Metabolic parameters linked by phenotype microarray to acid resistance profiles of poultry-associated Salmonella enterica.

Phenotype microarrays were analyzed for 51 datasets derived from Salmonella enterica. The top 4 serotypes associated with poultry products and one associated with turkey, respectively Typhimurium, Enteritidis, Heidelberg, Infantis and Senftenberg, were represented. Datasets were partitioned initially into two clusters based on ranking by values at pH 4.5 (PM10 A03). Negative control wells were used to establish 90 respiratory units as the point differentiating acid resistance from sensitive strains. Thus, 24 isolates that appeared most acid-resistant were compared initially to 27 that appeared most acid-sensitive (24 × 27 format). Paired cluster analysis was also done and it included the 7 most acid-resistant and -sensitive datasets (7 × 7 format). Statistical analyses of ranked data were then calculated in order of standard deviation, probability value by the Student's t-test and a measure of the magnitude of difference called effect size. Data were reported as significant if, by order of filtering, the following parameters were calculated: i) a standard deviation of 24 respiratory units or greater from all datasets for each chemical, ii) a probability value of less than or equal to 0.03 between clusters and iii) an effect size of at least 0.50 or greater between clusters. Results suggest that between 7.89% and 23.16% of 950 chemicals differentiated acid-resistant isolates from sensitive ones, depending on the format applied. Differences were more evident at the extremes of phenotype using the subset of data in the paired 7 × 7 format. Results thus provide a strategy for selecting compounds for additional research, which may impede the emergence of acid-resistant Salmonella enterica in food.

Reduction of Salmonella Enteritidis in the spleens of hens by bacterins that vary in fimbrial protein SefD.

The objective of this research was to determine whether variation in the presence of fimbrial protein SefD would impact efficacy of bacterins as measured by recovery of Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) from the spleens of hens. Two bacterins were prepared that varied in SefD content. Also, two adjuvants were tested, namely, water-in-oil and aluminum hydroxide gel (alum). Control groups for both adjuvant preparations included infected nonvaccinated hens and uninfected nonvaccinated hens. At 21 days postinfection, Salmonella Enteritidis was recovered from 69.7%, 53.1%, and 86.0% from the spleens of all hens vaccinated with bacterins lacking SefD, bacterins that included SefD, and infected nonvaccinated control hens, respectively. No Salmonella was recovered from uninfected nonvaccinates. Results from individual trials showed that both bacterins reduced positive spleens, but that the one with SefD was more efficacious. Alum adjuvant had fewer side effects on hens and egg production as compared to water-in-oil. However, adjuvant did not change the relative recovery of Salmonella Enteritidis from spleens. These results suggest that SefD is a promising target antigen for improving the efficacy of immunotherapy in hens, and is intended to reduce Salmonella Enteritidis in the food supply.

Recovery of Salmonella enterica serovar Enteritidis from hens initially infected with serovar Kentucky.

Salmonella enterica serovars Enteritidis and Kentucky differ greatly in epidemiology. We wanted to know if the non-pathogenic serotype Kentucky impacted the recovery of the pathogen Enteritidis from chickens. To explore this issue, 4 groups of hens were treated as follows: (i) hens were inoculated orally with Kentucky and injected intramuscularly 2 weeks later with Enteritidis, (ii) hens were contact infected with Kentucky and then with Enteritidis, (iii) hens were injected with Enteritidis only, and (iv) hens were contact infected with Enteritidis only. Hens exposed orally to serotype Kentucky received 10 exp9 CFU, and hens injected with serotype Enteritidis received 10 exp7 CFU intramuscularly. Contact infected hens were kept in rooms with deliberately infected hens. Droppings, cecal tonsils and 5 internal organs were sampled and cultured at 6, 13 and 20 days post-infection from the 4 groups. Egg production was monitored. Results suggest that non-pathogenic serotypes of Salmonella may mitigate recovery of Enteritidis from chickens exposed by contact. In summary, we show results from an initial experiment intended to investigate if multiple serotypes impact the ecology of pathogenic S. enterica on-farm.

Serotype and antimicrobial resistance patterns of Salmonella isolates from commercial birds and poultry environment in Mississippi.

