Selection and characterization of cellulose-deficient derivates of shiga toxin-producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) is known to have several defense mechanisms, one of which is the production of extracellular substances including cellulose. The goal of this study was to prepare pairs of STEC cultures for use in future studies designed to address the role of cellulose in protecting the cells of STEC for survival under adverse environmental conditions. Cells of STEC deficient in cellulose production were separated from cellulose-proficient wild-type cells. The identities of the two types of cells were confirmed using serotyping and pulsed-field gel electrophoresis (PFGE). Selected growth characteristics of the two types of cells were determined using three phenotype microarray plates, PM9, PM10, and PM11. The cellulose-deficient and cellulose-proficient cells in each STEC pair shared the same serotype and PFGE profile. The deficiency in cellulose production did not significantly (P > 0.05) affect the growth characteristics of STEC cells under 191 of the 210 tested growth conditions. Significant differences in growth between the two types of cells were observed only in the presence of two antibiotics, a short chain fatty acid, and high concentrations of osmolytes, as well as under extreme acidic and alkaline pH. These results suggest that deficiency in cellulose production did not alter the serological property, PFGE profile, and growth characteristics of selected STEC strains under optimal growth conditions. The STEC strains and their cellulose-deficient derivates could be useful for studying the role of cellulose in protecting the cells of STEC for survival under adverse environmental conditions.

Kinetics of growth and inactivation of Salmonella enterica serotype Typhimurium DT104 in pasteurised liquid egg products.

The potential impact of post-pasteurisation contamination of liquid egg products with the multi-antibiotic resistant pathogen Salmonella enterica serotype Typhimurium definitive type 104 (DT104) was assessed by determining the viability of this bacterium in whole egg, albumen and 10% w/w sugared and salted yolk incubated at 4-42 degrees C. Results indicated that populations of S. Typhimurium DT104 were slowly inactivated in all four products when stored at 4 degrees C. However, based on the typical shelf-lives of cold-stored liquid egg, less than 0.6 log-kill would be achieved in those products prior to their use. Incubation at temperatures pertaining to abuse situations (10, 15, 20 and 25 degrees C) revealed an increasing potential for growth of S. Typhimurium DT104 in whole egg, albumen and sugared yolk, as indicated by trends in growth rate, lag duration and maximum population density. At even higher temperatures (30, 37 and 42 degrees C), growth rates of S. Typhimurium DT104 in whole egg and sugared yolk continued to increase. The same was true for S. Typhimurium DT104 in albumen except that growth was not observed at 42 degrees C and instead populations were inactivated within 30 h. At no temperature tested was S. Typhimurium DT104 able to grow in salted yolk. The influence of these growth and inactivation patterns on the risk of salmonellosis in relation to product type and storage temperature is discussed.

Growth and survival of antibiotic-resistant Salmonella typhimurium DT104 in liquid egg products.

Since 11 September 2001, quality and food safety are no longer the concerns of only consumers, industry, regulatory agencies, or other government officials. Liquid foods that are prepared or stored in bulk, including liquid egg products, are considered to be at potential risk for sabotage. Because of their versatility, low price, and functional properties, many of these products are being marketed. Four of the most common products of this type are whole egg, egg albumen, 10% sugared yolk, and 10% salted yolk. Although all of the serotypes of Salmonella enterica may cause illness, multidrug-resistant Salmonella Typhimurium DT104 has become widespread and can cause severe illness that is difficult to treat. Studies were conducted to determine growth patterns of Salmonella Typhimurium DT104 in four commercial liquid egg products held at 4, 10, 20, 30, 37, and 42 degrees C for 0 to 384 h. All experiments were performed in duplicate and repeated twice. Standard methods were used to estimate cell numbers, and log CFU per gram of egg product was plotted against time. The number of cells of Salmonella Typhimurium DT104 increased to 8 to 9 log CFU/g in whole egg and 10% sugared yolk, increased by 1 log CFU/g in liquid albumen, but decreased by 3 log CFU/g in 10% salted yolk. Data from this study have been archived in the ComBase database to further assist policy makers or other scientists interested in Salmonella growth characteristics in liquid eggs. However, based on data generated in this study, Salmonella Typhimurium DT104 probably does not constitute a food threat agent in liquid eggs.

Detection of Salmonella enterica subpopulations by phenotype microarray antibiotic resistance patterns.

Three strains of Salmonella enterica serotype Enteritidis were compared to Salmonella enterica serotype Heidelberg, Salmonella enterica serotype Newport, and Salmonella enterica serovar Typhimurium for growth in the presence of 240 antibiotics arranged within a commercial high-throughput phenotype microarray. The results show that antibiotic resistances were different for subpopulations of serotype Enteritidis separated only by genetic drift.

