Differential carbon source utilization by Campylobacter jejuni 11168 in response to growth temperature variation.

Campylobacter spp. readily colonize the intestinal tracts of both human and avian species. While most often commensal organisms in birds, campylobacters remain the leading cause of bacterial gastroenteritis in humans. The association of campylobacters with poultry is well established as a primary route for human exposure. The difference in normal core body temperature between chickens (42 degrees C) and humans (37 degrees C) has been suggested to trigger potential colonization or virulence factors and investigators have demonstrated differential gene expression at the two temperatures. Campylobacter spp. exhibit unique nutritional requirements and have been thought to only utilize amino acids and Kreb cycle intermediates as carbon sources for growth. We evaluated the ability of the genome-sequenced strain of Campylobacter jejuni 11168 (GS) to oxidize 190 different substrates as sole carbon sources at 37 degrees C and 42 degrees C using phenotype microarray (PM) technology. Results indicate that the expected amino acids, l-serine, l-aspartic acid, l-asparagine, and l-glutamic acid were utilized in addition to a number of organic acids. In general, oxidation of the substrates was greater at 42 degrees C than at 37 degrees C with a few exceptions. By employing the PM method, we observed a number of potential false-positive reactions for substrates including the triose, dihydroxyacetone; and the pentose sugars, d-xylose, d-ribose, l-lyxose, and d- and l-arabinose. The presence of genes possibly responsible for utilization of pentose sugars is supported by the genomic sequence data, but actual utilization as sole carbon sources for active respiration has not been observed. A better understanding of the metabolic pathways and nutritional requirements of campylobacters could lead to improvements in culture media for detection and isolation of the pathogen and to future intervention methods to reduce human exposure.

In vitro penetration of egg yolks by Salmonella Enteritidis and Salmonella Heidelberg strains during thirty-six-hour ambient temperature storage.

Although Salmonella deposition inside yolks is uncommon in naturally contaminated eggs, migration through the vitelline membrane into the nutrient-rich yolk contents could enable rapid bacterial multiplication. Egg refrigeration restricts both penetration and growth, but a recently proposed national Salmonella Enteritidis control program would allow unrefrigerated ambient temperature storage of eggs on farms for up to 36 h. The present study used an in vitro egg contamination model to assess the ability of small numbers of 4 Salmonella Enteritidis strains and 4 Salmonella Heidelberg strains to penetrate the vitelline membrane and multiply inside yolks during 36 h of storage at either 20 or 30 degrees C. After inoculation onto the exterior surface of the vitelline membrane, all 8 Salmonella strains penetrated to the yolk contents (at a mean frequency of 45.1%), and most strains grew to significantly higher levels (with a mean (log)10 bacterial concentration of 2.2 cfu/mL) during incubation at 30 degrees C. Significant differences in penetration frequency and yolk multiplication were observed between individual strains and between serotypes (Salmonella Enteritidis > Salmonella Heidelberg for both parameters). Penetration and multiplication were significantly less frequent during incubation at 20 degrees C. These results demonstrate that controlling ambient temperatures during prerefrigeration storage may be an important adjunct to prompt refrigeration for limiting Salmonella growth in eggs and thereby for preventing egg-transmitted human illness.

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.

Evaluation of eggshell quality of hens infected with Salmonella enteritidis by application of compression.

Eggs collected from hens of different ages and that differed in infection status with Salmonella enteritidis were evaluated for the ability to resist cracking following application of maximum compression load from an Instron materials testing machine. Orally infected 24-wk-old hens that were prepeak produced eggs with significantly lower hardness units (HU) of shells compared with a paired control group (P < or = 0.01). However, 1 of 3 additional infection trials in hens at peak (29 wk) and older hens postpeak (58 wk) showed an increase in HU in one trial and no difference in the other 2 trials. Thus, Salmonella enteritidis may be able to alter HU in a manner that is influenced by multiple factors, which include the age of the hen and the strain used for infection. Hardness was overall a sensitive physiological barometer of age, because readings correlated positively (all R > 0.50) with hens entering peak production, regardless of infection status. Detection of a very low HU reading (<1.0) was indicative of a hairline crack in the egg, which increased in incidence from 0.01% preinfection to 0.08% postinfection. Two other clinical signs noted postinfection in hens were that i) daily egg production significantly increased in older hens, and ii) emaciation was evident in a few hens that were infected by contact. These results suggest that there may be supportive approaches to achieve reduction of S. enteritidis in table eggs that do not rely on culturing.