To obtain information about Salmonella from commercial birds and poultry environments within Mississippi, 50 Salmonella enterica isolates were collected and characterized by intergenic sequence ribotyping (ISR) serotyping and by determining antimicrobial resistance. ISR assigned serotype to all 50 Salmonella enterica isolates whereas the Kauffman-White-LeMinor antibody-based scheme assigned serotype to 48. Agreement between both methods was K = 89.58. Within the set, 12 serotypes were detected. The antimicrobial resistance patterns (ARP) of 12 serotypes, namely Enteritidis, Typhimurium, Kentucky, Bredeney, Mbandaka, Saintpaul, Montevideo, Cubana, Lille, Senftenberg, Johannesburg, and one serotype UN0094, were determined using minimum inhibitory concentration values. The antibiograms demonstrated differences between Salmonella serotypes and among isolates of the same serotype. All isolates were 100% susceptible to enrofloxacin and trimethoprim/sulfamethoxazole. The number of antimicrobials to which the isolates were resistant ranged from two to nine. Twenty-two different ARPs were identified and ARP1, with resistance to spectinomycin and sulfadimethoxine, was most frequently observed. Forty isolates (80%) were resistant to three or more antimicrobials and were thus designated multidrug resistant. Detection of a unique serotype, and variation in antibiograms within the set, demonstrates that it is important to survey isolates periodically from a region to follow epidemiologic trends.

Presence of Salmonella enteritidis and Salmonella gallinarum in commercial laying hens diagnosed with fowl typhoid disease in Colombia.

A severe outbreak of salmonellosis in commercial brown table egg layers first occurred in Colombia in 2006. From 2008 to 2012, 35 samples collected from commercial layers farms in the states of Cundinamarca, Santander, Bolivar, and San Andres, were positive for Salmonella enterica. Salmonella was isolated from liver and spleen (71.42%), pools of organs (liver, spleen, and ovarian follicles; 25.71%), and drag swabs (2.85%). Serotype was assigned using single nucleotide polymorphisms or DNA microarray hybridization. Sixteen strains of Salmonella Enteritidis, and 13 of Salmonella Gallinarum were identified. Seven strains yielded three unique sequences, and they were designated as UN0038, UN0052, and UN0054 by intergenic sequence ribotyping. These strains were later identified as Salmonella serotypes Isangi, Braenderup, and Yoruba, respectively, by DNA microarray hybridization. The discovery that a common human pathogen (Salmonella Enteritidis) was coisolated from farms with an avian pathogen (Salmonella Gallinarum) in similar commercial brown layer hens and in different regions indicates that it is important to investigate the dynamics of Salmonella infection and determine the serotypes circulating within the same ecologic niche.

Comparison of dkgB-linked intergenic sequence ribotyping to DNA microarray hybridization for assigning serotype to Salmonella enterica.

Two DNA-based methods were compared for the ability to assign serotype to 139 isolates of Salmonella enterica ssp. I. Intergenic sequence ribotyping (ISR) evaluated single nucleotide polymorphisms occurring in a 5S ribosomal gene region and flanking sequences bordering the gene dkgB. A DNA microarray hybridization method that assessed the presence and the absence of sets of genes was the second method. Serotype was assigned for 128 (92.1%) of submissions by the two DNA methods. ISR detected mixtures of serotypes within single colonies and it cost substantially less than Kauffmann-White serotyping and DNA microarray hybridization. Decreasing the cost of serotyping S. enterica while maintaining reliability may encourage routine testing and research.

Virulence and metabolic characteristics of Salmonella enterica serovar enteritidis strains with different sefD variants in hens.

Salmonella enterica serovar Enteritidis is one of a few Salmonella enterica serotypes that has SEF14 fimbriae encoded by the sef operon, which consists of 4 cotranscribed genes, sefABCD, regulated by sefR. A parental strain was used to construct a sefD mutant and its complement, and all 3 strains were compared for gene expression, metabolic properties, and virulence characteristics in hens. Transcription of sefD by wild type was suppressed at 42°C and absent for the mutant under conditions where the complemented mutant had 10(3) times higher transcription. Growth of the complemented mutant was restricted in comparison to that of the mutant and wild type. Hens infected with the wild type and mutant showed decreased blood calcium and egg production, but infection with the complemented mutant did not. Thus, the absence of sefD correlated with increased metabolic capacity and enhanced virulence of the pathogen. These results suggest that any contribution that sefD makes to egg contamination is either unknown or would be limited to early transmission from the environment to the host. Absence of sefD, either through mutation or by suppression of transcription at the body temperature of the host, may contribute to the virulence of Salmonella enterica by facilitating growth on a wide range of metabolites.