Interactions of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes plants cultivated in a gnotobiotic system

The growth and persistence of Escherichia coli O157:H7, Salmonella typhimurium and Listeria monocytogenes on a diverse range of plant types over extended cultivation periods was studied. When introduced on the seed of carrot, cress, lettuce, radish, spinach and tomato all the pathogens became rapidly established shortly after germination, attaining cell densities of the order of 5.5-6.5 log cfu/g. In general, Es. coli O157:H7 and L. monocytogenes became established and persisted at significantly higher levels on seedlings (9 days post-germination) than Salmonella. Es. coli O157:H7 became internalized in cress, lettuce, radish and spinach seedlings but was not recovered within the tissues of mature plants. Internalization of Salmonella was also observed in lettuce and radish but not cress or spinach seedlings. In contrast, L. monocytogenes did not internalize within seedlings but did persist on the surface of plants throughout the cultivation period. Co-inoculation of isolates recovered from the rhizosphere of plants did not significantly affect the numbers or persistence of human pathogens. The only exception was with Enterobacter cloacae, which reduced Es. coli O157:H7 Ph1 and L. monocytogenes levels by ca. 1 log cfu/g on lettuce. With the bioluminescent phenotype of Es. coli O157:H7 Ph1, it was demonstrated that the human pathogen became established on the roots of growing plants. Scanning electron micrographs of root seedlings suggested that Es. coli O157:H7 Ph1 preferentially colonized the root junctions of seedlings. It is proposed that such colonization sites enhanced the persistence of Es. coli O157:H7 on plants and facilitated internalization within developing seedlings. The results suggest that the risk associated with internalized human pathogens in salad vegetables at harvest is low. Nevertheless, the introduction of human pathogens at an early stage of plant development could enhance their persistence in the rhizosphere. The implications of the study with regards to on-farm food safety initiatives are discussed.     

Effectiveness of cleaners and sanitizers in killing Salmonella Newport in the gut of a free-living nematode, Caenorhabditis elegans

Caenorhabditis elegans, a free-living nematode found in soil, has been shown to ingest human enteric pathogens, thereby potentially serving as a vector for preharvest contamination of fruits and vegetables. A study was undertaken to evaluate the efficacy of cleaners and sanitizers in killing Salmonella enterica serotype Newport in the gut of C. elegans. Adult worms were fed nalidixic acid-adapted cells of Escherichia coli OP50 (control) or Salmonella Newport for 24 h, washed, placed on paper discs, and incubated at temperatures of 4 or 20 degrees C and relative humidities of 33 or 98% for 24 h. Two commercial cleaners (Enforce and K Foam Lo) and four sanitizers (2% acetic acid, 2% lactic acid, Sanova, and chlorine [50 and 200 microg/ml]) were applied to worms for 0, 2, or 10 min. Populations of E. coli and Salmonella Newport (CFU per worm) in untreated and treated worms were determined by sonicating worms in 0.1% peptone and surface plating suspensions of released cells on tryptic soy agar containing nalidixic acid. Populations of Salmonella Newport in worms exposed to 33 or 98% relative humidity at 4 degrees or 33% relative humidity at 20 degrees C were significantly (P < or = 0.05) lower than the number surviving exposure to 98% relative humidity at 20 degrees C. In general, treatment of desiccated worms with cleaners and sanitizers was effective in significantly (P < or = 0.05) reducing the number of ingested Salmonella Newport. Results indicate that temperature and relative humidity influence the survival of Salmonella Newport in the gut of C. elegans, and cleaners and sanitizers may not eliminate the pathogen.     

Iceberg lettuce as suggested source of a nationwide outbreak caused by two Salmonella serotypes, Newport and Reading, in Finland in 2008

A nationwide outbreak of Salmonella enterica serotypes Newport and Reading occurred between 17 October and 28 November 2008 in Finland. A total of 77 culture-confirmed Salmonella Newport and 30 Salmonella Reading cases, including one case with a double infection, were reported. All strains isolated from the patients were subtyped using serotyping, microbial resistance profiling, and pulsed-field gel electrophoresis (PFGE). Here, the PFGE patterns of the studied Salmonella Newport strains were identical, whereas four different PFGE profiles were found among the Salmonella Reading strains. Two elderly patients died within 2 weeks of the onset of symptoms. Three geographical clusters of cases with an epidemiological link were identified. The traceback investigation suggested that the factor connecting the cases was ready-chopped iceberg lettuce available for mass catering use. However, none of the tested food, environmental samples, or the samples taken from the staff of the processing plant contained Salmonella bacteria. Tracing back to outbreak sources with a short shelf life can be complex.     

