Charlotte Johansen

The microbial ecology of processing equipment in different fish industries - analysis of the microflora during processing and following cleaning and disinfection

The microflora adhering to the processing equipment during production and after cleaning and disinfecting procedures was identified in four different processing plants. A total of 1009 microorganisms was isolated from various-agar plates and identified. A stepwise procedure using simple phenotypic tests was used to identify the isolates and proved a fast way to group a large collection of microorganisms. Pseudomonas, Neisseriaceae, Enterobactericeae, Coryneform, Acinetobacter and lactic acid bacteria dominated the microflora of cold-smoked salmon plants, whereas the microflora in a plant processing semi-preserved herring consisted of Pseudomonas, Alcaligenes and Enterobactericeae. Psychrobacter, Staphylococcus and yeasts were found in a caviar processing plant. Overall, many microorganisms that are often isolated from fish were also isolated from the fish processing plants. However, some selection depending on processing parameters occurred, since halo- and osmo-tolerant organisms dominated in the caviar processing. After cleaning and disinfection, yeasts, Pseudomonas, Neisseriaceae and Alcaligenes remained in smokehouses, yeasts and Pseudomonas in the herring plant and Pseudomonas, Staphylococcus and yeasts in the caviar plant. The dominant adhering organisms after cleaning and disinfection were pseudomonads and yeasts independently of the microflora during processing. Knowledge of the adhering microflora is essential in the Good Hygienic Practises programme of food processing plants, as the development and design of improved cleaning and disinfecting procedures should target the microorganisms persisting and potentially contaminating the product.

An inhibitor of bacterial quorum sensing reduces mortalities caused by Vibriosis in rainbow trout (Oncorhynchus mykiss, Walbaum).

The fish pathogen Vibrio anguillarum produces quorum sensing signal molecules, N-acyl homoserine lactones (AHLs), which in several Gram-negative human and plant pathogenic bacteria regulate virulence factors. Expression of these factors can be blocked using specific quorum-sensing inhibitors (QSIs). The purpose of this study was to investigate the effect of a QSI, furanone C-30, on mortality of rainbow trout during challenge with V. anguillarum. Addition of 0.01 or 0.1 microM furanone C-30 to rainbow trout infected by cohabitation caused a significant reduction in accumulated mortality from 80-100% in challenge controls to 4-40% in treated groups. Furanone C-30 had no effect in an immersion challenge system, probably due to a very high water exchange and a rapid dilution of furanone C-30. Growth and survival of V. anguillarum were not affected by the concentrations of furanone C-30 used in the challenge experiments, thus avoiding selection for resistance. To elucidate the mechanism of disease control by furanone C-30, we determined its effect on the bacterial proteome, motility, and respiration. No effects were seen of furanone C-30 in any of these experiments. Although no cytotoxic effect on HeLa cells were observed, exposure to 1 microM (or higher) concentrations of furanone C-30 had detrimental effects on the rainbow trout. Our results indicate that QSIs can be used in non-antibiotic based control of fish diseases. However, they also underline the need for development of novel, less toxic QSI compounds and the need for understanding the exact mechanism(s) of action.

Shewanella putrefaciens adhesion and biofilm formation on food processing surfaces.

ABSTRACT Laboratory model systems were developed for studyingShewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25°C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 108 CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 102 CFU/cm2) than in a batch system (reaching 107 CFU/cm2), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4′,6′-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces.

Curvularia Haloperoxidase: Antimicrobial Activity and Potential Application as a Surface Disinfectant

ABSTRACT A presumed antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide, Curvularia haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The Curvularia haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp. and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of S. marcescens and S. epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia haloperoxidase system. The killing effect of the Curvularia haloperoxidase system was rapid, and 106 CFU of E. coli cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E. coli 10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the Curvularia haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.

Impact of salinomycin on the intestinal microflora of broiler chickens.

BackgroundThe ionophoric coccidiostat salinomycin is widely used in chicken feed. In the near future the use of ionophore coccidiostats may be banned as has been the case for other antimicrobial growth promoters. This study was conducted to examine the effect of salinomycin on Campylobacter jejuni infection and on the composition of the caecal microflora in broiler chickens.MethodsAn experimental infection study was carried out in isolators and the intestinal microflora was analyzed using quantitative cultivation, denaturant gradient gel electrophoresis (DGGE), cloning and sequencing.ResultsWe found no effect of salinomycin on C. jejuni but salinomycin significantly affected the composition of the microflora. In addition, salinomycin significantly reduced the prevalence of Clostridium perfringens and we observed a significant increase (62%) in the mean body weight of salinomycin treated chickens compared to un-treated controls.ConclusionTermination of the use of ionophore coccidiostats will not affect food safety related to campylobacter, but will increase the risk of necrotic enteritis in the broilers.

The combined inhibitory effect of lysozyme and low pH on growth of Listeria monocytogenes

Egg white lysozyme significantly inhibited growth of Listeria monocytogenes Scott A in tryptic soy broth (TSB) at 5°C and 25°C. The inhibition, which was mainly seen as a prolonged lag phase, was enhanced when the pH was lowered from 7.2 to 5.5. Lowering of the pH from 7.2 to 5.5 caused a lag phase of nine days at 5°C and addition of lysozyme (10,000 μ/ml) further increased the lag phase from 60 to 70 days. Similarly, lag phases at 25°C were 4 h (pH 5.5, no lysozyme) and 37 h (pH 5.5, 50,000 μ/ml lysozyme). Lowering of the pH from 7.2 to 5.5 did not, however, affect the lytic activity of the egg white lysozyme as measured by the decrease of absorbance of a suspension of live L. monocytogenes cells. The observed interactive inhibitory effect of lysozyme and low pH on growth is, therefore, suggested to be due to the growth retarding effect of low pH allowing enzymatic hydrolysis to exceed the rate of cell proliferation. The study shows that lysozyme may hold great potential for inhibition of L. monocytogenes in foods.