Survival of Campylobacter jejuni and Salmonella enterica Typhimurium in vacuum-packed, moisture-enhanced pork

The abilities of Campylobacter jejuni and Salmonella enterica Typhimurium to survive in vacuum-packaged, moisture-enhanced pork stored at 4 or 10°C were examined. Pork loins were surface inoculated with either C. jejuni or Salmonella Typhimurium and then moisture enhanced to a target of 10 or 20%. The enhanced pork loins were sliced 1 cm thick and vacuum packaged. A pork loin without moisture enhancement was sliced and vacuum packaged as a control. Samples were collected, plated, and the numbers of surviving organisms were determined periodically during storage at 4 and 10°C. The numbers of C. jejuni or Salmonella Typhimurium in samples with different moisture enhancement levels were similar (P > 0.05). No significant differences (P > 0.05) in C. jejuni counts were observed between samples at 10°C and those at 4°C. In contrast, the numbers of Salmonella Typhimurium in samples at 10°C had significantly (P < 0.05) increased (0.41 log CFU/g) from those at the refrigerated temperature of 4°C. Vacuum storage at 4 and 10°C for 28 days did not result in dramatic reductions in the mean numbers of C. jejuni and Salmonella Typhimurium. Our findings indicate that vacuum packaging under chilled conditions will not add substantially to safety for moisture-enhanced pork. Strict hygienic practices or the implementation of decontamination technologies is recommended.     

Survival characteristics of Salmonella enterica serovar Newport in the dairy farm environment

Multi-drug resistant (MDR) Salmonella enterica serovar Newport (S. Newport) has established a reservoir in dairy cattle. Infected herds suffer significant mortality in both adult and young animals, posing a considerable economic loss to producers. Land application of manure from infected animals may further spread the pathogen into the agroecosystem, causing public health concerns. Previous work by our group demonstrated that the organism persisted in manure and manured soil for 6 to 10 mo under laboratory conditions. In the present study, we determined the survival characteristics of MDR S. Newport in a dairy lagoon, compost pile, and soil of a grass field under natural conditions using environmental sentinel chambers with an initial concentration of S. Newport around 7 log₁₀ per gram. In the static compost pile at 64°C, S. Newport was eliminated within 18 h. In the dairy effluent lagoon, the pathogen survived for >137 d, whereas in the field soil, the organisms persisted for over 276 d. The survival of MDR S. Newport in both the lagoon and field soil followed a pattern of (1) an increase or plateau for a few days, (2) log-linear decline for 6 to 13 wk, and (3) a long tailing phase at low and variable concentration for 4 to 9 mo. Log reduction times (days required for 90% decrease in concentration) based on the log-linear decline phase were 7 d in the lagoon and 14 to 20 d in the soil. Conditions leading to faster inactivation during the initial phase do not necessarily translate into a quicker elimination of the pathogen. Regression models of the log-linear phase may be inaccurate for estimating complete pathogen elimination.     

Survival of Salmonella in spices and growth in cooked food

Contamination of spices with pathogens has been reported worldwide, and Salmonella might result in foodborne infections. In this study, we investigated the survival of Salmonella in black pepper and red pepper, and the growth of the surviving Salmonella in cooked food. Salmonella Enteritidis, Salmonella Weltevreden and Salmonella Senftenberg were inoculated into spices, and their survival during storage was examined. In black pepper, S. Enteritidis was no longer viable after storage for 28 days, but S. Weltevreden and S. Senftenberg remained viable. In red pepper, S. Weltevreden and S. Senftenberg survived for 28 days although S. Enteritidis was not viable after 7 days. Salmonella Weltevreden and Salmonella Senftenberg were inoculated into cooked food, and their survival during storage was determined. In potato salad, egg salad, namul and kimchi as cooked foods, both pathogens grew at 30 degrees C, but not at 10 degrees C. Our results indicate that cooked food should be stored at low temperature after addition of spices, such as black pepper and red pepper, following the cooking.     

