Application of antimicrobial-producing lactic acid bacteria to control pathogens in ready-to-use vegetables

Five psychrotrophic strains of lactic acid bacteria (Lactobacillus casei, Lact. plantarum and Pediococcus spp.) were isolated from 22 samples of commercial salads. These strains were shown to inhibit Aeromonas hydrophila, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus on MRS agar, in salads and in juice prepared from vegetable salads. Lactobacillus casei IMPCLC34 was most effective in reducing total mesophilic bacteria and the coliform group; Aer. hydrophila, Salm. typhimurium and Staph. aureus disappeared after 6 d of storage, while the counts for L. monocytogenes remained constant. The potential application of antimicrobial-producing lactic acid bacteria as biopreservatives of ready-to-use vegetables is suggested.     

Population dynamics of Aeromonas spp. in an urban river watershed

Density of Aeromonas spp. at one site in the Buffalo River and at four sites on its upstream tributaries was followed from June 1992–June 1993. Membrane filtration counts of Aeromonas during the summer ranged between 18 and 4000 ml⁻¹, which were one to two logs higher than faecal coliform and faecal streptococci densities. Aeromonas spp. in the Buffalo River, and faecal coliforms, faecal streptococci, and the heterotrophic plate count throughout the watershed, increased by approximately one log during summer rainstorms. However, Aeromonas spp. increased only by a factor of two during rainstorms at the upstream sites. Aeromonas spp. showed a strong positive correlation with both indicator bacteria and total suspended solids at the upstream sites during the summer but not the winter. Correlations between Aeromonas and indicator bacteria remained strong in the Buffalo River during the winter, signifying that different conditions exist in the Buffalo River and its upstream tributaries. The strong correlation between Aeromonas spp. and indicator bacteria in the Buffalo River suggest that, in the absence of media capable of the quantitative recovery of potentially pathogenic aeromonads, standard faecal coliform analyses may adequately assess public health risks from Aeromonas spp. in an urban river used for recreational purposes.     

Prevalence of bacterial faecal pathogens in separated and unseparated stored pig slurry

AIMS: To examine the prevalence and diversity of bacterial faecal pathogens in unseparated slurry, separated solids and liquid fractions from a commercial pig farm. METHODS: A total of 43 stored slurry specimens originating from a fattening house over the period February-April 2002 were analysed, consisting of unseparated (n = 14) slurry, separated solids (n = 16) and separated liquid (n = 13). Specimens were examined for the presence of five bacterial pathogens including Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli O157 and Yersinia enterocolitica. Selective enrichment and plating methods were employed for detection of Salmonella spp. and Campylobacter spp. and conventional selective plating techniques for the remaining genera. Antibiogram profiles to 12 antibiotic agents were obtained for all Salmonella isolates obtained. RESULTS: Salmonella spp. were identified in all components of the slurry specimens, whereas Campylobacter spp. was only recovered from the unseparated and separated liquid fractions. In both cases, the separated liquid fraction had the highest prevalence of pathogens and the separated solid fraction had the lowest prevalence. None of the slurry specimens examined were positive for E. coli O157:H7, Shigella spp. or Y. enterocolitica. Twenty-nine isolates of Salmonella were recovered from the slurry specimens, comprising seven serovars, of which Salmonella manhattan was the most prevalent, accounting for over half [15 of 29 (51.7%)] of all Salmonella isolates. Salmonella anatum, Salm. derby, Salm. give, Salm. heidelberg, Salm. simi and Salm. stanley serovars were also recovered. All Salmonella isolates were sensitive to ampicillin, augmentin (amoxicillin/clavulanic acid), chloramphenicol, ciprofloxacin, gentamicin, kanamycin and trimethoprim, but has variable resistance to tetracycline (100%), sulphonamides (84.6%), furazolidone (38.5%), nalidixic acid (15.4%) and streptomycin (15.4%). The majority (57.7%) of isolates displayed antibiotic resistance to at least two antibiotic agents, followed by 34.6% of isolates being resistant to three agents and the remainder (7.7%) being resistant to four antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a marked reduction in the prevalence of Campylobacter and Salmonella in the solids component of separated pig slurry. The adoption of control processes such as aeration of slurry prior to its spread onto agricultural land and newer approaches to pathogen reduction should be investigated, to reduce the transmission of pathogens from pig slurry to the environment.     

Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater

The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm, typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium, as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.     

