Saumya Bhaduri

Thermal destruction ofListeria monocytogenes in liver sausage slurry

Abstract Thermal destruction ofListeria monocytogenes was determined in a liver sausage slurry (1:1, liver sausage batter and water) using a submerged ampule technique.D-values forL. monocytogenes Scott A grown at 37°C were 8·9 min at 57·2°C, 2·4 min at 60·0°C, and 1·1 min at 62·8°C (Z=6·2°C) based on analysis of the linear portion of the survivor curves.D-values of 6·6, 1·6, and 0·4 min (Z=4·65°C) were obtained when the data were analyzed using non-linear techniques.L. monocytogenes strain V7 (D60=1.0min) was more thermosensitive than Scott A (D60=1·6min) or HO-VJ-S (D60=1·6min). When Scott A was grown at 19°C, there was a decrease in thermal resistance (D60=0·8min). These data indicate thatL. monocytogenes has a thermal resistance in liver sausage comparable to that observed in other food systems.

Survival of cold-stressed Campylobacter jejuni on ground chicken and chicken skin during frozen storage

ABSTRACT Campylobacter jejuni is prevalent in poultry, but the effect of combined refrigerated and frozen storage on its survival, conditions relevant to poultry processing and storage, has not been evaluated. Therefore, the effects of refrigeration at 4°C, freezing at −20°C, and a combination of refrigeration and freezing on the survival of C. jejuni in ground chicken and on chicken skin were examined. Samples were enumerated using tryptic soy agar containing sheeps blood and modified cefoperazone charcoal deoxycholate agar. Refrigerated storage alone for 3 to 7 days produced a reduction in cell counts of 0.34 to 0.81 log10 CFU/g in ground chicken and a reduction in cell counts of 0.31 to 0.63 log10 CFU/g on chicken skin. Declines were comparable for each sample type using either plating medium. Frozen storage, alone and with prerefrigeration, produced a reduction in cell counts of 0.56 to 1.57 log10 CFU/g in ground chicken and a reduction in cell counts of 1.38 to 3.39 log10 CFU/g on chicken skin over a 2-week period. The recovery of C. jejuni following freezing was similar on both plating media. The survival following frozen storage was greater in ground chicken than on chicken skin with or without prerefrigeration. Cell counts after freezing were lower on chicken skin samples that had been prerefrigerated for 7 days than in those that had been prerefrigerated for 0, 1, or 3 days. This was not observed for ground chicken samples, possibly due to their composition. C. jejuni survived storage at 4 and −20°C with either sample type. This study indicates that, individually or in combination, refrigeration and freezing are not a substitute for safe handling and proper cooking of poultry.

Prevalence of Pathogenic Yersinia enterocolitica Strains in Pigs in the United States

ABSTRACT Yersinia enterocolitica is considered an important food-borne pathogen impacting the pork production and processing industry in the United States. Since this bacterium is a commensal of swine, the primary goal of this study was to determine the prevalence of pathogenic Y. enterocolitica in pigs in the United Sates using feces as the sample source. A total of 2,793 fecal samples were tested for its presence in swine. Fecal samples were collected from late finisher pigs from 77 production sites in the 15 eastern and midwestern pork-producing states over a period of 27 weeks (6 September 2000 to 20 March 2001). The prevalence of ail-positive Y. enterocolitica was determined in samples using both a fluorogenic 5′ nuclease PCR assay and a culture method. The mean prevalence was 13.10% (366 of 2,793 fecal samples tested) when both PCR- and culture-positive results were combined. Forty-one of 77 premises (53.25%) contained at least one fecal sample positive for the ail sequence. The PCR assay indicated a contamination rate of 12.35% (345/2,793) compared to 4.08% (114/2,793) by the culture method. Of the 345 PCR-positive samples, 252 were culture negative, while of the 114 culture-positive samples, 21 were PCR negative. Among 77 premises, the PCR assay revealed a significantly (P < 0.05) higher percentage (46.75%, n = 36 sites) of samples positive for the pathogen (ail sequence) than the culture method (22.08%, n = 17 sites). Thus, higher sensitivity, with respect to number of samples and sites identified as positive for the PCR method compared with the culture method for detecting pathogenic Y. enterocolitica, was demonstrated in this study. The results support the hypothesis that swine are a reservoir for Y. enterocolitica strains potentially pathogenic for humans.