Potential of a plant-parasitic nematode to facilitate internal contamination of tomato plants by Salmonella

The objective of this study was to determine whether tomato plants infested with a plant-parasitic nematode, Meloidogne incognita, can internalize Salmonella. Tomato plants (Lycopersicon esculentum Mill. 'Rutgers') were grown in soil infested with M. incognita and/or inoculated with a six-serotype mixture of Salmonella enterica. M. incognita, upon wounding roots when parasitizing the tomato plant, does not result in the entry and survival of Salmonella. Analysis of roots, galls, stems, and leaves 2 and 4 weeks after inoculation of the soil failed to reveal the presence of Salmonella. Salmonella remained viable in soil for at least 4 weeks. The potential for the presence of Salmonella in the tissues of tomato fruits via root entrance facilitated by M. incognita appears to be remote.     

Competitive inhibition bacteria of bovine origin against Salmonella serovars

Studies were conducted to isolate bacteria inhibitory to Salmonella enterica serovar Typhimurium definitive type (DT) 104 in vitro from cattle not carrying Salmonella and to determine the inhibitory activity of the isolated bacteria through competitive growth in cattle feces artificially contaminated with Salmonella Typhimurium DT104 and S. enterica serovar Newport. Fecal samples (108) were obtained from dairy and beef cows. S. enterica serovars were isolated from 9.25% of the samples and included Salmonella Newport (4), Salmonella Bareilly (1), Salmonella Mbandaka (1), Salmonella Montevideo (1), Salmonella Meleagridis (1), and monophasic Salmonella (2). All four Salmonella Newport isolates were resistant to at least nine antibiotics. Of 1,097 bacterial isolates from cattle feces screened, 30 were inhibitory to Salmonella Typhimurium DT104 in vitro. The inhibitory isolates included 22 Escherichia coli, 6 Bacillus circulans, 1 Serratia fonticola, and 1 Enterobacter cloacae. Typing by pulsed-field gel electrophoresis showed 17 distinguishable profiles among the 22 E. coli. Competitive inhibition isolates did not significantly reduce Salmonella Typhimurium DT104 during 21 days of storage at 37 degrees C in cattle feces. B. circulans (10(5) CFU/g of inoculum) significantly reduced Salmonella Newport on days 3 and 5 and on day 21 with 10(8) CFU/g of inoculum at 37 degrees C. At 21degrees C, significant reductions of Salmonella Typhimurium DT104 occurred with 10(8) CFU of gram-negative competitive inhibition bacteria per g and 10(5) CFU of B. circulans per g on day 5 only. No significant reductions were observed with Salmonella Newport at 21 degrees C. The 25 competitive inhibition bacteria identified in this study offer a first step in identifying competitive inhibition bacteria that may reduce the level of intestinal carriage and fecal shedding of Salmonella Typhimurium DT104 and Salmonella Newport in cattle.     

Antimicrobial activity of apple, hibiscus, olive, and hydrogen peroxide formulations against Salmonella enterica on organic leafy greens

Salmonella enterica is one of the most common bacterial pathogens implicated in foodborne outbreaks involving fresh produce in the last decade. In an effort to discover natural antimicrobials for use on fresh produce, the objective of the present study was to evaluate the effectiveness of different antimicrobial plant extract-concentrate formulations on four types of organic leafy greens inoculated with S. enterica serovar Newport. The leafy greens tested included organic romaine and iceberg lettuce, and organic adult and baby spinach. Each leaf sample was washed, dip inoculated with Salmonella Newport (10(6) CFU/ml), and dried. Apple and olive extract formulations were prepared at 1, 3, and 5% concentrations, and hibiscus concentrates were prepared at 10, 20, and 30%. Inoculated leaves were immersed in the treatment solution for 2 min and individually incubated at 4°C. After incubation, samples were taken on days 0, 1, and 3 for enumeration of survivors. Our results showed that the antimicrobial activity was both concentration and time dependent. Olive extract exhibited the greatest antimicrobial activity, resulting in 2- to 3-log CFU/g reductions for each concentration and type of leafy green by day 3. Apple extract showed 1- to 2-log CFU/g reductions by day 3 on various leafy greens. Hibiscus concentrate showed an overall reduction of 1 log CFU/g for all leafy greens. The maximum reduction by hydrogen peroxide (3%) was about 1 log CFU/g. The antimicrobial activity was also tested on the background microflora of organic leafy greens, and reductions ranged from 0 to 2.8 log. This study demonstrates the potential of natural plant extract formulations to inactivate Salmonella Newport on organic leafy greens.     

