Mario Vaerewijck

Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle.

Aims: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle.

Grouping of pseudomonads by SDS-PAGE of whole-cell proteins.

Summary A numerical analysis was performed on one-dimensional whole-cell protein electrophoretic fingerprints of 210 bacterial reference strains of the pseudomonads. Within the authentic genus Pseudomonas, uniform and distinct patterns were found for strains of the species P. aeruginosa, P. agarici, P. alcaligenes, P. amygdali, P. caricapapayae, P. chlororaphis, P. cichorii, P. coronafaciens, P. corrugata, P. ficuserectae, P. fragi, P. mendocina, P. pertucinogena, P. tolaasii and P. viridif lava. A significant protein electrophoretic heterogeneity was observed within the species P. fluorescens, P. marginalis, P. pseudoalcaligenes, P. putida, P. stanieri and P. stutzeri. Only the type strain was included of the species P. lundensis, P. meliae, P. mucidolens, P. oleovorans, P. resinovorans, P. synxantha and P. taetrolens, and all of them occupied a separate position. Also the three investigated strains of the complex species P. syringae grouped together in a single cluster. Similar profiles were obtained for representatives of the species P. asplenii and P. fuscovaginae. Reference strains of a few species, previously classified in the genus Pseudomonas, and recently reclassified in the genera Acidovorax, Burkholderia, Brevundimonas, Comamonas, Hydrogenophaga and Ralstonia were included. The latter data demonstrated again that no genus-specific, but species-specific protein profiles were obtained. Twenty-nine fresh isolates from polluted sites, presumably belonging to the pseudomonads, were characterized using the SDS-PAGE database. These data demonstrated that the technique provides a useful tool for the identification of newly isolated strains.

Microscopic and Molecular Studies of the Diversity of Free-Living Protozoa in Meat-Cutting Plants

ABSTRACT The diversity of free-living protozoa in five meat-cutting plants was determined. Light microscopy after enrichment culturing was combined with sequencing of PCR-amplified, denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments, which was used as a fast screening method. The general results of the survey showed that a protozoan community of amoebae, ciliates, and flagellates was present in all of the plants. Protozoa were detected mainly in floor drains, in standing water on the floor, on soiled bars of cutting tables, on plastic pallets, and in out-of-use hot water knife sanitizers, but they were also detected on surfaces which come into direct contact with meat, such as conveyer belts, working surfaces of cutting tables, and needles of a meat tenderizer. After 7 days of incubation at refrigerator temperature, protozoa were detected in about one-half of the enrichment cultures. Based on microscopic observations, 61 morphospecies were found, and Bodo saltans, Bodo spp., Epistylis spp., Glaucoma scintillans, Petalomonas spp., Prodiscophrya collini, and Vannella sp. were the most frequently encountered identified organisms. Sequencing of DGGE bands resulted in identification of a total of 49 phylotypes, including representatives of the Amoebozoa, Chromalveolata, Excavata, Opisthokonta, and Rhizaria. Sequences of small heterotrophic flagellates were affiliated mainly with the Alveolata (Apicomplexa), Stramenopiles (Chrysophyceae), and Rhizaria (Cercozoa). This survey showed that there is high protozoan species richness in meat-cutting plants and that the species included species related to known hosts of food-borne pathogens.

Interactions of Foodborne Pathogens with Free-living Protozoa: Potential Consequences for Food Safety

Free-living protozoa (FLP) are ubiquitous in natural ecosystems where they play an important role in the reduction of bacterial biomass and the regeneration of nutrients. However, it has been shown that some species such as Acanthamoeba castellanii, Acanthamoeba polyphaga, and Tetrahymena pyriformis can act as hosts of pathogenic bacteria. There is a growing concern that FLP might contribute to the maintenance of bacterial pathogens in the environment. In addition to survival and/or replication of bacterial pathogens in FLP, resistance to antimicrobial agents and increased virulence of bacteria after passage through protozoa have been reported. This review presents an overview of FLP in food-associated environments and on foods, and discusses bacterial interactions with FLP, with focus on the foodborne pathogens Campylobacter jejuni, Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. The consequences of these microbial interactions to food safety are evaluated.

Occurrence and diversity of free-living protozoa on butterhead lettuce.

The occurrence and diversity of free-living protozoa (FLP) on butterhead lettuce (Lactuca sativa L.) was investigated using four different sampling techniques (washing, swabbing, homogenization, and excising). FLP were recovered from all leaf samples (n=64), and cultures were FLP-positive after 1 week. Identification of FLP was performed by light microscopy and sequencing of denaturing gradient gel electrophoresis (DGGE)-separated 18S rRNA gene fragments. Bodo saltans, Spumella (-like) spp. and Cercozoa were the most common heterotrophic nanoflagellates. Amoebae belonged mainly to the Vannellida and Tubulinida. Colpoda steinii and Cyclidium glaucoma were the most common ciliates. The total number of FLP on middle leaves estimated by the Most Probable Number method ranged from 9.3 × 10(2)MPN/g to 2.4 × 10(5)MPN/g leaf, with flagellates (92 MPN/g to 2.4 ×10(5)MPN/g) being more abundant than amoebae (<3 MPN/g to 9.3 × 10(3)MPN/g) and ciliates (<3 MPN/g to 9.3 × 10(2)MPN/g). Washing or rinsing leaves followed by spin-drying in a household salad spinner reduced the protozoan number with maximum one log unit. Our survey shows that FLP on lettuce leaves are a common and diverse but largely unexplored group of microorganisms.

