Jean Guard

Cell invasion of poultry-associated Salmonella enterica serovar Enteritidis isolates is associated with pathogenicity, motility and proteins secreted by the type III secretion system

Salmonella enterica serovar Enteritidis (S. Enteritidis) is a major cause of food-borne gastroenteritis in humans worldwide. Poultry and poultry products are considered the major vehicles of transmission to humans. Using cell invasiveness as a surrogate marker for pathogenicity, we tested the invasiveness of 53 poultry-associated isolates of S. Enteritidis in a well-differentiated intestinal epithelial cell model (Caco-2). The method allowed classification of the isolates into low (n = 7), medium (n = 18) and high (n = 30) invasiveness categories. Cell invasiveness of the isolates did not correlate with the presence of the virulence-associated gene spvB or the ability of the isolates to form biofilms. Testing of representative isolates with high and low invasiveness in a mouse model revealed that the former were more invasive in vivo and caused more and earlier mortalities, whereas the latter were significantly less invasive in vivo, causing few or no mortalities. Further characterization of representative isolates with low and high invasiveness showed that most of the isolates with low invasiveness had impaired motility and impaired secretion of either flagella-associated proteins (FlgK, FljB and FlgL) or type III secretion system (TTSS)-secreted proteins (SipA and SipD) encoded on Salmonella pathogenicity island-1. In addition, isolates with low invasiveness had impaired ability to invade and/or survive within chicken macrophages. These data suggest that not all isolates of S. Enteritidis recovered from poultry may be equally pathogenic, and that the pathogenicity of S. Enteritidis isolates is associated, in part, with both motility and secretion of TTSS effector proteins.

Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type.

BackgroundSalmonella Enteritidis is currently the worlds leading cause of salmonellosis, in part because of its ability to contaminate the internal contents of eggs. Previous analyses have shown that it is an exceptionally clonal serotype, which nonetheless generates considerable phenotypic heterogeneity. Due to its clonality, whole genome analysis is required to find genetic determinants that contribute to strain heterogeneity of Salmonella Enteritidis. Comparative whole genome mutational mapping of two PT13a strains that varied in the ability to contaminate eggs and to form biofilm was achieved using a high-density tiling platform with primers designed from a PT4 reference genome. Confirmatory Sanger sequencing was used on each putative SNP identified by mutational mapping to confirm its presence and location as compared to the reference sequence. High coverage pyrosequencing was used as a supporting technology to review results.ResultsA total of 250 confirmed SNPs were detected that differentiated the PT13a strains. From these 250 SNPS, 247 were in the chromosome and 3 were in the large virulence plasmid. SNPs ranged from single base pair substitutions to a deletion of 215 bp. A total of 15 SNPs (3 in egg-contaminating PT13a 21046 and 12 in biofilm forming PT13a 21027) altered coding sequences of 16 genes. Pyrosequencing of the two PT13a subpopulations detected 8.9% fewer SNPs than were detected by high-density tiling. Deletions and ribosomal gene differences were classes of SNPs not efficiently detected by pyrosequencing.ConclusionsThese results increase knowledge of evolutionary trends within Salmonella enterica that impact the safety of the food supply. Results may also facilitate designing 2nd generation vaccines, because gene targets were identified that differentiate subpopulations with variant phenotypes. High-throughput genome sequencing platforms should be assessed for the ability to detect classes of SNPs equivalently, because each platform has different advantages and limits of detection.

Prevalence of coliforms, Salmonella, Listeria, and Campylobacter associated with eggs and the environment of conventional cage and free-range egg production

There is a desire by US consumers for eggs produced by hens in alternative production systems. As the retail shell-egg market offers these products to accommodate consumer demands, additional information is needed to ensure processing methodologies result in safe eggs from all egg sources. A study was conducted to determine if there were differences in the prevalence of coliforms, Salmonella, Listeria, and Campylobacter on and within eggs and in the environment of a sister flock of conventional cage and free-range laying hens. Microbial sampling occurred approximately every 6 wk between 20 and 79 wk of age. A random sampling of typical coliform colonies produced 371 viable isolates for biochemical identification. Twenty-nine genera or species of bacteria were identified. There was a significantly greater (P < 0.0001) prevalence of Campylobacter in the free-range nest box swabs compared with that in the free-range grass and conventional cage swab samples (number of positives: 8 nest box, 1 grass, 0 cage). Seven isolates of Listeria innocua were detected with no significant difference in prevalence between the treatments. Isolates were associated with eggshells (2 free-range floor, 1 cage) and the free-range environment (2 nest box, 2 grass). There were 21 Salmonella isolates detected between all sample locations, with no significant difference in the prevalence of Salmonella detection between the treatments. Additional studies are needed to fully understand the effect of alternative production methods on the prevalence of pathogens and coliforms associated with nest-run eggs and the production environment.

Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen

ABSTRACT Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nalr strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis.

Salmonella Enteritidis Strains from Poultry Exhibit Differential Responses to Acid Stress, Oxidative Stress, and Survival in the Egg Albumen

Salmonella Enteritidis is the major foodborne pathogen that is primarily transmitted by contaminated chicken meat and eggs. We recently demonstrated that Salmonella Enteritidis strains from poultry differ in their ability to invade human intestinal cells and cause disease in orally challenged mice. Here we hypothesized that the differential virulence of Salmonella Enteritidis strains is due to the differential fitness in the adverse environments that may be encountered during infection in the host. The responses of a panel of six Salmonella Enteritidis strains to acid stress, oxidative stress, survival in egg albumen, and the ability to cause infection in chickens were analyzed. This analysis allowed classification of strains into two categories, stress-sensitive and stress-resistant, with the former showing significantly (p<0.05) reduced survival in acidic (gastric phase of infection) and oxidative (intestinal and systemic phase of infection) stress. Stress-sensitive strains also showed impaired intestinal colonization and systemic dissemination in orally inoculated chickens and failed to survive/grow in egg albumen. Comparative genomic hybridization microarray analysis revealed no differences at the discriminatory level of the whole gene content between stress-sensitive and stress-resistant strains. However, sequencing of rpoS, a stress-regulatory gene, revealed that one of the three stress-sensitive strains carried an insertion mutation in the rpoS resulting in truncation of σ(S). Finding that one of the stress-sensitive strains carried an easily identifiable small polymorphism within a stress-response gene suggests that the other strains may also have small polymorphisms elsewhere in the genome, which likely impact regulation of stress or virulence associated genes in some manner.

The Relationship Between the Numbers of Salmonella Enteritidis, Salmonella Heidelberg, or Salmonella Hadar Colonizing Reproductive Tissues of Experimentally Infected Laying Hens and Deposition Inside Eggs

Abstract Contamination of eggs by Salmonella Enteritidis has been a prominent cause of human illness for several decades and is the focus of a recently implemented national regulatory plan for egg-producing flocks in the United States. Salmonella Heidelberg has also been identified as an egg-transmitted pathogen. The deposition of Salmonella strains inside eggs is a consequence of reproductive tract colonization in infected laying hens, but prior research has not determined the relationship between the numbers of Salmonella that colonize reproductive organs and the associated frequency of egg contamination. In the present study, groups of laying hens in two trials were experimentally infected with large oral doses of strains of Salmonella Enteritidis (phage type 13a), Salmonella Heidelberg, or Salmonella Hadar. Reproductive tissues of selected hens were cultured to detect and enumerate Salmonella at 5 days postinoculation, and the interior contents of eggs laid between 6 and 25 days postinoculation were tested for contamination. Significantly more internally contaminated eggs were laid by hens infected with Salmonella Enteritidis (3.58%) than with strains of either Salmonella Heidelberg (0.47%) or Salmonella Hadar (0%). However, no significant differences were observed between Salmonella strains in either isolation frequency or the number of colony-forming units (CFU) isolated from ovaries or oviducts. Salmonella isolation frequencies ranged from 20.8% to 41.7% for ovaries and from 8.3% to 33.3% for oviducts. Mean Salmonella colonization levels ranged from 0.10 to 0.51 log CFU/g for ovaries and from 0.25 to 0.46 log CFU/g for oviducts. Although parallel rank-orders were observed for Salmonella enumeration (in both ovaries and oviducts) and egg contamination frequency, a statistically significant relationship could not be established between these two parameters of infection.

