Cross-contamination of mature Listeria monocytogenes biofilms from stainless steel surfaces to chicken broth before and after the application of chlorinated alkaline and enzymatic detergents.

The objectives of this study were, firstly, to compare a conventional (i.e., chlorinated alkaline) versus an alternative (chlorinated alkaline plus enzymatic) treatment effectivity for the elimination of biofilms from different L. monocytogenes strains (CECT 5672, CECT 935, S2-bac and EDG-e). Secondly, to evaluate the cross-contamination to chicken broth from non-treated and treated biofilms formed on stainless steel surfaces. Results showed that all L. monocytogenes strains were able to adhere and develop biofilms at approximately the same growth levels (≈5.82 log CFU/cm2). When non-treated biofilms were put into contact with the model food, obtained an average transference rate of potential global cross-contamination of 20.4%. Biofilms treated with the chlorinated alkaline detergent obtained transference rates similar to non-treated biofilms as a high number of residual cells (i.e., around 4 to 5 Log CFU/cm2) were present on the surface, except for EDG-e strain on which transference rate diminished to 0.45%, which was related to the protective matrix. Contrarily, the alternative treatment was shown to not produce cross-contamination to the chicken broth due to its high effectivity for biofilm control (<0.50% of transference) except for CECT 935 strain that had a different behavior. Therefore, changing to more intense cleaning treatments in the processing environments can reduce risk of cross-contamination.

Removal of Listeria monocytogenes biofilms on stainless steel surfaces through conventional and alternative cleaning solutions.

Conventional treatments are not effective enough to completely remove Listeria monocytogenes biofilms from surfaces, thus implying the presence of certain persistent bacterial forms. In this study, eleven treatments (i.e. two enzymatic agents applied at two different temperatures and concentrations, two alkaline cleaners and one acid detergent) were used to remove mature L. monocytogenes S2-bac biofilms. A combined treatment was then selected for its application to four different L. monocytogenes strains (i.e. CECT 5672, CECT 935, S2-bac, EDG-e). Effectivity of the treatments was evaluated quantitatively using TEMPO and qualitatively by direct epifluorescent microscopy (DEM). Bacterial detachment obtained after the application of acid, alkaline and chlorinated alkaline treatments were 6.03, 6.24 and 4.76 Log CFU/cm2, respectively. Enzymatic treatments applied at 50 °C obtained the greatest detachment and biocidal activity. The results derived from the observation of the remaining biofilm structure by DEM proved that conventional treatments were unable to completely remove conformed structures with the potential risk this entails. Last, the application of a combined treatment using a chlorinated alkaline cleaner followed by an enzymatic treatment enhanced the dispersal of the bacterial cells from surfaces, thus consolidating this as a good option to recommend for the 5-step cleaning procedure.

Dual-species biofilms formation between dominant microbiota isolated from a meat processing industry with Listeria monocytogenes and Salmonella enterica: Unraveling their ecological interactions.

Alternatives to combat the persistence of pathogens need to consider the microbiota established on industrial surfaces as they can influence the protection or replacement (i.e. reduction/inhibition) of pathogens. The objective of the present study was to determine the ecological interactions established in dual-species biofilms between Listeria monocytogenes and Salmonella enterica as target pathogens, and isolates recovered from a meat processing facility (i.e.Pseudomonas fluorescens, Pseudomonas fragi, Bacillus safensis, Bacillus megaterium, and Candida zeylanoides). Results showed different ecological relations in biofilms depending on the species evaluated. Pseudomonas spp. did not influence the growth of either pathogen, although tested species tended to protect the pathogens in the structures generated. B. megaterium and C. zeylanoides affected the two pathogens differently, demonstrating a reduction of L. monocytogenes adhered cells within the formed biofilm. B. safensis reduced or presented non-influence on S. enterica depending on the incubation conditions. Contrarily, B. safensis was the microorganism that demonstrated the highest replacement capacity for L. monocytogenes, reducing its growth by up to 4 log CFU/cm2. The in vitro study of bispecies biofilms is important for the food industry, helping to understand how they behave and to find an effective way to eliminate them.

New approach for the removal of mature biofilms formed by wild strains of Listeria monocytogenes isolated from food contact surfaces in an Iberian pig processing plant.