Sampling strategy, occurrence and diversity of free-living protozoa in domestic refrigerators

Aims:  Evaluation of a sampling method to recover free‐living protozoa (FLP) from plastic surfaces. Application of the method on different areas inside domestic refrigerators.

Persistence of Free-Living Protozoan Communities across Rearing Cycles in Commercial Poultry Houses

ABSTRACT The introduction and survival of zoonotic bacterial pathogens in poultry farming have been linked to bacterial association with free-living protozoa. To date, however, no information is available on the persistence of protozoan communities in these environments across consecutive rearing cycles and how it is affected by farm- and habitat-specific characteristics and management strategies. We therefore investigated the spatial and temporal dynamics of free-living protozoa in three habitats (pipeline, water, and miscellaneous samples) in three commercial poultry houses across three rearing cycles by using the molecular fingerprinting technique denaturing gradient gel electrophoresis (DGGE). Our study provides strong evidence for the long-term (ca. 6-month) persistence of protozoa in broiler houses across consecutive rearing cycles. Various free-living protozoa (flagellates, ciliates, and amoebae), including known vectors of bacterial pathogens, were observed during the down periods in between rearing cycles. In addition, multivariate analysis and variation partitioning showed that the protozoan community structure in the broiler houses showed almost no change across rearing cycles and remained highly habitat and farm specific. Unlike in natural environments, protozoan communities inside broiler houses are therefore not seasonal. Our results imply that currently used biosecurity measures (cleaning and disinfection) applied during the down periods are not effective against many protozoans and therefore cannot prevent potential cross-contamination of bacterial pathogens via free-living protozoa between rearing cycles.

Co-occurrence of free-living protozoa and foodborne pathogens on dishcloths: Implications for food safety

In the present study, the occurrence of free-living protozoa (FLP) and foodborne bacterial pathogens on dishcloths was investigated. Dishcloths form a potentially important source of cross-contamination with FLP and foodborne pathogens in food-related environments. First various protocols for recovering and quantifying FLP from dishcloths were assessed. The stomacher technique is recommended to recover flagellates and amoebae from dishcloths. Ciliates, however, were more efficiently recovered using centrifugation. For enumeration of free-living protozoa on dishcloths, the Most Probable Number method is a convenient method. Enrichment was used to assess FLP diversity on dishcloths (n=38). FLP were found on 89% of the examined dishcloths; 100% of these tested positive for amoebae, 71% for flagellates and 47% for ciliates. Diversity was dominated by amoebae: vahlkampfiids, vannellids, Acanthamoeba spp., Hyperamoeba sp. and Vermamoeba vermiformis were most common. The ciliate genus Colpoda was especially abundant on dishcloths while heterotrophic nanoflagellates mainly belonged to the genus Bodo, the glissomonads and cercomonads. The total number of FLP in used dishcloths ranged from 10 to 10(4) MPN/cm(2). Flagellates were the most abundant group, and ciliates the least abundant. Detergent use was identified as a prime determinant of FLP concentrations on used dishcloths. Bacterial load on dishcloths was high, with a mean total of aerobic bacteria of 7.47 log 10 cfu/cm(2). Escherichia coli was detected in 68% (26/38) of the used dishcloths, with concentrations up to 4 log 10 cfu/cm(2). Foodborne pathogens including Staphylococcus aureus (19/38), Arcobacter butzleri (5/38) and Salmonella enterica subsp. enterica ser. Halle (1/38) were also present. This study showed for the first time that FLP, including some opportunistic pathogens, are a common and diverse group on dishcloths. Moreover, important foodborne pathogens are also regularly recovered. This simultaneous occurrence makes dishcloths a potential risk factor for cross-contamination and a microbial niche for bacteria-FLP interactions.

Assessment of the efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and Tetrahymena spp.

The efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and two Tetrahymena spp. was determined based on the European Standard EN 1276:2009 suspension test. Trophozoite viability was assessed by determination of the membrane integrity using flow cytometry as a fast screening technique. Bovine serum albumin was added to simulate clean (0.3 g/liter) and dirty (3 g/liter) conditions. Benzalkonium chloride caused cell lysis at concentrations above 50 mg/liter under clean and dirty conditions. A concentration of 50 mg of free chlorine per liter had a strong biocidal effect on acanthamoebae and tetrahymenae after 15 min under clean and dirty conditions. Our results suggest that benzalkonium chloride and sodium hypochlorite were effective against the three microorganisms at concentrations commonly applied in the food industry.