Processing plant persistent strains of Listeria monocytogenes appear to have a lower virulence potential than clinical strains in selected virulence models

Listeria monocytogenes is an important foodborne bacterial pathogen that can colonize food processing equipment. One group of genetically similar L. monocytogenes strains (RAPD type 9) was recently shown to reside in several independent fish processing plants. Persistent strains are likely to contaminate food products, and it is important to determine their virulence potential to evaluate risk to consumers. We compared the behaviour of food processing persistent and clinical L. monocytogenes strains in four virulence models: Adhesion, invasion and intracellular growth was studied in an epithelial cell line, Caco-2; time to death in a nematode model, Caenorhabditis elegans and in a fruit fly model, Drosophila melanogaster and fecal shedding in a guinea pig model. All strains adhered to and grew in Caco-2 cells in similar levels. When exposed to 10(6) CFU/ml, two strains representing the persistent RAPD type 9 invaded Caco-2 cells in lower numbers (10(2)-10(3) CFU/ml) as compared to the four other strains (10(4)-10(6) CFU/ml), including food and human clinical strains. In the D. melanogaster model, the two RAPD type 9 strains were among the slowest to kill. Similarly, the time to reach 50% killed C. elegans worms was longer (110 h) for the RAPD type 9 strains than for the other four strains (80 h). The Scott A strain and one RAPD type 9 strain were suspended in whipping cream before being fed to guinea pigs and the persistent RAPD type 9 strain was isolated from feces in a lower level (approximately 10(2) CFU/g) than the Scott A strain (approximately 10(5) CFU/g) (P<0.05). The addition of NaCl has been shown to cause autoaggregation and increases adhesion of L. monocytogenes to plastic. However, growth in the presence of NaCl did not alter the behaviour of the tested L. monocytogenes strains in the virulence models. Overall, the two strains representing a very common fish processing plant persistent group (RAPD type 9) appear to have a lower virulence potential in all four virulence models than Scott A and a strain isolated from a clinical case of listeriosis.     

Genetic variability among isolates of Listeria monocytogenes from food products,clinical samples and processing environments,estimated by RAPD typing

RAPD analysis with four primers was used to examine the genetic relationship among 432 strains of Listeria monocytogenes isolated from clinical and veterinarian cases of listeriosis, dairy, vegetable, meat- and fish-based food items, environmental samples and samples collected from one transport terminal, one poultry-processing company and four Atlantic salmon-processing plants. The purpose of the study was to determine whether clinical isolates belonged to a specific genetic group, whether links could be made between food groups and clinical cases and whether specific genetic groups were associated with specific food products or processing units. There was great genetic variability among the isolates, which produced a total of 141 RAPD composites based on the RAPD analysis with four primers. The RAPD composites divided in two major clusters and clinical isolates were evenly distributed in both of them. None of the isolates from food products had the same RAPD composite as isolates from human patients, thus, no particular food commodity could be linked to clinical cases. Each food-processing environment was contaminated with more than one RAPD composite and the genetic variability found within each company was, in most cases, of approximately the same magnitude as the variability found when considering all the samples. In each plant, one or a few types persisted over time, indicating the presence of an established in-house flora. Our results indicate that most of the analysed cases of listeriosis were sporadic and, further, that these cases cannot be traced to a few specific food sources. We also found that no particular RAPD composite was better suited for survival in specific food types or food-processing environments, indicating that although differences may be found in virulence properties of individual strains, all L. monocytogenes must be treated as potentially harmful.     

Low technology water purification by bentonite clay flocculation as performed in Sudanese villages. Virological examinations

Viruses were removed from various types of water by flocculation with natural bentonite clays from the banks of the Nile as well as with pure bentonite. The flocculation technique employed corresponds to that used to clarify Nile water in Sudanese villages. If proper flocculation occurred 3 to 4 log₁₀ units of virus could be removed. Some of the conditions for floc formation were examined.     

