Agents Of Foodborne Diseases Research Articles

Cyclic-di-GMP Regulates Extracellular Polysaccharide Production, Biofilm Formation, and Rugose Colony Development by Vibrio vulnificus

Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3'-5')-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP.

ANTIFUNGAL PROPERTIES OF OLIVE LEAF EXTRACTS AND THEIR PHENOLIC COMPOUNDS

ABSTRACT Olive (Olea europaea L.) leaf extracts were obtained using water or different organic solvents such as acetone, methanol and ethyl acetate. Antimicrobial activities of the extracts and some phenolic components were investigated to screen against 30 fungal strains (Alternaria alternata, Aspergillus chevalieri, A. chrysogenum, A. elegans, A. flavus [three strains], A. fumigatus, A. nidulans, A. niger [two strains], A. oryzae, A. parasiticus[four strains], A. tamari, P. verrucosum, A. versicolor, A. wentii, Fusarium oxysporum, F. semitectum, Mucor racemosus, Neurospora crassa, Penicillium citrinum, P. echinulatum, P. griseofulvum, P. italicum, P. roqueforti and Rhizopus oligosporus) using the disc diffusion method. In this study, in terms of inhibition activity, it was determined that aqueous extract was the best as it completely inhibited the growth of 10 molds, followed by acetone and methanol extracts, which were effective against eight molds, and diethyl ether extract, which was effective against 7 out of 30 test fungi. The inhibition zones ranged from 7 to 21 mm. Comparing the sensitivity of the fungi with all crude olive leaf extracts and pure phenolic compounds, we found that A. parasiticus(4) was the most resistant strain while A. wentii was the most sensitive.PRACTICAL APPLICATIONSFoodborne diseases are still a major problem in the world, even in well‐developed countries. Fusarium spp., Aspergillus spp., Rhizopus spp. and Penicillium spp. have been reported as the causal agents of foodborne diseases and food spoilage. Raw and processed foods are open to contamination during the production, sale and distribution. Thus, at present, it is a necessity to the food industry to use chemical preservatives to prevent the growth of fungi in foods. Because of the economic impact of spoiled foods and consumer concerns over the safety of foods containing synthetic chemicals, considerable attention has been paid to naturally derived compounds or natural products. Recently, there has been considerable interest in using extracts from plants with antimicrobial activities to control pathogens or toxin‐producing microorganisms in foods. Olive leaves have been shown to inhibit or delay the rate of growth of a range of fungi; thus, they might be useful as natural preservatives.

An Overview of Vibrio vulnificus and Vibrio parahaemolyticus

ABSTRACT: The Vibrionaceae are environmentally ubiquitous to estuarine waters. Two species in particular, V. vulnificus and V. parahaemolyticus, are important human pathogens that are transmitted by the consumption of contaminated molluscan shellfish. This document provides a comprehensive review of the current state of knowledge about these important foodborne disease agents. Topics include the epidemiology of human disease; biotypes and virulence factors; cultural and molecular‐based detection methods; phenotyping and genotyping approaches; microbial ecology; and candidate control strategies. Recent international risk assessment efforts are also described. The reader will gain an understanding of why these organisms pose a public health risk and how improving our understanding of their behavior in the environment and the host can aid in reducing that risk in the future.

Influence of Peptone Source on Sporulation of Clostridium perfringens Type A

Clostridium perfringens is a foodborne disease agent that produces a sporulation-specific enterotoxin. To produce enterotoxin for experimental purposes or spores for challenge or physiological studies, the use of a convenient sporulation medium is required. The most commonly used is Duncan-Strong medium. Few isolates sporulate at high levels in this medium. We investigated the effectiveness of peptones from a variety of sources on the sporulation of this organism compared with the peptone in the original formulation, proteose peptone (control). Seven strains were used to screen 32 peptones, with starch or raffinose as the carbohydrate source. In most cases, raffinose was more effective than starch in stimulating sporulation, confirming our previous study. Two promising peptones, potato peptone, and Proteose Peptone no. 3, were selected and tested against 49 additional enterotoxin-positive and -negative strains, with raffinose as the carbohydrate. For 49 strains, 5 sporulated best (>10%) in the control peptone, 6 sporulated best in Peptone no. 3, and 23 sporulated best in the potato peptone. Of the 23 strains, 16 sporulated at levels 25% more than the control peptone. The increase in sporulation rates was reflected in the enterotoxin and heat-resistant spore levels. The methylxanthines caffeine and theobromine were effective in increasing the sporulation of less than half of 19 enterotoxin-positive strains. Our results suggest that the replacement of proteose peptone with potato peptone be considered if difficulty in obtaining spores of specific strains of C. perfringens is encountered.