Rate of change of composition of lettuce in response to nitrogen depletion or re‐supply

BACKGROUND: Lettuce accumulates high levels of nitrate when grown in winter in heated greenhouses. Temporarily removing nitrate from the nutrient solution may lower tissue nitrate. The composition of hydroponic lettuce was examined over a 6‐ to 13‐day interval of nitrogen depletion or re‐supply. RESULTS: Growth responses were delayed by 6 days or more after changing N supply, except root growth responded in 3–4 days. The total nitrogen increased or decreased for 6 days. When measured in the light, nitrate concentration changed 10‐fold in roots within 2 days. The change in leaf and petiole tissue was slower. Amino acids responded to a change in N‐supply within 1 day in leaf and root. All tissues of N‐depleted plants had more sugars compared to N‐sufficient plants within 2 days after the start of depletion. Nitrogen depletion had reversible effects on growth or tissue concentrations if applied for only 6 days. CONCLUSIONS: Tissue nitrate changed more rapidly than total nitrogen, or growth responses. However, nitrate changed more slowly in petiole than in leaf blade or roots, and larger plants have more biomass in petioles. Thus, clearing nitrate from lettuce is slower for large compared to smaller lettuce plants. Copyright © 2012 Society of Chemical Industry     

Uptake of arsenic species by turnip (Brassica rapa L.) and lettuce (Lactuca sativa L.) treated with roxarsone and its metabolites in chicken manure

Roxarsone is an organoarsenic feed additive that can be metabolised to other higher toxic arsenic (As) species in animal manure such as arsenate, arsenite, monomethylarsonic acid, dimethylarsinic acid, 3-amino-4-hydroxyphenylarsonic acid and other unknown As species. The accumulation, transport and distribution of As species in turnip (Brassica rapa L.) and lettuce (Lactuca sativa L.) amended with roxarsone and its metabolites in chicken manure were investigated. Results showed arsenite was the predominant As form, followed by arsenate in turnip and lettuce plants, and a low content of dimethylarsinic acid was detected only in lettuce roots. Compared with the control plants treated with chicken manure without roxarsone and its metabolites, the treatments containing roxarsone and its metabolites increased arsenite content by 2.0–3.2% in turnip shoots, by 6.6–6.7% in lettuce shoots, by 11–44% in turnip tubers and by 18–20% in lettuce roots at two growth stages. The enhanced proportion of arsenate content in turnip shoots, turnip tubers and lettuce roots was 4.3–14%, 20–35% and 70%, respectively, while dimethylarsinic acid content in lettuce roots increased 2.4 times. Results showed that the occurrence of dimethylarsinic acid in lettuce roots might be converted from the inorganic As species and the uptake of both inorganic and organic As compounds in turnip and lettuce plants would be enhanced by roxarsone and its metabolites in chicken manure. The pathway of roxarsone metabolites introduced into the human body via roxarsone → animal → manure → soil → crop was indicated.     

Persistence of Escherichia coli O157:H7, Salmonella Newport, and Salmonella Poona in the gut of a free-living nematode, Caenorhabditis elegans, and transmission to progeny and uninfected nematodes