Survival, elongation, and elevated tolerance of Salmonella enterica serovar Enteritidis at reduced water activity

Growing microorganisms on dry surfaces, which results in exposure to low water activity (a(w)), may change their normal morphology and physiological activity. In this study, the morphological changes and cell viability of Salmonella enterica serovar Enteritidis challenged to low a(w) were analyzed. The results indicated that exposure to reduced a(w) induced filamentation of the cells. The amount of filamentous cells at a(w) 0.94 was up to 90% of the total number of cells. Surviving filamentous cells maintained their membrane integrity after exposure to low a(w) for 21 days. Furthermore, cells prechallenged to low a(w), obtained with an ionic humectant, demonstrated higher resistance to sodium hypochlorite than control cells. These resistant cells are able to survive disinfection more efficiently and can therefore cause contamination of foods coming in contact with surfaces. This points to the need for increased attention to cleaning of surfaces in household environments and disinfection procedures in processing plants.     

Survival of mouse embryos after freezing in medium containing dimethylsulphoxide and yolk extract

The ability of the cryoprotective solution containing dimethylsulphoxide in combination with yolk extract (DYG 550) to protect mouse embryos at ultra low temperatures is reported. Of the embryos of 46.1% frozen to -196 C at various rates formed blastocysts in vitro compared to 41.2% and 23.2% survivals protected by 1M dimethylsulphoxide and 1M glycerol.     

Population changes and growth modeling of Salmonella enterica during alfalfa seed germination and early sprout development

Food Science and Biotechnology - This study examined the effects of alfalfa seed germination on growth of Salmonella enterica. We investigated the population changes of S. enterica during early...     

Survival of Vibrio parahaemolyticus during freezing

The ability of Vibrio parahaemolyticus to survive on frozen rock lobster tails was investigated. Rock lobster tails were artificially inoculated with the organism and were stored at ? 18°C. Vibrio died off within 1 week at ?18°C in lightly contaminated tails but a heavy infection survived for a few months at this temperature. Recovery depended on the degree of infection, the presence of other bacteria and the selectivity of the medium.     

Effect of the growth environment on the strain variability of Salmonella enterica kinetic behavior

Intra-species variability of microbial growth kinetic behavior is an event with important implications for food safety research. Aiming at the evaluation of the growth variability among Salmonella enterica strains as affected by the growth environment, the kinetic behavior of 60 isolates of the pathogen was assessed at 37 °C in tryptone soy broth of different pH values (4.3-7.0) and NaCl concentrations (0.5-6.0%). Maximum specific growth rate (μ_max) values corresponding to each strain and growth condition were estimated by means of absorbance detection times of serially decimally diluted cultures using the automated turbidimetric system Bioscreen C. A total of 9600 optical density curves were generated for the strains and the growth conditions tested. The variability of μ_max among the S. enterica strains was important and greater than that observed within the strains (i.e. among replicates). Moreover, strain variability increased as the growth conditions became more stressful both in terms of pH and NaCl. The coefficient of variation of μ_max among the tested strains at pH 7.0-0.5% NaCl was 6.1%, while at pH 4.3-0.5% NaCl and pH 7.0-6.0% NaCl was 11.8% and 23.5%, respectively. Beyond the scientific interest in understanding strain variability, the findings of this study should be useful in strain selection for exploitation in food safety challenge studies as well as in incorporating strain variability in predictive microbiology and microbial risk assessment.     

Salmonella enterica serovars from pigs on farms and after slaughter and validity of using bacteriologic data to define herd Salmonella status

The primary objective of this study was to evaluate the validity of using data obtained from slaughtered pigs for farm-level epidemiologic studies of Salmonella. The study involved groups of pigs from five farms. Salmonella isolates were obtained from on-farm samples, and a total of 370 on-farm and an additional 486 isolates from samples collected after commercial slaughter were subsequently tested. Preharvest samples included feces of individual animals from defined groups of nursery and finishing pigs on commercial farms and swabs from trucks. Postslaughter samples were cecal contents and mesenteric lymph node samples. The concordance between Salmonella serovars isolated from on-farm samples and those serovars isolated after slaughter varied widely among farms. Results of paired lymph node and cecal cultures were strongly associated (odds ratio, 7.0), but the agreement between on-farm and postslaughter results at the pig level was poor (kappa = 0.34). The results support recent findings that risk of exposure to Salmonella during transport and lairage remains a concern under contemporary industry conditions. The findings further imply that slaughter plant studies based on phenotyping of Salmonella alone (such as serovars) may not reliably indicate the Salmonella status of commercial swine farms.     