Salmonella Enteritidis Deposition in Eggs after Experimental Infection of Laying Hens with Different Oral Doses

The continuing attribution of human Salmonella Enteritidis infections to internally contaminated eggs has necessitated the commitment of substantial public and private resources to Salmonella Enteritidis testing and control programs in commercial laying flocks. Cost-effective risk-reduction requires a detailed and comprehensive understanding of how Salmonella Enteritidis infections in hens result in deposition of the pathogen inside eggs. The present study sought to resolve some incompletely defined aspects of the relationship between Salmonella Enteritidis oral-exposure dose levels in experimentally infected laying hens and the frequency and location of subsequent egg contamination. In two trials, groups of specific-pathogen-free hens were experimentally inoculated with oral doses of 10(4), 10(6), or 10(8) CFU of a phage type 4 Salmonella Enteritidis strain. Eggs were collected 5 to 23 days postinoculation, and the yolk and albumen of each egg were cultured separately to detect Salmonella Enteritidis contamination. Larger oral doses of Salmonella Enteritidis administered to hens were associated with significant increases in the frequencies of both yolk and albumen contamination. Moreover, Salmonella Enteritidis was found in the albumen of a far-higher proportion of contaminated eggs from hens given the largest dose than from the other two groups. Salmonella Enteritidis contamination was detected in 0.7% of yolk and 0.2% of albumen samples after inoculation of hens with 10(4) CFU, 4.0% of yolk and 1.7% of albumen samples after inoculation with 10(6) CFU, and 6.5% of yolk and 10.8% of albumen samples after inoculation with 10(8) CFU. These results demonstrate that oral-exposure doses of Salmonella Enteritidis for laying hens can significantly affect both the frequency and location of deposition of this pathogen inside eggs.

Colonization of avian reproductive-tract tissues by variant subpopulations of Salmonella Enteritidis.

Abstract Leghorn hens were infected with Salmonella Enteritidis cultures of known genomic content and subpopulation characteristics to determine comparative abilities to colonize the avian reproductive tract. Group 1 received phage-type (PT)4 22079, which is a dimorphic subpopulation that can both contaminate eggs and form biofilm. Group 2 received a 90:10 mixture of monomorphic PT13a strains 21027 and 21046, which produce biofilm or contaminate eggs, respectively. Group 3 received a 10:90 mixture of the same two PT13a strains, respectively. Trials were repeated three times and a total of 30 hens per treatment group were infected. Dosage was by oral gavage and was calculated as 8.6 ± 2.01 × 107 colony-forming units per hen. Liver, spleen, and three different sections of oviduct (ovary, upper oviduct, and lower oviduct) were cultured per bird. Results were that all three groups had livers and spleens that were mostly positive (90.0% and 94.4% of 270 hens cultured, respectively). Reproductive-tract organs yielded 75 positives from 270 hens (27.8%), and treatment groups ranged from a low of 6.7% to a high of 76.7% positive cultures in any one trial. There was no significant difference between the numbers of positive reproductive-tract samples between treatment groups due to variance. These results suggest that the status of the reproductive tract at the time of infection may impact recovery of culture-positive tissue and contribute to variance. It is suggested that Salmonella Enteritidis cultures that vary in subpopulation composition have subtle differences in colonization of reproductive tissue that contribute to variance in egg contamination. Culture of non–reproductive-tract organs such as the liver and spleen was overall more reliable for detection of infected hens. The spleen was especially useful for detection because of its small size. Further research is needed to determine how sex hormones influence the infection pathway that results in egg contamination.

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.

Multiplication of Salmonella Enteritidis in Egg Yolks after Inoculation outside, on, and inside Vitelline Membranes and Storage at Different Temperatures

Prompt refrigeration to restrict bacterial growth is important for reducing eggborne transmission of Salmonella enterica serovar Enteritidis (SE). The nutrient-rich yolk interior is a relatively infrequent location for initial SE deposition in eggs, but migration across the vitelline membrane can result in rapid bacterial multiplication during storage at warm temperatures. The objective of the present study was to measure the multiplication of SE in yolks after introduction at three different locations and subsequent storage at a range of temperatures. Using an in vitro egg contamination model, approximately 100 CFU of SE was inoculated either inside yolks, onto the exterior surface of vitelline membranes, or into the adjacent albumen. After storage of samples from each inoculation group at 10, 15, 20, and 25°C for 24 h, SE was enumerated in yolks. For all three inoculation locations, the final SE levels in yolks increased significantly with increasing storage temperatures. At all storage temperatures, significant differences in SE multiplication were observed between inoculation sites (yolk inoculation>vitelline membrane inoculation>albumen inoculation). At 25°C, final log concentrations of 7.759 CFU of SE per ml (yolk inoculation), 2.014 CFU/ml (vitelline membrane inoculation), and 0.757 CFU/ml (albumen inoculation) were attained in yolks after storage. These results demonstrate that, even when the initial site of SE deposition is outside the egg yolk, substantial multiplication supported by yolk nutrients can occur during the first day of storage and the risk of bacterial growth increases at higher ambient storage temperatures.