Essential oil composition and antibacterial activity of different extracts of Allium roseum L., a North African endemic species

This study deals with the valorisation of medicinal and aromatic plants of the Tunisian flora, in order to find new bioactive natural products. The essential oil constituents from the flowers of Allium roseum var. odoratissimum, a North African endemic species growing in the south-east of Tunisia, were extracted by hydrodistillation and analysed by GC–MS. The most important compounds were sulphurous compounds (2,4-dimethylthiophene, 2-propenyl methyl disulfide, 1-propenyl methyl disulfide, dimethyl trisulfide, and 3-methyl-thiopropionaldehyde or methional). After an exhaustive literature review dealing with other varieties of the same species, it seems that the variety odoratissimum is the only taxon characterized by trisulfide and methional contents. Methional is of particular importance because it is found at high concentration (17%). The antibacterial activity of seven extracts was evaluated by the diffusion method and by determining the inhibition zone. All extracts exhibited an antibacterial activity at different levels against strains reported as the causal agents of food-borne diseases. Results suggest the potential use of the plant as condiment and preservative in the food industry. La présente étude porte sur les possibilités de valorisation d'une plante aromatique et médicinale de la flore tunisienne dans le but de mettre au point de nouveaux produits bioactifs. Il s'agit d'Allium roseum var. odoratissimum, qui est une espèce endémique nord-africaine, très commune en Tunisie méridionale. Les constituants des huiles essentielles des fleurs de cette plante, extraites par hydrodistillation, ont été analysés par GC–MS. Les principaux constituants de ces huiles étaient des dérivés soufrés (2,4-diméthylthiophène, 2-propénylméthyle disulfide, 1-propénylméthyle disulfide, diméthyl trisulfide et 3-méthyl-thiopropionaldéhyde ou méthional). Ces résultats, analysés à la lumière des données bibliographiques à propos d'autres variétés de la même espèce, permettent de dire que la variété odoratissimum est le seul taxon qui est caractérisé par la présence des trisulfides et du méthional. Ce dernier a été détecté à une forte concentration (17%). L'activité antibactérienne des sept extraits testés a été étudiée par la méthode de diffusion et de détermination de la zone d'inhibition. Tous ces extraits ont montré une activité antibactérienne plus ou moins importante vis-à-vis des souches signalées. Ces résultats suggèrent la possibilité d'utiliser cette plante comme condiment et agent de conservation dans l'industrie alimentaire.

Serological and genetic diversity amongst Salmonella strains isolated in a salami processing line

Salmonella is one of the most important agents of foodborne disease in Brazil and in other countries, with meat and meat products being identified as important vehicles of salmonelosis. A total of 54 Salmonella strains isolated from a commercial salami processing line were first serotyped and then their antibiotic resistance and macro restriction profiles were determined. 11.1% of the strains showed resistance to 3 or more antibiotics with profile AmpCStxTe being the most frequent. PFGE generated 9 and 12 profiles with enzymes XbaI and SpeI, respectively. It was observed that different serotypes of Salmonella could be found in the different steps of the processing line. The genetic profile of the strains had low relationship indicating the genetic diversity of the tested strains.

MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars

Salmonella enterica subsp . enterica is one of the main causative agents of food-borne disease in man, and can also be the cause of serious systemic illness. Organisms belonging to this genus have traditionally been classified on the basis of the antigenic properties of the cell-surface lipopolysaccharide and of the phase 1 and phase 2 flagellar proteins. Primary isolation, biochemical identification, and serotyping are laborious and time consuming. Molecular identification based on suitable marker genes could be an attractive alternative to conventional bacteriological and serological methods. We have assessed the applicability of two housekeeping genes, gyrB, atpD, in combination with the flagellin genes fliC and fljB in multilocus sequence typing of Salmonella. Sequencing and comparative analysis of sequence data was performed on multiple strains from Austria, the United Kingdom, and Switzerland, representing all subspecies and 22 of the more prevalent non-typhoid S. enterica subsp. enterica serovars. A combination of these four marker genes allowed for a clear differentiation of all the strains analysed, indicating their applicability in molecular typing. The term MLST-v, for multilocus sequence typing based on virulence genes, is proposed to distinguish this approach from MLST based solely on housekeeping genes. An assortative recombination of the fliC gene was found in seven of the analysed serovars indicating multiple phylogenetic origin of these serovars.

Incidence of Clostridium perfringens in Broiler Chickens in the Czech Republic

Clostridium perfringens is a causative agent of human and animal foodborne diseases. It is known as a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen causing necrotic enteritis. The aim of the present study was to detect the incidence of C. perfringens in healthy broiler chickens. From May 2005 to September 2006, 609 samples of caecal content from broilers from 23 intensive poultry farms were analyzed. The samples were cultivated on TSC and blood agar, typical colonies were identified and biochemically confirmed. the total number of positive samples was 112 (18.39%). the samples were processed by the multiplex polymerase chain reaction method (PCR) for toxin genotyping. The presence of alpha, beta, beta2 and enterotoxin gene was detected. All C. perfringens isolates were classified as type A, four isolates had the cpb2 gene. In conclusion the prevalence of C. perfringens-positive farms is approximately 74% and the amount ranges about 104 cfu/g of caecal content.

Systematic Environmental Evaluations To Identify Food Safety Differences between Outbreak and Nonoutbreak Restaurants

Restaurants are important settings for foodborne disease transmission. The Environmental Health Specialists Network (EHS-Net) was established to identify underlying factors contributing to disease outbreaks and to translate those findings into improved prevention efforts. From June 2002 through June 2003, EHS-Net conducted systematic environmental evaluations in 22 restaurants in which outbreaks had occurred and 347 restaurants in which outbreaks had not occurred. Norovirus was the most common foodborne disease agent identified, accounting for 42% of all confirmed foodborne outbreaks during the study period. Handling of food by an infected person or carrier (65%) and bare-hand contact with food (35%) were the most commonly identified contributing factors. Outbreak and nonoutbreak restaurants were similar with respect to many characteristics. The major difference was in the presence of a certified kitchen manager (CKM); 32% of outbreak restaurants had a CKM, but 71% of nonoutbreak restaurants had a CKM (odds ratio of 0.2; 95% confidence interval of 0.1 to 0.5). CKMs were associated with the absence of bare-hand contact with foods as a contributing factor, fewer norovirus outbreaks, and the absence of outbreaks associated with Clostridium perfringens. However, neither the presence of a CKM nor the presence of policies regarding employee health significantly affected the identification of an infected person or carrier as a contributing factor. These findings suggest a lack of effective monitoring of employee illness or a lack of commitment to enforcing policies regarding ill food workers. Food safety certification of kitchen managers appears to be an important outbreak prevention measure, and managing food worker illnesses should be emphasized during food safety training programs.

Detection of Noroviruses in Foods: A Study on Virus Extraction Procedures in Foods Implicated in Outbreaks of Human Gastroenteritis

Disease outbreaks in which foods are epidemiologically implicated as the common source are frequently reported. Noroviruses and enteric hepatitis A viruses are among the most prevalent causative agents of foodborne diseases. However, the detection of these viruses in foods other than shellfish is often time-consuming and unsuccessful. In this study, three virus concentration methods were compared: polyethylene glycol (PEG) plus NaCl, ultracentrifugation, and ultrafiltration. Two RNA extraction methods, TRIzol and RNeasy Mini Kit (Qiagen), were compared for detection of viruses in whipped cream and lettuce (as representatives of the dairy and vegetable-fruit food groups, respectively). A seeding experiment with canine calicivirus was conducted to determine the efficiency of each virus extraction procedure. The PEG-NaCl-TRIzol method was most efficient for the detection of viruses in whipped cream and the ultracentrifugation–RNeasy–Mini Kit procedure was best for detection on lettuce. Based on the seeding experiments, food items implicated in norovirus-associated gastroenteritis outbreaks were subjected to the optimal procedure for a specific composition and matrix. No noroviruses were detected in the implicated food items, possibly because the concentration of virus on the food item was too low or because of the presence of inhibitory factors. For each food group, a specific procedure is optimal. Inhibitory factors should be controlled in these procedures because they influence virus detection in food.