Thermal resistance of Salmonella spp. and Listeria monocytogenes in liquid egg yolk and egg yolk products

The effectiveness of various pasteurization procedures in destroying Listeria monocytogenes and Salmonella enteritidis in liquid egg products was evaluated. Survivor studies were perfonned on individual strains of L. monocytogenes and L. innocua in commercially broken raw egg yolk samples after heating at 61.1, 63.3, and 64.4°C using submerged vials, and on Salmonella spp. at 60.0, 61.1, and 62.2°C. Surviving bacteria were enumerated on TSA and results expressed as D-values. The influence of aw -lowering ingredients such as salt and sugar on thermal resistance in yolk was investigated using a five-strain mixture of L. monocytogenes or a mixture of Salmonella spp. (four strains of S. enteritidis , one stain each of S. senftenberg and S. typhimurium ) at 61.1°C to 66.7°C. At 61.1°C (present minimum temperature for pasteurization of plain egg yolk), a 7-log-unit reduction of Salmonella took 1.4 to 2.4 min, whereas a 7-log-unit reduction of L. monocytogenes took 4.9 to 16.1 min. The D-value for L. monocytogenes at 64.4°C increased from 0.44 min in plain yolk to 8.26 min after a 21.5-min lag (total time to achieve 1-log-unit reduction was 30.7 min) in yolk with 10% salt and 5% sugar, and 27.3 min after a 10.5-min lag (total time 37.8 min for 1-log-unit reduction) in yolk with 20% salt. The D-value for Salmonella in egg yolk at 64.4°C was < 0.2 min, but when 10% salt was added, the D-value was 6.4 min. Aw -lowering solutes in liquid egg yolk increased the thermal resistance of Salmonella and L. monocytogenes .

Studies on Escherichia coli serotype O157:H7 strains containing a 60-MDa plasmid and on 60-MDa plasmid-cured derivatives.

Seventeen strains of Escherichia coli serotype O157:H7 producing Shiga-like toxin were examined for the presence of plasmids and for the ability to adhere to HEp-2 and Intestine 407 cells. All of the strains possessed a common 60-MDa plasmid. To determine the role of the 60-MDa plasmid, plasmid-cured strains were compared with the parent strains for their ability to produce pili and to adhere to epithelial cells in culture. The total cell lysate protein and outer-membrane protein (OMP) profiles were also compared. Both the parent strains and their plasmid-cured derivatives produced pili. Immunofluorescence assay results indicated that the plasmid-cured and parent strains adhered equally well to HEp-2 and Intestine 407 cells; overall adherence was greater with intestinal cells than HEp-2 cells. SDS-PAGE of polypeptides synthesised in an E. coli system in vitro showed that plasmid DNA encodes c. 35 proteins. SDS-PAGE of OMP preparations demonstrated that the 60-MDa plasmid appears to be involved in the synthesis of a 33-kDa OMP. Two strains cured of the 60-MDa plasmid, one that possessed no plasmids and one that still contained a 2.2-MDa plasmid, produced exopolysaccharide (EPS) when cultured on solid medium at 25 degrees C. Two other strains, which were cured of the 60-MDa plasmid but contained a 4.5-MDa plasmid, did not produce visible amounts of EPS. Gas chromatography analysis showed that the EPS consisted of fucose, glucose and galactose in an approximate molar ratio of 2.0:0.9:1.1 and also had 7% of a uronic acid sugar as part of its structure.