A study was undertaken to determine the persistence of Escherichia coli O157:H7 and salmonellae in the gut of a free-living nematode, Caenorhabditis elegans, as affected by temperature and relative humidity and to determine if infected worms transmit Salmonella enterica serotype Newport to progeny and uninfected worms. Worms were fed cells of a non-pathogenic strain of E. coli (OP50), E. coli O157:H7, S. enterica serotype Newport, and S. enterica serotype Poona, followed by incubating at 4, 20, or 37 degrees C for up to 5 days. Initial populations of ingested pathogens significantly increased by up to 2.93 log(10) cfu/worm within 1 day at 20 degrees C on K agar and remained constant for an additional 4 days. When worms were placed on Bacto agar, populations of ingested pathogens remained constant at 4 degrees C, decreased significantly at 20 degrees C, and increased significantly at 37 degrees C within 3 days. Worms fed E. coli OP50 or S. Newport were incubated at 4 or 20 degrees C at relative humidities of 33%, 75%, or 98% to determine survival characteristics of ingested bacteria. Fewer cells of the pathogens survived incubation at 33% relative humidity compared to higher relative humidities. Populations of ingested E. coli OP50 and S. Newport decreased by up to 1.65 and 3.44 log(10) cfu/worm, respectively, in worms incubated at 20 degrees C and 33% relative humidity. Placement together on K agar of adult worms, labeled with green fluorescent protein (gfp) in the pharynx area, that had ingested gfp-labeled S. Newport and uninfected wild type worms resulted in transfer of the pathogen to gut of wild type worms. S. Newport was isolated from C. elegans two generations removed from exposure to the pathogen. Results of these studies show that C. elegans may serve as a temporary reservoir of foodborne pathogens, and could perhaps be a vector for contaminating preharvest fruits and vegetables, thus potentially increasing the risk of enteric infections associated with consumption of raw produce.     

Migration of Caenorhabditis elegans to manure and manure compost and potential for transport of Salmonella newport to fruits and vegetables

A study was done to determine if a free-living, bacterivorous nematode, Caenorhabditis elegans, migrates to bovine manure, turkey manure, composted bovine manure, composted turkey manure, and manure-amended soil inoculated with Salmonella Newport. Movement of the worm to lettuce, strawberries, and carrots was also studied. C. elegans moved most rapidly to turkey manure and strawberries, with 35% and 60% of worms, respectively, associating with samples within 30 min. Survival and reproduction of C. elegans in test materials were not affected by the presence of S. newport. Bovine manure and bovine manure compost inoculated with S. newport (8.6 log10 CFU/g) were separately placed in the bottom of a glass jar and covered with a layer of soil (5 cm) inoculated (50 worms/g) or not inoculated with C. elegans. A piece of lettuce, strawberry, or carrot was placed on top of the soil before jars were sealed and held at 20 degrees C for up to 10 days. In the system using soil inoculated with C. elegans, S. newport initially in bovine manure was detected on the surface of lettuce, strawberry, and carrot samples within 3, 1, and 1 days, respectively. The pathogen was detected on lettuce, strawberry, and carrot within 1, 7, and 1 days, respectively, when initially present in bovine manure compost. With one exception, the pathogen was not detected on the produce over the 10-day incubation period when C. elegans was not present in the soil. Results indicate that C. elegans has the potential for transporting S. newport in soil to the surface of preharvest fruits and vegetables in contact with soil.     

Association of Escherichia coli O157:H7 with preharvest leaf lettuce upon exposure to contaminated irrigation water.

Recent foodborne outbreaks have linked infection by enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 to the consumption of contaminated lettuce. Contamination via food handler error and on-the-farm contamination are thought to be responsible for several outbreaks. Though recent studies have examined the application of EHEC to store-bought lettuce, little is known about the attachment of EHEC to growing plants. We investigated the association of lettuce seedlings with EHEC O157:H7 strains implicated in lettuce or fruit outbreaks using hydroponic and soil model systems. EHEC strains that express the green fluorescent protein were observed by stereomicroscopy and confocal laser scanning microscopy to determine adherence patterns on growing lettuce seedlings. Bacteria adhered preferentially to plant roots in both model systems and to seed coats in the hydroponic system. Two of five nonpathogenic E. coli strains showed decreased adherence to seedling roots in the hydroponic system. EHEC was associated with plants in as few as 3 days in soil, and contamination levels were dose-dependent. EHEC levels associated with young plants inoculated with a low dose suggested that the bacteria had multiplied. These data suggest that preharvest crop contamination via contaminated irrigation water can occur through plant roots.     

Evidence for internalization of Escherichia coli into the aerial parts of maize via the root system

Escherichia coli introduced into the hydroponic growing medium of maize plants was detected 48 h later in the shoot. Decapitation of root tips or severing of the plant root system at the root-shoot junction enhanced bacterial internalization. The density of the bacteria in shoots of plants with damaged roots or removed root systems was 27.8 and 23.9 times higher than that in plants with intact roots, respectively. The concentration of viable cells in the hydroponic solution decreased over time from 9.3 x 10(6) CFU/ml at the time of inoculation to 8.5 x 10(1) CFU/ml 4 days thereafter. The number of E. coli cells associated with the roots also decreased with time, but a significant decline appeared only at 4 days postinoculation. At the time of sampling for E. coli presence in the shoot, 10(2) CFU/ml was present in the nutrient solution and 8 x 10(3) CFU/g was associated with the roots. The present study is the first to demonstrate internalization of E. coli via the root in a monocotyledonous plant.     