Antiviral activities of purified compounds from Youngia japonica (L.) DC (Asteraceae, Compositae)

The ethanol extract of a biannual medicinal herb, Youngia japonica (commonly known as Oriental hawk's beard) was reported previously to have potent antiviral activity against respiratory syncytial virus (RSV) cultured in HEp-2 cells. Three anti-microbial agents, namely 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and luteolin-7- O-glucoside were subsequently purified and chemically characterized from the ethanol extract of Youngia japonica. The two dicaffeoylquinic acids exhibited prominent anti-RSV with 50% inhibitory concentration (IC 50) of 0.5 μg/ml in vitro. Luteolin-7- O-glucoside together with the two dicaffeoylquinic acids were also manifested to have some antibacterial activity towards the causal agents of food-borne disease, namely Vibrio cholerae and Vibrio parahaemolyticus at the concentration of 2 mg/ml. Bacillus cereus was sensitive to 3,4-dicaffeoylquinic acid and 3,5-dicaffeoylquinic acid only, but not to luteolin-7- O-glucoside.

Vibrio vulnificus Secretes a Broad-Specificity Metalloprotease Capable of Interfering with Blood Homeostasis through Prothrombin Activation and Fibrinolysis

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhagic complications. In this study, we purified and characterized an extracellular metalloprotease (designated as vEP) having prothrombin activation and fibrinolytic activities from V. vulnificus ATCC 29307. vEP could cleave various blood clotting-associated proteins such as prothrombin, plasminogen, fibrinogen, and factor Xa, and the cleavage could be stimulated by addition of 1 mM Mn2+ in the reaction. The cleavage of prothrombin produced active thrombin capable of converting fibrinogen to fibrin. The formation of active thrombin appeared to be transient, with further cleavage resulting in a loss of activity. The cleavage of plasminogen, however, did not produce an active plasmin. vEP could cleave all three major chains of fibrinogen without forming a clot. It could cleave fibrin polymer formed by thrombin as well as the cross-linked fibrin formed by factor XIIIa. In addition, vEP could also cleave plasma proteins such as bovine serum albumin and gamma globulin, and its broad specificity is reflected in the cleavage sites, which include Asp207-Phe208 and Thr272-Ala273 bonds in prothrombin and a Tyr80-Leu81 bond in plasminogen. Taken together, the data suggest that vEP is a broad-specificity protease that could function as a prothrombin activator and a fibrinolytic enzyme to interfere with blood homeostasis as part of the mechanism associated with the pathogenicity of V. vulnificus in humans and thereby facilitate the development of systemic infection.

Induction of interleukin‐8 production via nuclear factor‐κB activation in human intestinal epithelial cells infected with Vibrio vulnificus

