from mutant 35A1 and wild-type strain. These differences were located, mainly in the fatty acid region and in the polysaccharide composition. This study contributes to understanding more the resistance/sensitivity of Ent. faecalis to (P)-DvnRV41, a subclass IIa bacteriocin.

Evaluation of efficacy of disinfectants against Salmonella from the feed industry.

AIMS: To evaluate disinfectants against Salmonella under conditions relevant for the feed industry. MATERIALS AND RESULTS: A survey on the use of disinfectants in feed industry showed that a range of different types was used. Nine disinfectants, reflecting the most commonly used active ingredients, were tested for bactericidal activity on Salmonella isolated from the feed industry. All disinfectants were efficient against Salmonella in suspension. The bactericidal effect varied considerably between different types of active compounds on bacteria dried on surfaces or grown as biofilm. Tenside-based disinfectants and hypochlorite were found to have low bactericidal activity and the efficiency was significantly reduced when the ratio of amount disinfectant per cell decreased. It was shown that concentrations of 70-80% ethanol were effective against Salmonella. Among the disinfectants tested a product containing 70% ethanol was most efficient followed by Virkon S. CONCLUSIONS: Many disinfectants had low bactericidal activity against Salmonella at surfaces while Virkon S and a product containing 70% ethanol were most effective. Another advantage of ethanol-based disinfectants is evaporation of ethanol, resulting in low residual water after use. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of the disinfectants found to be efficient against surface associated Salmonella, may assist the industry in combating Salmonella.

Characterization of Serratia marcescens surviving in disinfecting footbaths.

AIM: To determine if disinfecting footbaths in the food industry were contaminated with bacteria and to characterize some of the bacteria present. METHODS AND RESULTS: Bacterial strains were isolated from disinfecting footbaths containing TEGO 103G (amphoteric disinfectant) or TP-99 (alkyl amino acetate-based disinfectant) in five of six dairy factories. Fourteen strains identified as Cedecea spp. by their fatty acid composition were further characterized. Results from Rapid ID 32 E API analysis and 16S-rDNA-sequencing showed that all strains were Serratia marcescens. Unlike S. marcescens ATCC 13880, the isolates from disinfecting footbaths were not killed (<5 log10 reduction) by the recommended in-use concentration of TEGO 103G, TEGO 51 or benzalkonium chloride. Survival and multiplication in tap water with an in-use concentration of TEGO 103G was demonstrated for one of the strains. All strains were killed by the in-use concentrations of commercial disinfectants based on peracetic acid, hypochlorite, quaternary ammonium compounds and alkyl amino acetate (TP-99). There were no indications of cross-resistance between disinfectants and antibiotics. CONCLUSION: Serratia marcescens may survive and multiply in disinfecting footbaths containing TEGO 103G or alkyl amino acetate because of disinfectant resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: Disinfecting footbaths may act as contamination sources in food factories and should not be used without regular hygienic monitoring.

Inhibition of Listeria monocytogenes in chicken cold cuts by addition of sakacin P and sakacin P-producing Lactobacillus sakei.

AIMS: To evaluate the potential of sakacin P and sakacin P-producing Lactobacillus sakei for the inhibition of growth of Listeria monocytogenes in chicken cold cuts, by answering the following questions. (i) Is sakacin P actually produced in food? (ii) Is sakacin P produced in situ responsible for the inhibiting effect? (iii) How stable is sakacin P in food? METHODS AND RESULTS: Listeria monocytogenes, a Lact. sakei strain and/or the bacteriocin sakacin P were added to chicken cold cuts, vacuum packed and incubated at 4 or 10 degrees C for 4 weeks. Each of two isogenic Lact. sakei strains, one producing sakacin P and the other not, had an inhibiting effect on the growth of L. monocytogenes. The effect of these two isogenic strains on the growth of L. monocytogenes was indistinguishable, even though sakacin P was produced in the product by one of the two Lact. sakei strains. The addition of purified sakacin P had an inhibiting effect on the growth of L. monocytogenes. A high dosage of sakacin P (3.5 microg x g(-1)) had a bacteriostatic effect throughout the storage period of 4 weeks, while a low dosage (12 ng x g(-1)) permitted initial growth, but at a slow rate. After 4 weeks of storage, the number of L. monocytogenes in the samples with a low dosage of sakacin P was 2 logs below that in the untreated control. When using a high dosage of sakacin P, the bacteriocin was detected in samples stored for up to 6 weeks. CONCLUSIONS: (i) Sakacin P is produced by a Lact. sakei strain when growing on vacuum-packed chicken cold cuts. (ii) Inhibiting effects of Lact. sakei, other than sakacin P, are active in inhibiting the growth of L. monocytogenes growing on chicken cold cuts. (iii) Sakacin P is stable on chicken cold cuts over a period of 4 weeks. SIGNIFICANCE AND IMPACT OF THE STUDY: Both sakacin P and Lact. sakei were found to have potential for use in the control of L. monocytogenes in chicken cold cuts.

Production of sakacin P by Lactobacillus sakei in a completely defined medium.

In order to investigate factors influencing the production of the bacteriocin, sakacin P, Lactobacillus sakei CCUG 42687 was grown in a completely defined medium (DML-B) with 33 components. Although the maximum sakacin P concentration obtained was higher on a complex medium due to higher cell mass, the production per cell mass was higher in DML-B. Sakacin P was produced at 4-30 degrees C, with the highest specific production at low temperatures. More sakacin P was produced at uncontrolled pH compared with cultivation at pH 6.3. Tween-80 had a positive effect on sakacin P production, while addition of sodium chloride and trace metals had negative effects. The decrease in sakacin P concentration during the late growth and stationary phases was shown to be cell-independent and promoted at high temperature and pH. Some differences in production levels of sakacin P were found among six strains of Lactobacillus sakei tested.

Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42687.

The effects of process conditions and growth kinetics on the production of the bacteriocin sakacin P by Lactobacillus sakei CCUG 42687 have been studied in pH-controlled fermentations. The fermentations could be divided into phases based on the growth kinetics, phase one being a short period of exponential growth, and three subsequent ones being phases of with decreasing specific growth rate. Sakacin P production was maximal at 20 degrees C. At higher temperatures (25-30 degrees C) the production ceased at lower cell masses, when less glucose was consumed, resulting in much lower sakacin P concentrations. With similar media and pH, the maximum sakacin P concentration at 20 degrees C was seven times higher than that at 30 degrees C. The growth rate increased with increasing concentrations of yeast extract, and the maximum concentration and specific production rate of sakacin P increased concomitantly. Increasing tryptone concentrations also had a positive influence upon sakacin P production, though the effect was significantly lower than that of yeast extract. The maximum sakacin P concentration obtained in this study was 20.5 mg l(-1). On the basis of the growth and production kinetics, possible metabolic regulation of bacteriocin synthesis is discussed, e.g. the effects of availability of essential amino acids, other nutrients, and energy.