Survival and activity of Salmonella typhimurium and Escherichia coli in tropical freshwater

The survival of Salmonella typhimurium LT2 and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Numbers were determined by acridine orange staining and a Coulter counter. Population activity was determined by microautoradiography, cell respiration, frequency of dividing cells, and by nucleic acid composition. Numbers of Salm. typhimurium and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, frequency of dividing cells, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 24 h, E. coli was more active than Salm. typhimurium , as measured by nucleic acid composition, and frequency of dividing cells. Both E. coli and Salm. typhimurium survived and remained active in this tropical rain forest watershed for more than 5 d, suggesting that Salm. typhimurium may be of prolonged public health significance once it is introduced into tropical surface waters. As E. coli was active and survived for a long time in this natural environment, it would seem to be unsuitable as an indicator of recent faecal contamination in tropical waters.     

The effect of slurry storage and anaerobic digestion on survival of pathogenic bacteria

The decline in viable numbers of Salmonella typhimurium, Yersinia enterocolitica and Listeria monocytogenes in beef cattle slurry is temperature-dependent; they decline more rapidly at 17°C than at 4°C. Mesophilic anaerobic digestion caused an initial rapid decline in the viable numbers of Escherichia coli, Salm. typhimurium, Y. enterocolitica and L. monocytogenes. This was followed by a period in which the viable numbers were not reduced by 90%. The T₉₀ values of E. coli, Salm. typhimurium and Y. enterocolitica ranged from 0.7 to 0.9 d during batch digestion and 1.1 to 2.5 d during semi-continuous digestion. Listeria monocytogenes had a significantly higher mean T₉₀ value during semi-continuous digestion (35.7 d) than batch digestion (12.3 d). Anaerobic digestion had little effect in reducing the viable numbers of Campylobacter jejuni.     

Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection

AIMS: To compare PCR combined with enrichment media with the standard microbiological techniques (SMT) and to determine the most sensitive method for the detection of Salmonella and the identification of Salm. typhimurium (ST), Salm. enteritidis (SE), Salm. gallinarum (SG) and Salm. pullorum (SP). METHODS AND RESULTS: We analysed 87 samples from poultry using PCR and SMT, PCR being performed from non-selective (NS) and Rappaport-Vassiliadis (RV) media. PCR-NS was less sensitive than PCR-RV and SMT for the detection and identification of Salmonella. PCR-RV detected more positive samples of Salmonella sp. than SMT but both these methods showed similar sensitivity regarding the identification of Salmonella serovars. CONCLUSIONS: PCR-RV was more sensitive and decreased the time necessary to detect and identify Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR-RV is a powerful tool for the rapid and accurate detection and identification of Salmonella and can be implemented in diagnostic and food analysis laboratories.     

Use of the polymerase chain reaction for Salmonella detection

J. KWANG, E.T. LITTLEDIKE AND J.E. KEEN. 1996. A primer set of oligonucleotides (S18 and S19) from the _omp C gene of Salmonella has been evaluated for specific detection of Salmonella by polymerase chain reaction (PCR). This primer set successfully amplified 40 Salmonella serovars (60 isolates), but not 24 non-Salmonella bacteria (42 isolates) that have been tested so far. The uniqueness of these primer sequences was also confirmed. The sensitivity of PCR detection in extracted chromosomal DNA for Salm. typhimurium was 1 pg. The sensitivity for boiled whole bacteria was 400 cells. The detection of Salm. typhimurium in ground beef samples required 4–6 h enrichment with an initial inocula of 100 bacteria.     

Isolation of human faecal bifidobacteria which reduce signs of Salmonella infection when orogastrically dosed to mice

AIMS: The aim of the study was to isolate human bifidobacteria that inhibit growth of Salmonella typhimurium in vitro, and provide protection against Salmonella infection in mice. METHODS AND RESULTS: A total of 92 micro-organisms, which displayed antagonist activity against Salm. typhimurium in vitro, were isolated from human faecal material. Based on their Gram stain status, cultures were pooled and tested for anti-Salmonella activity. The Gram-variable group was the most active. From that group, three bifidobacteria (Laftitrade markB22, B74 and B97) individually showed good pathogen inhibition in vivo. CONCLUSION: Oral administration of certain human bifidobacteria provides protection against Salmonella infection in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that certain bifidobacteria may be used as a prophylaxis for reduced incidence and severity of Salmonella infections.     

Salmonella in animal slurry can be destroyed by aeration at low temperatures

Cattle and other animals infected by Salmonella can emit high numbers of these bacteria. To determine an effective means for reducing this bacterial group in animal slurry, samples were subjected to aeration in laboratory experiments and in farm-scale slurry tanks. A clear reduction in Salmonella levels was found in laboratory experiments at temperatures from 4 to 40 °C. Aeration in farm-scale slurry tanks increased the temperature above the ambient temperatures (often less than 0 °C) to maxima ranging between 19 and 40 °C. Farm-scale aeration resulted in similar reductions in Salmonella as those achieved in laboratory experiments. Thus, reductions, ranging from greater than 99% of the initial number to no detectable Salmonella , could be reached after 2–5 weeks using aeration processes with cattle slurries contaminated by Salm. infantis or pig slurry contaminated by Salm. typhimurium . These results suggest that farmers can control the spread of Salmonella from slurry to agricultural fields. The reduction mechanisms remain unknown, though the increase in pH (to 7·6–9·0) found in slurries after aeration might exert a decreasing effect on these bacteria.     