Vibrio vulnificus, a Gram-negative estuarine bacterium, is a causative agent of food-borne diseases, such as life-threatening septicaemia and wound infection disease. V. vulnificus penetrating into the epithelial barrier stimulates an inflammatory response in the adjacent mucosa. Therefore, interaction between V. vulnificus and epithelial cells is important for understanding of both the immunology of mucosal surfaces and V. vulnificus. In this study, we investigated the effect and action mechanism of V. vulnificus infection on production of interleukin (IL)-8, a proinflammatory cytokine, in human intestinal epithelial INT-407 cells. V. vulnificus infection significantly induced IL-8 production in a time- and multiplicity of infection (MOI)-dependent manner, as determined by human IL-8 enzyme-linked immunosorbent assay (ELISA). In addition, V. vulnificus infection significantly increased IL-8 mRNA levels in INT-407 cells, indicating that the increased IL-8 production by V. vulnificus occurred at the transcriptional level. V. vulnificus infection also enhanced IL-8 gene promoter activity in INT-407 cells transiently transfected with IL-8 promoter constructs, but this effect was impaired in INT-407 cells transfected with IL-8 promoter constructs deleted or mutated of a kappaB site. V. vulnificus infection increased the nuclear factor-kappaB (NF-kappaB) binding activity to a kappaB site and the degradation of IkappaB-alpha protein in a time- and a MOI-dependent manner. Furthermore, BAY11-7082, an inhibitor of NF-kappaB activation, significantly reduced the IL-8 production, NF-kappaB binding activity and IkappaB-alpha degradation induced by V. vulnificus infection. Taken together, these results indicate clearly that V. vulnificus infection significantly induces IL-8 production in human intestinal epithelial cells via NF-kappaB activation.

A Rapid and Simple PCR Analysis Indicates There Are Two Subgroups of Vibrio vulnificus Which Correlate with Clinical or Environmental Isolation

Vibrio vulnificus is an estuarine bacterium which is the causative agent of both food-borne disease and wound infection. Although V. vulnificus is commonly found in molluscan shellfish at high numbers, the incidence of disease is relatively low, leading to the hypothesis that not all strains of V. vulnificus are equally virulent. Unfortunately, there is currently no easy test to identify virulent strains of this species. We have previously identified a 200 bp randomly amplified polymorphic DNA (RAPD) PCR amplicon associated with clinical isolates. DNA sequence data from this locus in six clinical and four environmental isolates showed that the strains could be divided into two groups, which we termed C-type (correlates with clinical origin) and E-type (correlates with environmental origin). We designed PCR primers that could distinguish between the two groups, and typed 55 randomly selected strains. We found that 90% of the C-type strains were clinical isolates, while 93% of environmental isolates were classified as E-type. The region directly downstream of this locus contained a heptanucleotide sequence repeated various times depending on the strain. Using a PCR-based assay to detect the repeat number present in a given strain, we found a statistically significant correlation with the C/E type classification and the number of repeats. The data reported here are consistent with the existence of two genotypes of V. vulnificus, with the C-type being a strong indicator of potential virulence.

RpoS involvement and requirement for exogenous nutrient for osmotically induced cross protection in Vibrio vulnificus

Vibrio vulnificus is an opportunistic human pathogen which is the causative agent of food-borne disease and wound infections. V. vulnificus is able to adapt to a variety of potentially stressful environmental changes, such as osmotic, nutrient, and temperature variations in estuarine environments, as well as oxidative, osmotic, and acidity differences following infection of a human host. After exposure to sub-lethal levels of a particular environmental stress, many bacteria become resistant to unrelated stresses, a phenomenon termed cross protection. In this study, we examined the ability of osmotic shock to cross protect V. vulnificus to high temperature as well as oxidative stress. Log phase cells of V. vulnificus strain C7184o were cross protected by prior osmotic shock to both heat and oxidative challenge, but only when exogenous nutrient was present during the osmotic upshift. Further, and unlike other bacteria, nutrient starvation alone did not result in cross protection against either stress. When small amounts of nutrient were present during osmotic shock, cross protection to an otherwise lethal heat challenge developed extremely rapidly, with significant protection seen within 10 min. Cross protection to oxidative stress was slower to develop, requiring several hours. Although stationary phase alone conferred some cross protection to heat and oxidative stress, the alternate sigma factor RpoS was required for complete cross protection of log phase cells to oxidative stress but not for resistance to heat challenge. Together these findings suggest that the cross protective response in V. vulnificus is complex and appears to involve multiple mechanisms.