One of the main objectives of the food industry is to guarantee food safety by providing innocuous food products. Therefore, this sector must consider all the possible biotic or abiotic contamination routes from the entry of raw materials to the release of the final product. Currently, one important problem in this regard is the presence of biofilms on food contact surfaces which can transmit pathogens such as L. monocytogenes. In industrial conditions biofilms are found in a mature state, so it is essential that when carrying out removal effectiveness studies in vitro the tests are realized with models that produce these structures in a similarly mature state. The main objective of this study was to evaluate the effectiveness of an alternative treatment (i.e. enzymatic detergent that include natural antimicrobial agents) and a conventional treatment (i.e. chlorinated alkaline) for the elimination of mature L. monocytogenes biofilms. The results showed a cell detachment from the formed mature biofilms with an effectivity of between 74.75%-97.73% and 53.94%-94.02% for the enzymatic treatment and the chlorinated alkaline detergent, respectively. On a qualitative level, it was observed that the dispersion in the structure was much higher for the enzymatic treatment than for the chlorinated alkaline, which continued to show obvious structure integrity. All this leads to the conclusion that treatments with an enzymatic detergent have a significantly greater impact on the removal of mature L. monocytogenes biofilms, although a further disinfection process would be needed, enhancing even more the treatment effectivity. This may imply that the industrial approach to addressing this problem should be modified to include new perspectives that are more effective than traditional ones.

Detection of Salmonella Typhimurium and Listeria monocytogenes biofilm cells exposed to different drying and pre-enrichment times using conventional and rapid methods.

The capacity of real-time PCR (RT-PCR), the VIDAS immunoassay system, and the conventional count method for detecting Salmonella enterica serovar Typhimurium and Listeria monocytogenes biofilm cells was evaluated in this study. After biofilm formation, tests were performed under different drying times (0, 6, 12, 24, and 72 h) and pre-enrichment times (0, 6, 18, and 25 h). The direct epifluorescence microscopic results demonstrated that Salmonella Typhimurium and L. monocytogenes biofilm cells can remain viable for 72 h under drying conditions. Pre-enrichment time and type of medium played an essential role in the detection of both microorganisms after drying. Furthermore, RT-PCR was more sensitive than VIDAS and the conventional method for detecting Salmonella Typhimurium and L. monocytogenes cells at different drying times and without pre-enrichment (0 h), with a detection range between 102 and 107 CFU/mL. TSBYE-T80 used as a pre-enrichment medium was effective for detecting both bacteria and was more effective than Demi Fraser-T80 medium for detecting L. monocytogenes. Therefore, pre-enrichment is recommended to avoid false positives and false negatives due to the presence of dead cells or a very low initial concentration of cells after drying.

Quantification of mature Listeria monocytogenes biofilm cells formed by an in vitro model: A comparison of different methods.

The presence of biofilms in food industrial environments is one of the main causes associated with food product contamination by L. monocytogenes. Biofilm control in the food industry is very relevant to public health and finding reliable and realistic quantification methods is essential. The aim of this study is to compare five L. monocytogenes biofilm quantification methods - conventional plate count, TEMPO, DEM, VIDAS and qPCR - and to examine a biodetector to visually detect biofilms in industrial settings. Results show that depending on the biofilm matrix production, the recovery of cells that conform the biofilm can be low and therefore, if it is an indirect method, microbial counts can be underestimated. At a species level, the methods that did not present significant differences were plate count, TEMPO (P = 0.998), DEM and qPCR (P = 0.508), so correlation studies were performed which established high correlation for plate count and TEMPO, but not for DEM and qPCR. The VIDAS method was adjusted so that it could quantify the biofilms, but the standard curve only allowed counts from 7 Log CFU cm-2. Results also revealed that the different strains of L. monocytogenes possess different biofilm-forming abilities, although it was not possible to correlate the capacity to produce these structures with the distinct serotypes. Last, visually detecting biofilms on stainless steel coupons proved that in industrial environments nowadays they can be rapidly and qualitatively detected so that relevant decisions can immediately be taken.

Monitoring volatile and nonvolatile amines in dried and salted roes of tuna (Thunnus thynnus L.) during manufacture and storage.

Dried and salted roe, obtained from the reproductive organs of female tuna (Thunnus tynnus L.), is a typical fish-based food in the Mediterranean area of Spain. In the present study, we monitored the formation of volatile amines (trimethyamine nitrogen [TMA-N] and total basic volatile nitrogen [TBVN]) and nonvolatile amines (biogenic amines) in dried and salted tuna roe after processing and storage for 8 weeks at 4, 20, and 30 degrees C. The salting and drying process significantly increased the TBVN, cadaverine, tyramine, phenylethylamine, and tryptamine contents, and bacteria with histamine decarboxylase activity were detected both in raw and in dried and salted tuna roes. During storage of tuna roe, TMA-N and TBVN levels increased significantly after the fourth week of storage at 30 degrees C, whereas biogenic amine contents remained more or less constant. However, samples stored at 30 degrees C showed histamine formation after the first week of storage, with a concentration of < 50 ppm. The volatile and nonvolatile amine concentrations in tuna roe were below the consumer safety limit, with the exception of the total biogenic amine level in roe stored at 30 degrees C, which exceeded the European Community's recommended limit (300 ppm). These results indicate that in properly stored tuna roe, histamine formation will not represent a serious health risk to consumers unless the tuna roe has previously been mishandled.