On the different growth conditions affecting silver antimicrobial efficacy on Listeria monocytogenes and Salmonella enterica

Silver is known to inhibit microorganisms and therefore it is an ideal candidate for its incorporation in a wide variety of materials for food applications. However, there is still a need for understanding how silver prolonged exposure to bacterial contamination affects the bioavailability of the active silver species. In the present study, growth curves of Listeria monocytogenes and Salmonella enterica were performed for 3-5days in Tryptic Soy Broth (TSB) and M9 minimal medium (M9) in the presence of silver ions and silver solutions previously in contact with the growth media. The cultivability of the bacteria under these conditions was correlated with the viability of the bacterial populations as measured by flow cytometry analysis (FC) using a LIVE/DEAD BacLight kit. It was found that, after a period where viable counts were not detected, bacterial populations recovered and were able to proliferate in most cases. The resuscitation of the cultures was explained by both the existence of a resilient fraction of bacteria in a compromised state and the parallel inactivation of the silver species. This inactivation was found to be highly influenced by time dependant chemical reactions taking place in the environment of exposure, producing differences of at least 3 fold between results for nutrient rich environments and results for limiting environments. This study points out the need for understanding these chemical interactions and bacterial mechanisms of adaptation and may have relevance in the design of silver-based antimicrobial systems for food-related applications.     

Survival of Bifidobacterium longum 1941 microencapsulated with proteins and sugars after freezing and freeze drying

Five types of proteins and three types of sugars were examined for their effectiveness in protecting B. longum after freeze drying, including their acid and bile tolerance, surface hydrophobicity, retention of β-glucosidase, lactate dehydrogenase and adenosine triphosphatase. Sodium caseinate 12%, whey protein concentrate 12%, sodium caseinate:whey protein concentrate 6%:6%, skim milk 12%, or soy protein isolate 12% was combined with glycerol (3% w/v), mannitol (3% w/v) or maltodextrin (3% w/v). Fifteen emulsion systems containing sugars were obtained. Concentrated B. longum 1941 was incorporated into each emulsion system at a ratio of 1:4 (bacteria:emulsion). All the mixtures were then freeze dried. Water activity (a_w) of freeze dried microcapsules was in the range of 0.30 to 0.35. WPC–CAS GLY provided high stability of bacteria (99.2%) during freezing, while high stability of cells after freeze drying and during exposure to acid and bile environment was achieved when CAS–MAN was applied (97.4%, 81.6% and 99.3%, respectively). High retention of β-glu of freeze-dried bacteria was achieved using SM–MAN as protectant (94.6%). ATPase and LDH were successfully retained by SM–GLY (94.9 and 83.6%, respectively) but there was no significant difference in protection effect using CAS–MAN (93.8 and 82.6%, respectively). Overall, milk proteins were superior to SPI and sugar alcohols provided more protection than MD.     

Survival of E. coli and Salmonella after Chilling and Freezing in Liquid Media

Predictive microbiology requires a sound basis of observed results, and factors that contribute to death of bacterial cells, before accurate equations can be developed. The effect of chilling and freezing strains of Escherichia coli and salmonella serotypes in nutrient broth, a noninhi-bitory liquid medium, was investigated. The phase of growth, small changes in composition of test medium, and sub-cultures made after primary isolation, influenced survival. Therefore, such influences must be considered when attempting to extrapolate results Erom pure cultures on laboratory media, to predict behavior of similar organisms in foods during chilling and freezing.     

Interaction of Salmonella enterica subspecies enterica serovar Typhimurium and mung bean (Phaseolus aureus) plants

The effect of Salmonella enterica subspecies enterica serovar Typhimurium, a zoonotic serovar, on mung bean (Phaseolus aureus) cultivar Pant Mung-3 plants was studied. Inoculation of mung bean seeds with Salmonella Typhimurium (7.2 x 10(5) CFU/ml) reduced sprouting rate (P < 0.07). This effect was more pronounced at higher levels of contamination. In the soil inoculated with Salmonella Typhimurium (7.2 x 10(6) CFU/g), germination was retarded and the number of defective sprouts was also significantly higher (P < 0.002). Salmonella Typhimurium grew inside germinating seeds and plant tissues and persisted in seedlings, adult plants, and harvested seedlings dried and stored at room temperature (30 degrees C) up to 45 days. Phaseolus aureus plants grown in sterile soil was resistant to Salmonella Typhimurium infection at 15 days of age and cleared Salmonella from all the aerial parts within 3 h of infection. However, Salmonella Typhimurium could be reisolated from the basal area of the stem and from soil even after 45 days of exposure to the pathogen.     