Survival and growth of Salmonella baildon in shredded lettuce and diced tomatoes, and effectiveness of chlorinated water as a sanitizer

An outbreak of salmonellosis associated with diced tomatoes occurred in the United States in 1999. Experiments were done to determine the efficacy of chlorine in killing Salmonella baildon, the causative serotype, inoculated onto shredded lettuce and diced tomatoes, and to determine survival characteristics of the organism on these produce items stored at 4 degrees C for up to 12 days and on tomatoes stored at 21 or 30 degrees C for up to 72 h. Populations of S. baildon in lettuce and tomatoes (pH 4.51 +/- 0.02) inoculated with 3.60 log10 and 3.86 log10 cfu/g, respectively, were reduced by less than 1 log when the produce was immersed for 40 s in a 120 or 200 microg/ml free chlorine solution. Produce inoculated with 0.60-0.86 log10 cfu/g was positive for the pathogen after treatment with 200 microg/ml chlorine. Initial populations of 3.28 and 3.40 log10 cfu/g of lettuce and tomatoes, respectively, decreased by about 2 log10 cfu/g during storage for 12 days at 4 degrees C. One of six samples of lettuce initially containing 0.28 log10 cfu of S. baildon per gram was positive after storage for 12 days, but the pathogen was not detected in tomatoes analyzed within 15 min of inoculation with 0.40 log10 cfu/g. While the number of viable cells decreased during storage at 4 degrees C, initial populations of 0.28 log10 cfu/g of shredded lettuce and 3.40 log10 cfu/g of diced tomatoes are not reduced to undetectable levels during storage at 4 degrees C for 12 days. Tolerance of S. baildon to an acidic pH (4.5) was not influenced by the pH (4.5, 5.8, or 7.2) of the medium in which it was grown, suggesting that this strain possesses unusual resistance to acid pH. The pathogen grew in diced tomatoes (pH 4.40 +/- 0.01) from an initial population of 0.79 log10 cfu/g to 5.32 and 7.00 log10 cfu/g within 24 h at 21 and 30 degrees C, respectively.     

Microbiological study of ready-to-eat salad vegetables from retail establishments uncovers a national outbreak of salmonellosis

The increasing availability of bagged prepared salad vegetables reflects consumer demand for fresh, healthy, convenient, and additive-free foods that are safe and nutritious. During May and June 2001 a study of retail bagged prepared ready-to-eat salad vegetables was undertaken to determine the microbiological quality of these vegetables. Examination of the salad vegetables revealed that the vast majority (3,826 of 3,852 samples; 99.3%) were of satisfactory or acceptable microbiological quality according to Public Health Laboratory Service microbiological guidelines, while 20 (0.5%) samples were of unsatisfactory microbiological quality. Unsatisfactory quality was due to Escherichia coli and Listeria spp. (not Listeria monocytogenes) levels in excess of 10(2) CFU/g. However, six (0.2%) samples were of unacceptable microbiological quality because of the presence of Salmonella (Salmonella Newport PT33 [one sample], Salmonella Umbilo [three samples], and Salmonella Durban [one sample]) or because of a L. monocytogenes level of 660 CFU/g, which indicates a health risk. In each case, the retailer involved and the UK Food Standards Agency were immediately informed, and full investigations were undertaken. Nineteen cases of Salmonella Newport PT33 infection were subsequently identified throughout England and Wales. The outbreak strain of Salmonella Newport PT33 isolated from the salad and from humans had a unique plasmid profile. Campylobacter spp. and E. coli O157 were not detected in any of the samples examined. The presence of Salmonella, as well as high levels of L. monocytogenes, is unacceptable. However, minimally processed cut and packaged salad is exposed to a range of conditions during growth, harvest, preparation, and distribution, and it is possible that these conditions may increase the potential for microbial contamination, highlighting the necessity for the implementation of good hygiene practices from farm to fork to prevent contamination and/or bacterial growth in these salad products.     