Protein hydrolysis and proteinase activity during the ripening of salted anchovy (Engraulis encrasicholus l.). A microassay method for determining the protein hydrolysis.

The protein hydrolysis and proteinase activity during the ripening of salted anchovy were studied. A rapid, simple, and inexpensive microassay method for determining the protein hydrolysis by trinitrobenzenesulfonic acid (TNBS) has been developed. A linear relationship was observed between proteolysis determination by the TNBS method and ripening time in the fish muscle and in the brine (r = 0.99). A linear relationship was also observed between the ratio nonprotein nitrogen and total nitrogen (NPN/TN) and ripening time (r = 0.98). Proteolysis by the TNBS method and NPN/TN determination could be considered as objective methods to follow and assess the ripening process of an anchovy. A value of proteolysis by the TNBS method of 240 mM leucine in the fish muscle and/or 200 mM leucine in the brine would indicate the ripening point. The crude enzyme prepared of fish muscle and brine showed that alkaline proteinases dominate.

Halotolerant and halophilic histamine-forming bacteria isolated during the ripening of salted anchovies (Engraulis encrasicholus).

This study was performed to investigate halotolerant and halophilic histamine-producing bacteria isolated during the ripening of salted anchovies. Of the isolates obtained during the ripening of anchovies, 1.37% showed histamine-forming activity, most of them (70%) belonging to the Staphylococcus genus. S. epidermidis showed a powerful histamine-forming activity, producing more than 1,000 microg/ml in the presence of 3% and 10% NaCl. Another powerful histamine-producing bacterium isolated during the ripening of salted anchovies was S. capitis. It was able to produce about 400 microg/ml of histamine in 10% NaCl under experimental conditions. Most of these species might be expected to be found as a result of contamination of fish during capture and subsequent unhygienic handling. However, no increase in histamine content was found in any batches through the ripening process. Histamine content always was acceptable in accordance with the maximum allowable levels of histamine fixed by the Spanish and European Union regulations.

Evaluation of three decarboxylating agar media to detect histamine and tyramine-producing bacteria in ripened sausages.

Histidine- and tyrosine-decarboxylase activity of 175 strains of bacteria isolated from eight retail samples of Spanish ripened sausages was tested in three decarboxylating agars (Niven medium, Joosten and Northolt medium and modified decarboxylating agar of Maijala) and confirmed by an enzymic method (histamine) and thin-layer chromatography (tyramine). Enterobacteria and pseudomonads showed the highest percentage of positive responses to histamine and tyramine in the three decarboxylating agars, but only enterobacteria were subsequently confirmed as histamine-producing. Confirmed tyramine-producing strains were all identified as enterococci or lactic acid bacteria. The medium described by Joosten and Northolt was more sensitive and faster at detecting tyramine-producing microorganisms. However, all three media failed to detect one histamine-positive strain of lactic acid bacteria used as a control.

Incidence of histamine-forming bacteria and histamine content in scombroid fish species from retail markets in the Barcelona area.

Incidence and diversity of histidine decarboxylating bacteria were determined in samples of tunafish, bonito and mackerel purchased at different retail markets. Histamine-forming bacteria occurred in a low proportion and always accounted for less than 0.1% of the total bacterial load in the fish samples studied. Similarly, histamine content in fish samples also was low ( < 25 ppm) and all of them met current histamine standards established by the European Union. Histamine was found in 83.3% of the tested tunafish samples with an average of 8.9 ppm. In contrast, none of mackerel samples and only 2 out of 12 of bonito showed detectable amounts of histamine. Morganella morganii and Klebsiella oxytoca were the most active histamine formers under experimental conditions, and produced on average 2765 and 1415 ppm of histamine, respectively, after incubation at 37 degrees C for 18 h. Some new histamine formers, such as Plesiomonas shigelloides, Enterobacter intermedium, Serratia marcescens, Serratia plymuthica and Serratia fonticola, have been identified. Especially Plesiomonas shigelloides would have an important role within histidine decarboxylating bacteria because it was the sole histamine former isolated that has frequently been associated with the marine aquatic environment. However, only 8-340 ppm of histamine was formed by these strains in laboratory trials.

Effect of chill and freezing temperatures on survival of Vibrio parahaemolyticus inoculated in homogenates of oyster meat.