Thermal resistance of Listeria monocytogenes and Salmonella spp. in liquid egg white

Survival of a five-strain mixture of Listeria monocytogenes and a six-strain mixture of Salmonella enteritidis , S. typhimurium , and S. senftenberg (not 775W) in liquid egg white was determined by a submerged-vial technique at 51.5°C and 53.2°C with 0.875% added H2O2 and at 55.5°C, 56.6°C, and 57.7°C with no additions. Survival at a range of pH values at 56.6°C also was determined. Surviving bacteria were counted on tryptic soy agar and results expressed as D-values; log-unit reductions in counts in 3.5 min or 6.2 min were calculated from these D-values. Plate pasteurization of commercially broken egg white (pH 8.8) inoculated with a single strain of L. innocua or S. senftenberg also was performed. Heating under currently approved pasteurization conditions, 51.5°C for 3.5 min with hydrogen peroxide, 55.6°C for 6.2 min, or 56.7°C for 3.5 min, resulted in a less than 3-1og unit reduction of viable Salmonella spp. and a less than 0.5-1og unit reduction of L. monocytogenes . At 53.2°C with peroxide, plate pasteurization resulted in a 3.44-1og unit reduction of S. senftenberg in 3.5 min. At 57.7°C with no peroxide, the D-value for Salmonella spp. was 0.78 min when heated in submerged vials, and plate pasteurization reduced viable numbers by 3.64 log units in 3.5 min. Destruction of Listeria under these conditions was still less than 1 log unit. Variation in the pH of the egg white from 7.8 to 9.3 resulted in D-values for Salmonella spp. at 56.6°C of 3.60 min to 1.08 min, respectively. D-values for L. monocytogenes under these conditions ranged from 10.4 min at pH 7.8 to 20.9 min at pH 9.3. The reduced heat sensitivity of Salmonella spp. at lower pH values should be considered in reevaluating pasteurization procedures.

Prevalence of Yersinia enterocolitica in market weight hogs in the United States.

Pigs are the major animal reservoir for Yersinia enterocolitica strains, which are potentially pathogenic for humans. The goals of this study were (i) to estimate the individual animal and on-farm prevalences of Y. enterocolitica in hogs based on tonsil samples collected during National Animal Health Monitoring System Swine 2002 study and (ii) to use these data with data previously published for fecal samples to determine on-farm risk factors for Y. enterocolitica. Tonsil swabs (1,218) and fecal samples (2,847) were collected on 124 farms located in the top 17 pork-producing states. Ten percent of tonsils (122 of 1,218 samples) were positive in irgasan-tiracillin-chlorate (ITC) enrichment broth by real-time PCR, but only 5.6% of samples (68 of 1,218) were positive after subculture on the more selective cefsulodin-irgasan-novobiocin (CIN) agar. For tonsils, the on-farm prevalence based on real-time PCR detection of the ail gene in ITC enrichment broth cultures was 32% (32 of 100 premises sampled); the prevalence based on subculture in CIN agar was 19.6% (20 of 102 premises). Results of bacteriological isolation and real-time PCR analysis of tonsils and feces were combined to estimate prevalence (individual animal and farm), which was subsequently correlated with 40 farm management practices. Four factors and their accompanying odds ratios (ORs) were identified in the final regression model: location in a central state (OR = 0.3), vaccination for Escherichia coli (OR = 3.0), percentage of deaths due to scours (OR = 3.5), and presence of meat or bone meal in grower-finisher diet (OR = 4.1).

Clonality and antibiotic susceptibility of Yersinia enterocolitica isolated from U.S. market weight hogs.