Regulation system for protease production in Vibrio vulnificus

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to consumption of raw seafoods. This human pathogen secretes a metalloprotease (VVP) that evokes enhancement of the vascular permeability and disruption of the capillaries. Production of microbial proteases is generally induced at early stationary phase of its growth. This cell density dependent regulation of VVP production in V. vulnificus known to be the quorum-sensing. When V. vulnificus was cultivated in Luria–Bertani (LB) medium, accumulation of the autoinducer, the signal molecule operating the quorum-sensing system, was detected. Moreover, expression of the vvp gene encoding VVP was found to be closely related with expression of the luxS gene that encode the synthase of the autoinducer precursor ( luxS). These findings may indicate VVP production is controlled by the quorum-sensing system in LB medium. Futhermore, this system functioned more effectively at 26 °C than at 37 °C. When incubated at 37 °C in human serum supplemented with ferric chloride, production of VVP and expression of vvp increased in proportion to the concentration of ferric ion; whereas, expression of luxS was not increased. This suggests that VVP production in human serum containing ferric ion may be regulated mainly by the system other than the quorum-sensing system.

Analysis of the action of compounds that inhibit the germination of spores of Bacillus species

To determine the mechanism of action of inhibitors of the germination of spores of Bacillus species, and where these inhibitors act in the germination process. Spores of various Bacillus species are significant agents of food spoilage and food-borne disease, and inhibition of spore germination is a potential means of reducing such problems. Germination of the following spores was studied: (i) wild-type B. subtilis spores; (ii) B. subtilis spores with a nutrient receptor variant allowing recognition of a novel germinant; (iii) B. subtilis spores with elevated levels of either the variant nutrient receptor or its wild-type allele; (iv) B. subtilis spores lacking all nutrient receptors and (v) wild-type B. megaterium spores. Spores were germinated with a variety of nutrient germinants, Ca2+-dipicolinic acid (DPA) and dodecylamine for B. subtilis spores, and KBr for B. megaterium spores. Compounds tested as inhibitors of germination included alkyl alcohols, a phenol derivative, a fatty acid, ion channel blockers, enzyme inhibitors and several other compounds. Assays used to assess rates of spore germination monitored: (i) the fall in optical density at 600 nm of spore suspensions; (ii) the release of the dormant spore's large depot of DPA; (iii) hydrolysis of the dormant spore's peptidoglycan cortex and (iv) generation of CFU from spores that lacked all nutrient receptors. The results with B. subtilis spores allowed the assignment of inhibitory compounds into two general groups: (i) those that inhibited the action of, or response to, one nutrient receptor and (ii) those that blocked the action of, or response to, several or all of the nutrient receptors. Some of the compounds in groups 1 and 2 also blocked action of at least one cortex lytic enzyme, however, this does not appear to be the primary site of their action in inhibiting spore germination. The inhibitors had rather different effects on germination of B. subtilis spores with nutrients or non-nutrients, consistent with previous work indicating that germination of B. subtilis spores by non-nutrients does not involve the spore's nutrient receptors. In particular, none of the compounds tested inhibited spore germination with dodecylamine, and only three compounds inhibited Ca2+-DPA germination. In contrast, all compounds had very similar effects on the germination of B. megaterium spores with either glucose or KBr. The effects of the inhibitors tested on spores of both Bacillus species were largely reversible. This work indicates that inhibitors of B. subtilis spore germination fall into two classes: (i) compounds (most alkyl alcohols, N-ethylmaleimide, nifedipine, phenols, potassium sorbate) that inhibit the action of, or response to, primarily one nutrient receptor and (ii) compounds [amiloride, HgCl2, octanoic acid, octanol, phenylmethylsulphonylfluoride (PMSF), quinine, tetracaine, tosyl-l-arginine methyl ester, trifluoperazine] that inhibit the action of, or response to, several nutrient receptors. Action of these inhibitors, is reversible. The similar effects of inhibitors on B. megaterium spore germination by glucose or KBr indicate that inorganic salts likely trigger germination by activating one or more nutrient receptors. The lack of effect of all inhibitors on dodecylamine germination suggests that this compound stimulates germination by creating channels in the spore's inner membrane allowing DPA release. This work provides new insight into the steps in spore germination that are inhibited by various chemicals, and the mechanism of action of these inhibitors. The work also provides new insights into the process of spore germination itself.

Effect of feeding the ionophores monensin and laidlomycin propionate and the antimicrobial bambermycin to sheep experimentally infected with E. coli O157:H7 and Salmonella typhimurium.