Survival of Salmonella Newport in oysters

Salmonella enterica is the leading cause of laboratory-confirmed foodborne illness in the United States and raw shellfish consumption is a commonly implicated source of gastrointestinal pathogens. A 2005 epidemiological study done in our laboratory by Brands et al., showed that oysters in the United States are contaminated with Salmonella, and in particular, a specific strain of the Newport serovar. This work sought to further investigate the host-microbe interactions between Salmonella Newport and oysters. A procedure was developed to reliably and repeatedly expose oysters to enteric bacteria and quantify the subsequent levels of bacterial survival. The results show that 10 days after an exposure to Salmonella Newport, an average concentration of 3.7 × 10³ CFU/g remains within the oyster meat, and even after 60 days there still can be more than 10² CFU/g remaining. However, the strain of Newport that predominated in the market survey done by Brands et al. does not survive within oysters or the estuarine environment better than any other strains of Salmonella we tested. Using this same methodology, we compared Salmonella Newport's ability to survive within oysters to a non-pathogenic strain of E. coli and found that after 10 days the concentration of Salmonella was 200-times greater than that of E. coli. We also compared those same strains of Salmonella and E. coli in a depuration process to determine if a constant 120 L/h flux of clean seawater could significantly reduce the concentration of bacteria within oysters and found that after 3 days the oysters retained over 10⁴ CFU/g of Salmonella while the oysters exposed to the non-pathogenic strain of E. coli contained 100-times less bacteria. Overall, the results of this study demonstrate that any of the clinically relevant serovars of Salmonella can survive within oysters for significant periods of time after just one exposure event. Based on the drastic differences in survivability between Salmonella and a non-pathogenic relative, the results of this study also suggest that unidentified virulence factors may play a role in Salmonella's interactions with oysters.     

Survival of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes on inoculated walnut kernels during storage

The survival of single strains or cocktails of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes was evaluated on walnut kernels. Kernels were separately inoculated with an aqueous preparation of the pathogens at 3 to 10 log CFU/g, dried for 7 days, and then stored at 23°C for 3 weeks to more than 1 year. A rapid decrease of 1 to greater than 4 log CFU/g was observed as the inoculum dried. In some cases, the time of storage at 23°C did not influence bacterial levels, and in other cases the calculated rates of decline for Salmonella (0.05 to 0.35 log CFU/g per month) and E. coli O157:H7 (0.21 to 0.86 log CFU/g per month) overlapped and were both lower than the range of calculated declines for L. monocytogenes (1.1 to 1.3 log CFU/g per month). In a separate study, kernels were inoculated with Salmonella Enteritidis PT 30 at 4.2 log CFU/g, dried (final level, 1.9 log CFU/g), and stored at -20, 4, and 23°C for 1 year. Salmonella Enteritidis PT 30 declined at a rate of 0.10 log CFU/g per month at 23°C; storage time did not significantly affect levels on kernels stored at -20 or 4°C. These results indicate the long-term viability of Salmonella, E. coli O157:H7, and L. monocytogenes on walnut kernels and support inclusion of these organisms in hazard assessments.     

Modeling the growth rate and lag time of different strains of Salmonella enterica and Listeria monocytogenes in ready-to-eat lettuce

The growth parameters (growth rate, μ and lag time, λ) of three different strains each of Salmonella enterica and Listeria monocytogenes in minimally processed lettuce (MPL) and their changes as a function of temperature were modeled. MPL were packed under modified atmosphere (5% O₂, 15% CO₂ and 80% N₂), stored at 7–30 °C and samples collected at different time intervals were enumerated for S. enterica and L. monocytogenes. Growth curves and equations describing the relationship between μ and λ as a function of temperature were constructed using the DMFit Excel add-in and through linear regression, respectively. The predicted growth parameters for the pathogens observed in this study were compared to ComBase, Pathogen modeling program (PMP) and data from the literature. High R² values (0.97 and 0.93) were observed for average growth curves of different strains of pathogens grown on MPL. Secondary models of μ and λ for both pathogens followed a linear trend with high R² values (>0.90). Root mean square error (RMSE) showed that the models obtained are accurate and suitable for modeling the growth of S. enterica and L. monocytogenes in MP lettuce. The current study provides growth models for these foodborne pathogens that can be used in microbial risk assessment.     