Pathotyping of Salmonella enterica by analysis of single-nucleotide polymorphisms in cyaA and flanking 23S ribosomal sequences.

The egg-contaminating phenotype of Salmonella enterica serotype Enteritidis was linked to single-nucleotide polymorphisms (SNPs) occurring in cyaA, which encodes adenylate cyclase that produces cAMP and pyrophosphate from ATP. Ribotyping indicated that SNPs in cyaA were linked to polymorphisms occurring in the rrlC and rrlA 23S ribosomal subunits. Phylogenetic analysis of cyaA discriminated between Salmonella enterica serotypes and within serotype Enteritidis. Serotypes Typhimurium, Heidelberg and Enteritidis produced one, three and six cyaA allelic variants, respectively, among the set of 56 isolates examined. Asparagine(702) of CyaA was converted to serine in a biofilm-producing isolate. Statistical analysis was applied to 42 other genes encoding proteins between 800 and 1000 amino acids (aa). Results show that the 848 aa CyaA of serovar Enteritidis evolved by nucleotide substitutions that did not significantly alter the purine-to-pyrimidine nucleotide substitution ratio, which was a characteristic of large genes that was positively correlated with increasing gene size. In summary, these analyses link SNPs occurring in the rrlC-rrlA genomic fragment of S. enterica to genetic drift within S. Enteritidis that is associated with egg contamination.

Impact of commercial processing on the microbiology of shell eggs.

Shell egg microbiology has been studied extensively, but little information is available on how modern U.S. processing conditions impact microbial populations. As regulations are being drafted for the industry, such information can be important for determining processing steps critical to product safety. Five different shell egg surface microbial populations (aerobic bacteria, yeasts and molds, Enterobacteriaceae, Escherichia coli, and Salmonella) were monitored at 12 points along the processing line (accumulator, prewash rinse, washer 1, washer 2, sanitizer, dryer, oiler, scales, two packer head lanes, rewash entrance, and rewash exit). Three commercial facilities were each visited three times, a total of 990 eggs were sampled, and 5,220 microbiological samples were subsequently analyzed. Although variations existed in concentrations of microorganisms recovered from each plant, the patterns of fluctuation for each population were similar at each plant. On average, aerobes, yeasts and molds, Enterobacteriaceae, and E. coli prevalence were reduced by 30, 20, 50, and 30%, respectively, by the end of processing. The microbial concentrations (log CFU per milliliter) in the egg rinse collected from packer head lanes were decreased by 3.3, 1.3, 1.3, and 0.5, respectively, when compared with those of rinses collected from eggs at the accumulator. Salmonella was recovered from 0 to 48% of pooled samples in the three repetitions. Higher concentrations of Salmonella were recovered from preprocessed than from in-process or ready-to-pack eggs. These data indicate that current commercial practices decrease microbial contamination of egg shell surfaces.

Identification of Enterobacteriaceae from washed and unwashed commercial shell eggs.

To evaluate the effect of processing on the safety and quality of retail shell eggs, a storage study was conducted with unwashed and commercially washed eggs. This work demonstrated that commercial processing decreased microbial contamination of eggshells. To know which species persisted during storage on washed or unwashed eggs, Enterobacteriaceae isolates were selected and identified biochemically. For each of three replications, shell eggs were purchased from a commercial processing plant, transported back to the laboratory, and stored at 4 degrees C. Once a week for 6 weeks, 12 eggs for each treatment (washed and unwashed control) were rinsed in sterile phosphate-buffered saline. A 1-ml aliquot of each sample was plated onto violet red bile glucose agar with overlay and incubated at 37 degrees C for 24 h. Following incubation, plates were observed for colonies characteristic of the family Enterobacteriaceae. A maximum of 10 isolates per positive sample were streaked for isolation before being identified to the genus or species level using commercially available biochemical strips. Although most of the isolates from the unwashed control eggs belonged to the genera Escherichia or Enterobacter, many other genera and species were identified. These included Citrobacter, Klebsiella, Kluyvera, Pantoea, Providencia, Rahnella, Salmonella, Serratia, and Yersinia. Non-Enterobacteriaceae also recovered from the unwashed egg samples included Xanthomonas and Flavimonas. Very few washed egg samples were contaminated with any of these bacteria. These data provide useful information on the effectiveness of processing in removing microorganisms from commercial shell eggs.

Comparison of weep and carcass rinses for recovery of Campylobacter from retail broiler carcasses.