Sequestration of Plant-Derived Phenolglucosides by Larvae of the Leaf Beetle <Emphasis Type="Italic">Chrysomela lapponica</Emphasis>: Thioglucosides as Mechanistic Probes

Feeding larvae of Chrysomela lapponica (Coleoptera: Chrysomelidae) acquire characteristic O-glucosides from the leaves of their food plants. The glucosides are selectively channeled from the gut to the defensive gland. Subsequent enzymatic transformations generate a blend of different defensive compounds, e.g., salicylaldehyde and two series of 2-methylbutyl and isobutyryl esters. By using systematically modified and hydrolysis-resistant thioglucosides as structural mimics of the plant-derived glucosides, e.g., salicin and its o-, m-, and p-isomers 1, 2, and 3; o-, m-, and p-cresols 5, 6, 7; along with thioglucosides of 2-phenylethanol 9 and (3Z)-hexenol 10, we demonstrated that the larvae of C. lapponica are able to sequester a broad range of structurally different thioglucosides with comparable efficiency. This sharply contrasts with the sequestration habitus previously observed in Chrysomela populi and Phratora vitellinae, which secrete almost pure salicylaldehyde and posses a highly specific transport mechanism for salicin (Kuhn et al., Proc. Natl. Acad. Sci. USA 101:13808–13813, 2004). Also, neither C. lapponica nor C. populi sequester in their gland the thioglucoside of 8-hydroxygeraniol, the mimic of the glucoside specifically transported by larvae secreting iridoid monoterpenes (Phaedon cochleariae, Gastrophysa viridula). Accordingly, leaf beetle larvae possess selective membrane carriers in their gut and their defensive systems that match the orientation of the functional groups of glucosides from their food plants probably by embedding the substrate in a network of hydrogen bonds inside the membrane carriers. The synthesis and the spectroscopic properties of the test compounds along with a comparative evaluation of the transport capabilities of larvae of C. populi and C. lapponica are described. Electronic Supplementary Material Supplementary material is available at and is accessible for authorized users.     

Ex Vivo Fluorescence Confocal Microscopy (FCM) Ensures Representative Tissue in Prostate Cancer Biobanking: A Feasibility Study

Background: Biobanking of prostate carcinoma is particularly challenging due to the actual cancer within the organ often without clear margins. Frozen sections are to date the only way to examine the biobank material for its tumor content. We used ex vivo fluorescence confocal microscopy (FCM) to analyze biobank samples prior to cryoasservation. Methods: 127 punch biopsies were acquired from prostatectomy-specimens from 40 patients. These biopsies were analyzed with a Vivascope 2500-G4 prior to their transfer to the biobank. In difficult cases, larger samples of the prostatectomy specimens were FCM scanned in order to locate tumor foci. After patient acquisition, all samples were taken from the biobank and analyzed. We compared the results of the FCM examinations with the results of conventional histology and measured the DNA content. Results: With upstream FCM, the tumor content of biobank samples could be determined with high confidence. The detection rate of representative biobank samples was increased due to the rapid feedback. The biobank samples were suitable for further molecular analysis. Conclusion: FCM allows for the first time lossless microscopic analysis of biobank samples prior to their cryoasservation and guarantees representative tumor and normal tissue for further molecular analysis.     

Increased Presence of Complement Factors and Mast Cells in Alveolar Bone and Tooth Resorption

Periodontitis is the inflammatory destruction of the tooth-surrounding and -supporting tissue, resulting at worst in tooth loss. Another locally aggressive disease of the oral cavity is tooth resorption (TR). This is associated with the destruction of the dental mineralized tissue. However, the underlying pathomechanisms remain unknown. The complement system, as well as mast cells (MCs), are known to be involved in osteoclastogenesis and bone loss. The complement factors C3 and C5 were previously identified as key players in periodontal disease. Therefore, we hypothesize that complement factors and MCs might play a role in alveolar bone and tooth resorption. To investigate this, we used the cat as a model because of the naturally occurring high prevalence of both these disorders in this species. Teeth, gingiva samples and serum were collected from domestic cats, which had an appointment for dental treatment under anesthesia, as well as from healthy cats. Histological analyses, immunohistochemical staining and the CH-50 and AH-50 assays revealed increased numbers of osteoclasts and MCs, as well as complement activity in cats with TR. Calcifications score in the gingiva was highest in animals that suffer from TR. This indicates that MCs and the complement system are involved in the destruction of the mineralized tissue in this condition.     