Escherichia coli O157:H7 and Salmonella are widely recognized as important agents of foodborne disease with worldwide distribution. The use of ionophores in feeding growing ruminants is widespread in the United States and has attracted recent interest due to the apparent temporal relationship between initial ionophore use and the increase in human E. coli O157:H7 cases. Two experiments were conducted to evaluate the effects of short-term feeding of ionophores on fecal shedding, intestinal concentrations, and antimicrobial susceptibility of E. coli O157:H7 and S. typhimurium in growing lambs. Sixteen lambs were used in each experiment, four lambs per treatment group: monensin, laidlomycin propionate, bambermycin, and a control treatment. Lambs were fed a grain and hay (50:50) diet with their respective ionophore for 12 d before experimental inoculation with E. coli O157:H7 or S. typhimurium. Animals were maintained on their respective diets an additional 12 d, and fecal shedding of inoculated pathogens was monitored daily. Lambs were killed and tissues and contents were sampled from the rumen, cecum, and rectum. No differences (P > 0.05) in fecal shedding of Salmonella or E. coli O157:H7 were observed due to treatment. Occurrence of Salmonella or E. coli in luminal contents and tissue samples from the rumen, cecum, and rectum did not differ (P > 0.05) among treatments. Feeding monensin decreased (P < 0.05) the incidence of scours in sheep infected with Salmonella compared with the other treatments. No differences in antimicrobial susceptibility were found in any of Salmonella or E. coli O157:H7 isolates. Results from these studies indicate that short-term ionophore feeding had very limited effects on E. coli and Salmonella shedding or on antimicrobial susceptibility in experimentally infected lambs.

Diagnóstico e investigación epidemiológica de un brote de toxiinfección alimentaria causado por Clostridium perfringens

Clostridium perfringens is a classical agent of food-borne disease but because of the mildness and self-limiting nature of the illness, many cases are undiagnosed. This study describes the investigation of an outbreak of diarrhea due to C. perfringens in a public restaurant. An epidemiological survey was performed and the restaurant was inspected. The specific attack rates for the items on the menu were calculated. Odds ratios were calculated to investigate the independent association between each item and the disease using a logistic regression model. Investigation of C. perfringens toxin in the feces of four symptomatic subjects and one exposed but asymptomatic subject was performed by the reverse passive latex agglutination test. The overall attack rate was 70.8%. The main symptoms were diarrhea (100%) and abdominal pain (94%). Significant differences were found in specific attack rates for consumption of different menu items. However, the independent contribution of each item was significant only for consumption of "ravioli with cheese sauce". Fecal detection of C. perfringens enterotoxin was positive in the four symptomatic subjects and negative in the exposed but asymptomatic subject. The overall attack rate in this outbreak was high. The clinical symptomatology was similar to previously published data. The epidemiological analysis revealed "ravioli with cheese sauce" to be responsible for transmission of the disease and clinical investigation together with the fecal enterotoxin detection established C. perfringens as the etiological agent.

Durable and Regenerable Antibacterial Finishing of Fabrics with a New Hydantoin Derivative

Durable and regenerable antibacterial fabrics were prepared by using an innovative chemical technology employing a precursor biocidal agent, dimethylol dimethylhydantoin (DMDMH), in a chemical finishing process. The method resulted in significant add-on rates of hydantoin groups on cellulose and established a durable antimicrobial functionality, once the grafted heterocyclic compounds were chlorinated by diluted chlorine bleaching. Both cotton fabrics and polyester/cotton fabrics exposed to treatment baths containing from 2 to 10% of DMDMH acquired a powerful inactivating capacity against a wide range of food-borne and water-borne infectious disease agents. The biocidal functions are regenerable by regular laundry exposure to chlorine bleach and can withstand over 50 standard machine washes without appreciable deterioration. In addition to their powerful antimicrobial efficacy, the fabrics exhibited improved wrinkle resistance and maintained appropriate mechanical properties, making them ideal for medical and hygienic textile applications. In this article we report the results from biocidal tests and durability evaluations and provide data characterizing physical attributes of the treated fabrics.