Campylobacter is frequently recovered from broiler carcasses. Carcass rinsing is a commonly used procedure for isolating Campylobacter from poultry. A viscous fluid, or weep, exudes from broiler carcasses that have been packaged. This fluid can contain bacteria that were attached to the carcass and represents a potential means of detecting Campylobacter-contaminated carcasses through cultural analysis. Experiments were conducted to compare the efficacy of a weep sampling method with that of a carcass rinse method. For both trials, retail carcasses were purchased. Packages were opened, and 0.1-ml aliquots of weep fluid from the retail packages were plated onto Campy-cefex agar. Carcasses were removed from the package and rinsed in 100 ml of sterile water. Next, 0.1-ml aliquots of the rinsate were plated onto Campy-cefex agar and incubated. In a second experiment, samples were both directly plated and enriched in Bolton enrichment broth. In the first experiment, 35 of 60 carcass rinses tested positive for Campylobacter, while 29 of 60 weep samples yielded Campylobacter isolates with levels of 1.0 and 1.1 log CFU/ml, respectively. In the second experiment, Campylobacter was recovered from 9 of 40 rinse samples and from 13 of 40 weep samples by direct plating, while the organism was recovered from 28 of 40 rinses samples and from 23 of 40 carcass samples by enrichment. There was no significant difference between the two methods with respect to Campylobacter prevalence as determined by the chi-square test. Campylobacter levels recovered by both methods averaged 0.9 log CFU/ml. The sampling of weep fluid was a simple, effective means of detecting this important human enteropathogen on broiler carcasses.

Genotype analyses of Campylobacter isolated from the gastrointestinal tracts and the reproductive tracts of broiler breeder roosters.

Campylobacter is considered to be the leading bacterial etiologic agent of acute gastroenteritis in humans. Evidence implicates poultry as a major source of the organism for human illness; however, the pathways involved in Campylobacter contamination of poultry flocks, horizontal transmission and/or vertical transmission, remain unclear. Recent evidence implicates breeders as a potential source for Campylobacter contamination of the subsequent broiler offspring. In this investigation, Campylobacter isolated from feces, cloacal swabs, ceca, semen, and vas deferens of 12 breeder broiler roosters were genotyped by both flagellin A short variable region (flaA SVR) DNA sequence analysis and repetitive element (rep)-polymerase chain reaction (PCR). In 9 of 12 roosters, Campylobacter was isolated from multiple sites sampled. Comparison of multiple isolates obtained from individual roosters revealed variable results. In five of the nine roosters, all Campylobacter isolated demonstrated closely related flaA SVR DNA sequences as well as rep-PCR patterns; isolates from these roosters were collected from both the gastrointestinal and the reproductive tracts or from the gastrointestinal tract alone. The remaining four roosters had Campylobacter that were distinct by both typing methods. Isolates from two of these four roosters originated from both the gastrointestinal and the reproductive tracts. Isolates from the remaining two roosters originated from only the reproductive tract. Comparisons of all Campylobacter isolates recovered from a distinct sample type within either the reproductive tract or the gastrointestinal tract (feces, semen, cloaca, vas deferens, or ceca) were quite diverse. No relationship between the genotypes and the sample type could be ascertained. Further investigation is needed to determine the route of contamination and if the presence of Campylobacter within the rooster leads to contamination of the broiler offspring via the fertilized egg.

Immunological Evaluation of Pasteurized, Deaerated Human Milk.

Retention of immunoglobulin A (IgA) and IgA specific for enteropathogenic Escherichia coli (EPEC) was determined in human milk that was processed and stored under refrigeration or frozen conditions. Human milk was subjected to three different processing conditions: (a) deaerated, vacuum packaged in metalized polyester bags and pasteurized (64°C for 6 min, 10 s); (b) vacuum packaged and pasteurized; (c) vacuum packaged. Samples were stored at 4°C for 7 d and at -20°C for 28 d. An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to EPEC was developed and used to monitor these antibody levels, while radial immunodiffusion (RID) plates were used to provide information on total IgA of the milk samples. In addition, the dissolved oxygen content of the samples was monitored. It was found that pasteurization and deaeration did not adversely affect the IgA levels, and levels of total IgA and IgA specific for EPEC remained stable or increased slightly during storage in most samples regardless of the storage temperatures.

Microbiological Examination of Pasteurized, Deaerated Human Milk.

Three composites of human milk samples were subjected to different processing conditions: (1) deaerated, vacuum packaged in metalized polyester bags and pasteurized at 56°C for 8 min; (2) vacuum packaged and pasteurized; (3) vacuum packaged. On days 0, 4, 7, 14, 28, 64, and 96 of storage, each treatment was analyzed for dissolved oxygen content and viable microflora. On days 0, 4, and 14, randomly selected isolates from each treatment were identified to the species level. Heat treatment of the milk samples reduced the number of viable microorganisms and resulted in a shift in the type of bacteria in the milk. Pasteurized samples contained primarily non-pathogenic skin commensals, while non-pasteurized samples were populated by species of Pseudomonas and other Gram negative microorganisms, including potential pathogens. Deaeration did not affect either the number or types of microorganisms surviving the heat treatment.