Structural and physical effects of aroma compound binding to native starch granules

The interaction and physical/structural effects of aroma compounds, at high concentrations on dry native starch granules were studied using eight selected model compounds: acetaldehyde, dimethyl sulphide, diacetyl, allyl isothiocyanate, ethyl butyrate, citral, octanol and butyric acid. The maize, potato and pea starches used represent different typical structural and chemical starch characteristics. Retention of the different aroma compounds varied from a few to one hundred percent and starch was found to induce as well as reduce aroma evaporation depending on the aroma compound and the starch type. As deduced from DSC, powder XRD and SEM analyses, citral, butyric acid and octanol exerted specific effects on the starch granules manifested in local melting of crystalline layers and partial disruption of the granular meso structure. The most prominent effect was obtained with citral that generated surface wrinkles on B‐ and C‐type polymorphic granules and aggregation of A‐type polymorphic granules, decreased the melting temperature and suppressed the crystallinity of the starch.     

Antilisterial activity of a Carnobacterium piscicola isolated from Brazilian smoked fish (surubim [Pseudoplatystoma sp.]) and its activity against a persistent strain of Listeria monocytogenes isolated from surubim

Data on the prevalence and growth of Listeria monocytogenes in lightly preserved fish products from subtropical and tropical regions are very scarce. Our research describes L. monocytogenes that was detected in 5% of the packages of cold-smoked surubim, a native Brazilian freshwater fish that we analyzed, and shows that the strains isolated were of the same random amplified polymorphic DNA subtype as the strains that were isolated from the same factory 4 years earlier. A bacteriocinogenic strain of Carnobacterium piscicola (strain C2), isolated from vacuum-packed cold-smoked surubim, and two C. piscicola strains, isolated from vacuum-packed, cold-smoked salmon, were capable of limiting or completely inhibiting the growth of an L. monocytogenes (strain V2) isolated from surubim in fish peptone model systems incubated at 10 degrees C. Monocultures of L. monocytogenes reached 108 CFU/ml (g), whereas the growth of L. monocytogenes was completely inhibited by C. piscicola C2. The bacteriocinogenic C. piscicola A9b+ and its nonbacteriocinogenic mutant A9b- reduced maximum Listeria levels by 2 to 3 log units. Both bacteriocinogenic C. piscicola strains prevented listerial growth in cold-smoked fish juices (surubim and salmon). Although the carnobacteria grew poorly on cold-smoked surubim at 10 degrees C, the strains were able to reduce maximum Listeria counts by 1 to 3 log units in an artificially inoculated product (surubim). We conclude that Brazilian smoked fish products harbor L. monocytogenes and should be stabilized against the growth of the organism. C. piscicola C2 has the potential for use as a bioprotective culture in surubim and other lightly preserved fish, but further studies are required to optimize its effect.     

Desiccation of adhering and biofilm Listeria monocytogenes on stainless steel: Survival and transfer to salmon products

The foodborne bacterial pathogen, Listeria monocytogenes, commonly contaminates foods during processing, where the microorganisms are potentially subjected to low relative humidity (RH) conditions for extended periods of time. The objective of this study was to examine survival during desiccation (43% RH and 15 °C) of biofilm L. monocytogenes N53-1 cells on stainless steel coupons and to assess subsequent transfer to salmon products. Formation of static biofilm (2 days at 100% RH and 15 °C) prior to desiccation for 23 days significantly (P < 0.05) improved survival of cells desiccated in initial low salt concentrations (0.5%) compared to the survival for non-biofilm cells also desiccated in low salt, indicating the protective effect of the biofilm matrix. Osmoadaptation of cells in 5% NaCl before formation of the static biofilm significantly (P < 0.05) increased long-term desiccation survival (49 days) irrespectively of the initial salt levels (0.5% and 5% NaCl). The efficiency of transfer (EOT) of desiccated biofilm cells was significantly (P < 0.05) lower than EOTs for desiccated non-biofilm bacteria, however, as biofilm formation enhanced desiccation survival more bacteria were still transferred to smoked and fresh salmon. In conclusion, the current work shows the protective effect of biofilm formation, salt and osmoadaptation on the desiccation survival of L. monocytogenes, which in turn increases the potential for cross-contamination during food processing.