Pigs are the only known animal reservoir of Yersinia enterocolitica strains pathogenic to humans. In this study 106 ail-positive pathogenic Y. enterocolitica isolates, previously recovered from 2793 swine fecal samples (3.8%) collected during National Animal Health Monitoring Systems Swine 2000 study, were examined. The presence of the previously described virulence plasmid, expression of plasmid-associated virulence determinants, and serotype were correlated with genotype, expression of YopE protein, and antibiotic susceptibility. Pulsed-field gel electrophoresis using the enzyme XbaI showed that O:3 and O:5 isolates were highly clonal within a serotype regardless of geographic origin. Antimicrobial resistance profiles of 106 isolates of serotypes O:3 and O:5 were evaluated by agar disk diffusion methodology to 16 different antibiotics. All isolates were susceptible to 13 of the 16 tested antimicrobials; resistance was noted to ampicillin, cephalothin, and tetracycline. The presence of the ail gene, virulence plasmid, the expression of virulence determinants, and serotypes indicate that these isolates from U.S. swine are potentially capable of causing human foodborne illness.

A comparison of sample preparation methods for PCR detection of pathogenic Yersinia enterocolitica from ground pork using swabbing and slurry homogenate techniques.

Two sample preparation methods for multiplex polymerase chain reaction (PCR) for detection of plasmid-bearing virulent Yersinia enterocolitica (YEP(+)) from ground pork were compared. Two sets of ground pork samples were inoculated with 10, 1, and 0.5 CFU/cm(2) of a YEP(+) strain, one set was swabbed and the second set was dispersed into a slurry homogenate. Both swab and slurry homogenate samples were enriched in sterile Whirl Pak bags containing modified trypticase soy broth for 48 h at 12 degrees C. From the enriched swab samples, the bacterial cells were pelleted, washed, boiled in sterile distilled water, and treated with proteinase K to prepare cell lysates to use as a DNA template. Since slurry homogenate samples contained food material, DNA extraction was performed using a commercial kit. The DNA from cell lysates and from extracted slurry homogenate samples were evaluated as templates for multiplex PCR employing primers for the chromosomal ail and plasmid virF genes. The enrichment of the YEP(+) strain was more efficient using the sponge-swabbed samples than the slurry homogenate samples at all three inoculum levels tested. It was necessary to dilute the DNA extracted from slurry homogenate to determine the optimal concentration of each sample for PCR amplification. No amplification signal was detected using undiluted DNA, possibly due to DNA inhibitors present in the slurry homogenate that were not removed in the process of extraction. However, DNA could be detected in undiluted cell lysates from swab samples. Thus, the cell lysates from swab samples are more advantageous than DNA extracted from ground pork slurry homogenate samples for the PCR assay.

Enrichment, isolation, and virulence of freeze-stressed plasmid-bearing virulent strains of Yersinia enterocolitica on pork.

The influence of freeze stress at -20 degrees C on the enrichment, isolation, detection, presence of virulence plasmid, and expression of virulence of plasmid-bearing Yersinia enterocolitica (YEP+) inoculated on pork chop medallions was assessed. Pork chop medallions (10 cm2) artificially contaminated with 10, 1, and 0.5 CFU/cm2 of YEP+ strains (serotype O:3) were placed in sterile petri dishes at -20 degrees C for 24 h. The medallions were swabbed when frozen, after thawing at room temperature for 1.5 h and after thawing at 4 degrees C for 18 h. Swabs were enriched and YEP+ were detected and isolated using the Congo red-binding and low-calcium-response assays. The YEP+ were isolated under all conditions on pork chop medallions inoculated with 10 CFU/cm2 and at a level of 1 CFU/cm2 when thawed at room temperature and at 4 degrees C but not from frozen pork chop medallions. The YEP+ were not isolated from pork chop medallions inoculated with 0.5 CFU/cm2 and then frozen, whereas YEP+ were recovered when inoculated at this level from pork chop medallions not subjected to freezing. Virulence of the strains isolated from frozen pork chop medallions was confirmed by PCR and the expression of plasmid-associated phenotypes. These results indicate that YEP+ subjected to freezing on pork are potentially capable of causing foodborne illness and that freezing is not a substitute for safe handling and proper cooking of pork.