Survival of Vibrio parahaemolyticus was determined in oyster meat homogenates at various temperatures. (4 degrees C, 0 degrees C, -18 degrees C and -24 degrees C) and bacterial levels (10(2), 10(4), 10(5) and 10(7) ml-1). In all cases, the numbers of V. parahaemolyticus were a logarithmic function of log time. This study indicates that high numbers of V. parahaemolyticus can be inactivated at low temperatures. The time of total inactivation depends on the initial number of micro-organisms and incubation temperature. It is possible to use this information to determine the storage time necessary to reduce V. parahaemolyticus hazards in fish.

A Modification of Lerke Enzymic Test for Histamine Quantification.

A modification of Lerke enzymic method for histamine determination has been realized. We have observed a modification of the optimum wavelength depending on incubation time at constant temperature of 37°C. A wavelength of 580 nm is recommended, with a incubation time of 15 min. At these conditions the linearity was observed among 1 and 25 ppm (µg/g) of histamine. This technique is a reliable method of screening in food samples and bacterial strains, with low sample preparations requirements, a short incubation time and a low cost.

Histidine, Lysine and Ornithine Decarboxylase Bacteria in Spanish Salted Semi-preserved Anchovies.

We have studied the histidine decarboxylase activity in 118 strains of bacteria isolated from commercial samples of Spanish semi-preserved anchovies. The lysine and ornithine qualitative decarboxylase activity was also studied. The microorganism that presented the highest histamine activity was Morganella morganii , with 2123.26 ± 414.00 ppm of histamine after 24 h of incubation at 37°C. Two strains of Bacillus spp. and a strain of Staphylococcus xylosus were isolated with the capacity to form 10.54 and 110.00 ppm of histamine, respectively. However, the histidine decarboxylase activity of Bacillus spp. is not likely to be significant to human health. The microbic species with capacity to form histamine and those with capacity to form other biogenic amines were similar. Therefore, the prevention of the proliferation of microorganisms able to form histamine would also mean avoiding amine accumulation that leads to histamine food poisoning. The Niven medium was an efficient test to valutate the histamine production of isolated strains after an incubation of 24 h at 37°C and using a backwards technique for quantification and detecting the false positives. This incubation time should be longer (48 h) when Bacillus is detected, with the finality to eliminate false negatives on the initial screening. The application of the enzymic technique for histamine quantification was excellent. In our research, we have observed that the number of microorganisms is an important factor in the accumulation of histamine, but other factors exist which also influence such accumulation, probably depending on the kind of enzyme decarboxylase.

Evolution of histidine decarboxylase bacterial groups during the ripening of Spanish semi-preserved anchovies.

We have studied the count evolutions of total aerobic mesophilic microorganisms, psychotrophic microorganisms, enterobacteria, faecal coliforms, sulphite-reducing bacteria and vibrio in spanish semi-preserved anchovies. These microorganisms are a sanitary index of the product and may produce high concentrations of histamine in both fresh and processed fish. The influence of NaCl concentration, redox potential, oxygen concentration and pH on bacterial growth have also been studied. With the exception of the sulphite-reducers and vibrio, the counts of the different bacterial groups decreased during the first two weeks of ripening, but later stabilized. The faecal coliforms did not appear in the culture media after these first two weeks, and the enterobacteria, what initially did not appear after first two weeks too, are detected at final phases probably for the final manipulation of elaboration processes. The count of the sulfite-reducers remained unchanged during the whole ripening process. Vibrio were not detected in any of the samples studied. NaCl and oxygen concentrations were the main factors influencing the decreasing bacterial counts. According to our results, the accumulation of high histamine concentrations in salted fish could be due to poor quality of the raw material, to inadequate handling or to other causes during its shelf life. The relationship with the histamine activity is probably due more to the presence of the halophilic or halotolerant microorganisms.

Determination of histamine in fish using an enzymic method.

The present paper describes a quick and simple enzymic method for evaluating histamine content in fish. After preparing a perchloric acid extract, it was neutralized to pH 6.8 with KOH. The action of a diamine oxidase on the histamine caused the formation of hydrogen peroxide. The addition of a second enzyme, a peroxidase, in the presence of hydrogen peroxide and a chromogen (leuco crystal violet) in reduced form (colourless) facilitated its oxidation into crystal violet (coloured). Following a two-hour incubation period, absorbance was measured at 596 nm. The correlation between histamine level and absorbance was acceptable in the concentration range from 3 to 30 mg/kg of histamine (r = 0.9946; p < 0.001). The accuracy of the method, expressed by CV, varied between 2.6% and 4.9%, and the recovery between 95.7% and 100.1%, depending on the concentration of analyte in the sample. The diamine oxidase used was very selective in relation to the histamine. Only the presence of tyramine, when histamine concentration was low (< 10 mg/kg), tended to interfere to a slight degree in the technique. Using a regression analysis, no statistically significant differences were found between the results obtained from the analysis of 18 tuna fish samples by HPLC and the enzymic method (r = 0.9867; p < 0.001).