Use of Polymerase Chain Reaction and Restriction Enzyme Analysis to directly detect and identify Salmonella typhimurium in food

A primer set of oligonucleotides (Salm 3 and Salm 4) from the inv A gene of Salmonellae has been evaluated for the specific detection of Salmonella spp. by the polymerase chain reaction (PCR). This primer set amplified 33 Salmonella serovars but did not amplify 16 non- Salmonella bacteria. Moreover, after PCR amplification, it was possible to identify Salm. typhimurium by restriction enzyme analysis. The PCR-RE method developed could represent a helpful tool for detecting Salmonella spp., and for directly and rapidly identifying Salm. typhimurium in food.     

Modulation of Salmonella infection by the lectins of Canavalia ensiformis (Con A) and Galanthus nivalis (GNA) in a rat model in vivo

The plant lectins, Concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA) have been prefed to rats for 3 d pre- and 6 d postinfection with Salmonella typhimurium S986 or Salm. enteritidis 857. Con A significantly increased numbers of Salm. typhimurium S986 in the large intestine and in faeces, and severely impaired growth of the rats, more severely than is the case of infection with Salmonella typhimurium alone. Con A had much less effect on rats infected with Salm. enteritidis 857 only showing a significant increase in numbers in the colon, accompanied by intermittent increases of Salmonella in the faeces during the study. GNA significantly reduced pathogen numbers in the lower part of the small bowel and the large intestine of rats infected with Salm. typhimurium S986 and significantly improved rat growth. GNA had little effect on infection by Salm. enteritidis 857 with slight decreases in Salmonella numbers in the small intestine and large intestine and transient increases in the faeces.     

Seasonal effects on the survival characteristics of Salmonella Typhimurium and Salmonella Derby in pig slurry during storage

AIMS: Studies were carried out to investigate the survival of Salmonella Typhimurium and Salmonella Derby in pig slurry during summer and winter seasons. METHODS AND RESULTS: Pig slurry samples collected from a commercial fattening house were inoculated with a broth culture of Salmonella Typhimurium and Salmonella Derby, each at a level of log(10) 5.0 CFU ml(-1) and log(10) 2.0 CFU ml(-1). At the higher inoculum level, S. Typhimurium and S. Derby survived for 34 and 23 days, respectively in the summer, and 58 and 46 days, respectively in the winter. Survival at the lower inoculum level for S. Typhimurium and S. Derby was 19 and 16 days, respectively, in the summer and 24 days for both in the winter. CONCLUSIONS: The survival of S. Typhimurium and S. Derby observed in this study indicates that a 2-month holding period of pig slurry, prior to land spreading, may be adequate if separate storage facilities are provided. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite difficulties correlating laboratory studies with on-farm conditions, pig slurry may not represent a major source of transmission of Salmonella spp. in the farm environment in Ireland.     

The effect of copper on the death rate of Salmonella typhimurium DT104:30 in food substrates acidified with organic acids

AIMS: The objective of this study was to investigate the effect of copper ions on the survival of Salmonella typhimurium DT104:30 in acidified liquid food substrates. METHODS AND RESULTS: The decimal reduction time (Dvalue) of Salm. typhimurium DT104:30 was determined in acidified liquid pig feed (LPF) and skimmed milk (SM) containing a range of copper concentrations (0-50 ppm). As copper concentration increased, the death rate of Salm. typhimurium DT104:30 increased. In LPF acidified with 150 mmol l-1 lactic acid the presence of 50 ppm copper resulted in a 10-fold increase in the death rate. CONCLUSIONS: The presence of copper salts in acidic liquid food substrates significantly increases the death rate of Salm. typhimurium DT104:30. SIGNIFICANCE AND IMPACT OF THE STUDY: This finding could influence policy on levels of copper in pig feed.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37 degrees C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135 degrees C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100 degrees C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of (TSBA) after 24 h at 37 degrees C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve.     