Transcriptional and functional responses of Escherichia coli O157:H7 growing in the lettuce rhizoplane

Lettuce and spinach are increasingly implicated in foodborne illness outbreaks due to contamination by Escherichia coli O157:H7. While this bacterium has been shown to colonize and survive on lettuce leaf surfaces, little is known about its interaction with the roots of growing lettuce plants. In these studies, a microarray analyses, mutant construction and confocal microscopy were used to gain an understanding of structure and function of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots. After three days of interaction with lettuce roots, 94 and 109 E. coli O157:H7 genes were significantly up- and down-regulated at least 1.5 fold, respectively. While genes involved in biofilm modulation (ycfR and ybiM) were significantly up-regulated, 40 of 109 (37%) of genes involved in protein synthesis were significantly repressed. E. coli O157:H7 was 2 logs less efficient in lettuce root colonization than was E. coli K12. We also unambiguously showed that a ΔycfR mutant of E. coli O157:H7 was unable to attach to or colonize lettuce roots. Taken together these results indicate that bacterial genes involved in attachment and biofilm formation are likely important for contamination of lettuce plants with Shiga toxin-producing E. coli strains.     

Interaction of Escherichia coli with growing salad spinach plants

In this study, the interaction of a bioluminescence-labeled Escherichia coli strain with growing spinach plants was assessed. Through bioluminescence profiles, the direct visualization of E. coli growing around the roots of developing seedlings was accomplished. Subsequent in situ glucuronidase (GUS) staining of seedlings confirmed that E. coli had become internalized within root tissue and, to a limited extent, within hypocotyls. When inoculated seeds were sown in soil microcosms and cultivated for 42 days, E. coli was recovered from the external surfaces of spinach roots and leaves as well as from surface-sterilized roots. When 20-day-old spinach seedlings (from uninoculated seeds) were transferred to soil inoculated with E. coli, the bacterium became established on the plant surface, but internalization into the inner root tissue was restricted. However, for seedlings transferred to a hydroponic system containing 10(2) or 10(3) CFU of E. coli per ml of the circulating nutrient solution, the bacterium was recovered from surface-sterilized roots, indicating that it had been internalized. Differences between E. coli interactions in the soil and those in the hydroponic system may be attributed to greater accessibility of the roots in the latter model. Alternatively, the presence of a competitive microflora in soil may have restricted root colonization by E. coli. The implications of this study's findings with regard to the microbiological safety of minimally processed vegetables are discussed.     

Inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce by chlorine dioxide gas

The purpose of this investigation was to study inactivation kinetics of inoculated Escherichia coli O157:H7 and Salmonella enterica on lettuce leaves by ClO(2) gas at different concentrations (0.5, 1.0, 1.5, 3.0, and 5.0 mg l(-1)) for 10 min and to determine the effect of ClO(2) gas on the quality and shelf life of lettuce during storage at 4 degrees C for 7 days. One hundred microliters of each targeted organism was separately spot-inoculated onto the surface (5 cm(2)) of lettuce (approximately 8-9 log CFU ml(-1)), air-dried, and treated with ClO(2) gas at 22 degrees C and 90-95% relative humidity for 10 min. Surviving bacterial populations on lettuce were determined using a membrane transferring method, which included a non-selective medium followed by a selective medium. The inactivation kinetics of E. coli O157:H7 and S. enterica was determined using first-order kinetics to establish D-values and z-values. The D-values of E. coli and S. enterica were 2.9+/-0.1 and 3.8+/-0.5 min, respectively, at 5.0 mg l(-1) ClO(2) gas. The z-values of E. coli and S. enterica were 16.2+/-2.4 and 21.4+/-0.5 mg l(-1), respectively. A 5 log CFU reduction (recommended by the United States Food and Drug Administration) for E. coli and S. enterica could be achieved with 5.0 mg l(-1) ClO(2) gas for 14.5 and 19.0 min, respectively. Treatment with ClO(2) gas significantly reduced inherent microflora on lettuce and microbial counts remained significantly (p<0.05) lower than the uninoculated control during storage at 4 degrees C for 7 days. However, treatment with ClO(2) gas had a significantly (p<0.05) negative impact on visual leaf quality. These results showed that treatment with ClO(2) gas significantly reduced selected pathogens and inherent microorganisms on lettuce; however, the processing conditions would likely need to be altered for consumer acceptance.     