Salmonella enterica multilocus sequence typing and its correlation with serotypes

Salmonella enterica is a foodborne pathogen of significant public health concern worldwide. In Thailand, S. enterica has also been ranked among the top five most significant bacterial agents of foodborne illnesses by the Ministry of Public Health. Conventionally, biochemical tests and antigen-antibody agglutination have been used to identify and subtype S. enterica, respectively. The objective of this study was to identify the serotypes of 180 S. enterica isolates. Multilocus sequence typing (MLST) was used to deduce the S. enterica serotypes based on sequence type (ST) correlation as shown in the MLST database (http://mlst.warwick.ac.uk/mlst/). Initially, MLST was used to confirm serotypes of 53 previously identified isolates of S. Enteritidis, S. Typhimurium, S. Hadar, S. Virchow, and S. Infantis isolated in Thailand. MLST and serotype correlation confirmed 52 (of 53) known isolates. MLST was performed in 127 S. enterica isolates of unknown serotypes from various sources. Serotypes of all 127 S. enterica isolates were successfully deduced based on STs. With MLST and PCR-based identification, we have shown that the majority of isolates are of monophasic S. Typhimurium (ST34; 43 isolates) and serotype Rissen (ST469; 37 isolates), in agreement with the top serotypes commonly found in Thailand based on the WHO National Salmonella and Shigella Center. We have also confirmed that MLST is a powerful Salmonella subtyping method which could be used not only as a tracking tool for an outbreak investigation at nucleotide level but also as a serotype predictor for making correlations with food safety regulations.     

Prevalence and characterization of Salmonella enterica serovar Weltevreden from imported seafood

During 2001-2005, 210 Salmonella enterica strains were isolated from seafood samples imported into US. Strains of S. enterica serovar Weltevreden were the most predominantly found among the 64 different serovars isolated. A total of 37 Salmonella Weltevreden isolates were characterized by pulsed-field gel electrophoresis (PFGE), plasmid profiles and antibiotic susceptibility to assess genetic diversity. Our results showed a low frequency of antibiotic resistance; 35 of the 37 isolates were sensitive to ampicillin, tetracycline, chloramphenicol, gentamicin, sulfisoxazole, streptomycin and kanamycin. Only two isolates, from samples originating in the Philippines and India, showed resistance to ampicillin and tetracycline and to streptomycin, sulfisoxazole and tetracycline, respectively. Of the 37 isolates, two isolates did not carry any plasmid and 35 isolates harbored several small and mega-plasmids. These isolates were differentiated into 10 distinct types based on plasmid profiles. Four different PFGE clusters were obtained with a genetic similarity of 66-76%. Four groups of isolates (formed by two or three isolates each) showed 100% similarity in the PFGE profiles. One of these groups included strains isolated in Vietnam in 2003, 2004 and 2005 from fish and shrimp. The other groups included strains isolated in Vietnam, Indonesia and Thailand in 2000, 2004 and 2005 from snail, shrimp and fish. Our findings show genetic diversity and temporal persistence of S. enterica serovar Weltevreden in recently monitored seafood imports.     

Survival of Pseudomonas fluorescens and Salmonella typhimurium after electron beam and gamma irradiation of refrigerated beef

The radurization effects of gamma ray and electron beam irradiation at 1.5 and 3.0 kGy on beef steaks inoculated with Salmonella Typhimurium and Pseudomonas fluorescens were investigated during 8 days of storage at 5 degrees C. Total bacterial counts and numbers of Salmonella Typhimurium and P. fluorescens were analyzed at 2-day intervals. Total bacterial counts of samples irradiated by both gamma rays and electron beam were significantly (P < 0.05) reduced by 3.8 to 5.3 log CFU/g. Salmonella Typhimurium was not detectable during the experimental period. P. fluorescens counts of beef samples irradiated by gamma rays at both 1.5 and 3.0 kGy were not detected; however, P. fluorescens in samples irradiated by electron beam at 1.5 and 3.0 kGy was recovered after 2 days, and bacterial counts reached 7.8 and 6.9 log CFU/g, respectively. Both gamma ray and electron beam irradiation reduced total bacterial counts initially, possibly extending shelf life. Irradiation was very effective in destroying Salmonella Typhimurium; however, P. fluorescens was not completely eliminated by electron beam irradiation. Consequently, gamma ray irradiation was more effective than electron beam irradiation in the destruction of P. fluorescens.