Effect of Saccharomyces boulardii against experimental oral infection with Salmonella typhimurium and Shigella flexneri in conventional and gnotobiotic mice

A.C.P. RODRIGUES, R.M. NARDI, E.A. BAMBIRRA, E.C. VIEIRA AND J.R. NICOLI. 1996. Saccharomyces boulardii was shown to be capable of inhibiting multiplication of enteropathogenic bacteria in vitro and is currently used for its anti-diarrhoea properties. We studied the capacity of this yeast to antagonize Salmonella typhimurium and Shigella flexneri in the intestinal tract of conventional or gnotobiotic NMRI mice. Conventional animals were given daily 10 mg doses of S. boulardii , whereas germ-free animals were given a single 10 mg dose. Both groups were challenged orally 5 d later with the pathogenic bacteria (10⁸ or 10² viable cells, respectively). Control groups were treated with saline instead of S. boulardii. Mortality and/or histopathological data showed a protective effect against the pathogenic bacteria in yeast-treated mice. Saccharomyces boulardii colonized the digestive tract of gnotobiotic mice and the number of viable cells ranged around 10¹⁰ g^-1 of faeces. In experimental and control gnotobiotic animals, Salm. typhimurium and Sh. flexneri became rapidly established at a level of about 10¹⁰ viable cells g^-1 of faeces and remained at high levels until the animals died or were sacrificed. The protection against Salm. typhimurium and Sh. flexneri obtained in conventional and/or gnotobiotic mice previously associated with S. boulardii is not due to the reduction of the bacterial populations in the intestines.     

Survival of dehydrated cells of Salmonella typhimurium LT2 at high temperatures

Cells of Salmonella typhimurium LT2 were dehydrated on hydrophobic membranes (Millipore FGLP2500) placed in a controlled atmosphere chamber held at 57% equilibrium relative humidity (ERH) and 37°C. Dehydration for 48 h under the above conditions increased the heat resistance of Salm. typhimurium LT2 when measured as the surviving fraction after a heat challenge of 135°C for 30 min. Results also showed that little or no death occurred during heat challenges of 1 h at temperatures of up to 100°C. The survival of Salm. typhimurium LT2 was measured as the ability to form colonies on solid media tryptone soy broth plus 1.2% agar (TSBA) after 24 h at 37°C. Incorporation of sodium pyruvate, at a concentration of 0.2% into the recovery medium, did not enhance the recovery of heated Salm. typhimurium LT2. Dehydrated cells of S. typhimurium LT2 showed a triphasic death curve. Increasing the period of dehydration from 48 h to 34 d, reduced initial numbers due to die off but did not alter the shape of the subsequent survival curve. and accepted 22 June 1989     

Distribution of pathogen inhibition in the Lactobacillus isolates of a commercial probiotic consortium

Pure strains of Lactobacillus ssp. isolated from a commercial probiotic consortium were checked in a double layer solid medium for their inhibition activities against selected pathogenic bacteria including serotypes of Listeria monocytogenes, Escherichia coli and Salmonella. The antagonistic properties of the Lactobacillus strains may be related to the production of bacteriocin-like compounds. All the pathogens tested were inhibited by one or a few strains of Lactobacillus, the best inhibition was observed against L. monocytogenes but the inhibition was also satisfactory against E. coli, Salm. typhimurium and Salm. enteritidis.     

Removal and inactivation of indicator bacteriophages in fresh waters

AIMS: The removal and inactivation of faecal coliform (FC) bacteria, enterococci (ENT), sulphite-reducing clostridia (SRC), somatic coliphages, F-specific RNA bacteriophages and bacteriophages infecting Bacteroides fragilis in fresh waters. METHODS AND RESULTS: Removal was studied in two areas of a river. The results showed different removal of each group of microbes. Faecal coliform bacteria were removed faster than any other, whereas SRC and bacteriophages infecting Bact. fragilis were the most persistent. Inactivation was measured by 'in situ' experiments, which showed significant differences in survival of the different groups of bacterial and bacteriophage indicators. The SRC and bacteriophages were more resistant than faecal coliforms and enterococci, with the exception of F-specific RNA bacteriophages in the summer. Inactivation experiments with pure cultures of bacteriophages confirmed that phage B40-8 of Bact. fragilis was the most resistant. CONCLUSIONS: Bacteria and bacteriophages show different resistance to natural inactivation. The use of phages allows information to be obtained in addition to that provided by bacterial indicators. Somatic coliphages and phages infecting Bact. fragilis might supply that indicator function. SIGNIFICANCE AND IMPACT OF THE STUDY: Confirmation was obtained that bacteriophages provided additional information to that provided by bacterial indicators to monitor the natural inactivation of viruses and/or pathogens.     

Serum survival and plasmid possession by strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow

Strains of Salmonella enteritidis, Salm. typhimurium and Salm. virchow , carrying different numbers of plasmids, were examined for the ability to multiply in sera. Viable counts were performed to monitor the kinetics of growth of bacteria when in human, chicken and turkey sera. The presence of plasmids in Salm. enteritidis, Salm. typhimurium and Salm. virchow reduced considerably the ability of strains of these serotypes to multiply in serum. SDS-PAGE was used to show that growth of Salm. enteritidis in serum did not involve changes in outer membrane proteins or lipopolysaccharide. It was concluded that the carriage of plasmids may be disadvantageous for the survival in serum of certain common salmonella serotypes.