Differential killing activity of cetylpyridinium chloride with or without bacto neutralizing buffer quench against firmly adhered Salmonella gaminara and Shigella sonnei on cut lettuce stored at 4 degrees C

Cetylpyridinium chloride (CPC) activity was quenched with Bacto neutralizing buffer on inoculated cut iceberg lettuce. This protocol permitted comparison of the numbers of Salmonella Gaminara- or Shigella sonnei-inoculated cells on lettuce that survived 1 min of CPC treatment. Cut lettuce was inoculated with about 6 log of Salmonella or 9 log of Shigella and stored in Whirl-Pak bags at 4 degrees C for up to 4 days. Loosely adhered pathogen cells were washed off before CPC treatment. Firmly adhered cells of Salmonella Gaminara or S. sonnei on cut iceberg lettuce survived treatment with CPC even at the 0.4% CPC level if the CPC activity was quenched after 1 min by adding Bacto neutralizing buffer. The results confirm that there is extended killing activity of residual CPC against Salmonella Gaminara or S. sonnei if the residual CPC remaining in contact with the lettuce after the initial 1-min wash is not quenched. The CPC treatment was useful in reducing the numbers of these target pathogens on lettuce.     

Effect of rearing temperature on the fecundity and development of Euzopherodes vapidella Mann (Lepidoptera: Pyralidae), a pest of stored yam

The fecundity and development of the yam moth, Euzopherodes vapidella Mann on Dioscorea alata L. was investigated in the laboratory at four different temperatures, 20, 24, 29 and 33 degrees C. The mean fecundity per female at 20, 24, 29 and 33 degrees C was 51.8+/-3.5, 102.4+/-3.8, 123.3+/-4.4 and 124.4 +/-4.4 eggs, respectively. Hatchability of eggs was highest at 29 degrees C and lowest at 20 degrees C. The mean developmental time at 20, 24, 29 and 33 degrees C was 12.1+/-0.6, 6.2+/-0.3, 3.0+/-0.0 and 2.7+/-0.1 days for the egg, 23.6+/-1.1, 20.0+/-0.9, 15.4+/-0.7 and 12.9+/-0.4 days for the larval stages, 13.0+/-0.03, 8.9+/-0.02, 7.9+/-0.02, and 6.4+/-0.03 days for the pupa and 48.7+/-3.5, 35.1+/-2.3, 26.3+/-1.2 and 22.0+/-1.0 days for the period from egg to adult emergence, respectively. The developmental threshold for the egg stage was estimated as 16.8 degrees C with thresholds of 8.0, 6.2 and 11.4 degrees C for larvae, pupae, and egg to adult emergence, respectively. Storage of yam tubers at low temperatures (but higher than 12 degrees C to avoid damage to tubers) will significantly retard the development of E. vapidella and therefore help in their control. Adult males ranged from 0.50 to 0.65cm in length and females from 0.70 to 0.90cm.     

Inhibitory effects of competitive exclusion and fructooligosaccharide, singly and in combination, on Salmonella colonization of chicks

The inhibitory effects of competitive exclusion (CE) and 0.1% concentration of fructooligosaccharide (FOS), singly and in combination, on Salmonella colonization of chicks were investigated. Moreover, quantitation of the major cecal flora (Bifidobacterium, Bacteroides, Lactobacillus, and Escherichia coli) was performed. One-day-old birds were divided into four groups: (i) control, (ii) CE, (iii) FOS, and (iv) CE plus FOS. Chicks received Salmonella Enteritidis at 7 days (experiment 1) or 21 days (experiment 2). Birds in each group were killed at 1 day, 7 days, and 14 days after inoculation of Salmonella Enteritidis for count of salmonella in cecal contents. In experiment 1, the mean number of Salmonella Enteritidis in the chicks inoculated with CE was significantly decreased compared with the other three groups at 1 day postinoculation. In experiment 2, the mean numbers of Salmonella Enteritidis in the chicks of the FOS group and the FOS plus CE group were significantly decreased compared with the control group at 1 day and 7 days postinoculation. On 7- and 21-day-old chicks, few changes on number of total bacteria, Bifidobacterium, Bacteroides, Lactobacillus, and E. coli were observed in the cecal contents of treated groups compared with the control group. Low-dose feeding of FOS in the diet of chicks with a CE treatment may result in reduced susceptibility to Salmonella colonization but may not lead to a shift in the intestinal gut microflora on 7- and 21-day-old chicks.