Re-sequencing of a virulent strain of Campylobacter jejuni NCTC11168 reveals potential virulence factors.

In vitro passage of Campylobacter jejuni strains results in phenotypic changes and a general loss of virulence, as is the case with the genome-sequenced strain C. jejuni NCTC11168. Re-sequencing of a virulent strain of NCTC11168 identified 41 SNPs or indels involving 20 genes, four intergenic regions and three pseudogenes. The genes include six motility genes, two chemotaxis genes, three hypothetical genes and a capsule biosynthesis gene, which might have a critical role in C. jejuni virulence. Additionally, we found an insertion in both Cj0676 and Cj1470c, pseudogenes in avirulent NCTC11168, but functional proteins in virulent NCTC11168.

The Campylobacter jejuni Dps homologue is important for in vitro biofilm formation and cecal colonization of poultry and may serve as a protective antigen for vaccination.

In this work, we investigated the Campylobacter jejuni dps (DNA binding protein from starved cells) gene for a role in biofilm formation and cecal colonization in poultry. In vitro biofilm formation assays were conducted with stationary-phase cells in cell culture plates under microaerophilic conditions. These studies demonstrated a significant (>50%) reduction in biofilm formation by the C. jejuni dps mutant compared to that by the wild-type strain. Studies in poultry also demonstrated the importance of the dps gene in host colonization by C. jejuni. Real-time PCR analysis of mRNA extracted from the cecal contents of poultry infected with wild-type C. jejuni indicated that the dps gene is upregulated 20-fold during poultry colonization. Cecal colonization was greater than 5 log CFU lower in chicks infected with the dps mutant than chicks infected with the wild-type C. jejuni strain. Moreover, the dps mutant failed to colonize 75% of the chicks following challenge with 10(5) CFU. Preliminary studies were conducted in chicks by parenteral vaccination with a recombinant Dps protein or through oral vaccination with a recombinant attenuated Salmonella enterica strain synthesizing the C. jejuni Dps protein. No reduction in C. jejuni was noted in chicks vaccinated with the parenteral recombinant protein, whereas, a 2.5-log-unit reduction of C. jejuni was achieved in chicks vaccinated with the attenuated Salmonella vector after homologous challenge. Taken together, this work demonstrated the importance of Dps for biofilm formation and poultry colonization, and the study also provides a basis for continued work using the Dps protein as a vaccine antigen when delivered through a Salmonella vaccine vector.

Investigations of Salmonella enterica serovar newport infections of oysters by using immunohistochemistry and knockout mutagenesis.

The consumption of raw oysters is an important risk factor in the acquisition of food-borne disease, with Salmonella being one of a number of pathogens that have been found in market oysters. Previous work by our lab found that Salmonella was capable of surviving in oysters for over 2 months under laboratory conditions, and this study sought to further investigate Salmonella's tissue affinity and mechanism of persistence within the oysters. Immunohistochemistry was used to show that Salmonella was capable of breaching the epithelial barriers, infecting the deeper connective tissues of the oysters, and evading destruction by the oysters' phagocytic hemocytes. To further investigate the mechanism of these infections, genes vital to the function of Salmonella's two main type III secretion systems were disrupted and the survivability of these knockout mutants within oysters was assayed. When the Salmonella pathogenicity island 1 and 2 mutant strains were exposed to oysters, there were no detectable deficiencies in their abilities to survive, suggesting that Salmonella's long-term infection of oysters does not rely upon these two important pathogenicity islands and must be due to some other, currently unknown, mechanism.

A Campylobacter jejuni Dps homolog has a role in intracellular survival and in the development of campylobacterosis in neonate piglets.

Iron acquisition is an absolute requirement by most microorganisms for host survival. In this work, we investigated the Campylobacter jejuni iron binding Dps protein for a potential role in virulence. In vitro assays using J774A.1 macrophage-like cells demonstrated a 2.5 log reduction in C. jejuni survival of the Dps mutant and a reduction of four logs in invasion of HEp-2 epithelial cells compared to the wild-type strain. To examine the role of the dps gene in host pathogenesis, the piglet model was used in C. jejuni challenge studies. In vivo inoculation studies of newborn piglets with wild-type C. jejuni demonstrated an 11-fold upregulation of the dps gene and intestinal lesion production typical of campylobacteriosis in humans. In contrast, piglets inoculated with the dps mutant were not colonized and remained normal throughout the study period. Mucosal lesion production was restored in piglets inoculated with the complemented Dps mutant strain. Based on these results, we conclude that the C. jejuni Dps homolog is a virulence factor in the production of campylobacteriosis, and warrants further investigation.

Survival of Salmonella Newport in oysters.

Salmonella enterica is the leading cause of laboratory-confirmed foodborne illness in the United States and raw shellfish consumption is a commonly implicated source of gastrointestinal pathogens. A 2005 epidemiological study done in our laboratory by Brands et al., showed that oysters in the United States are contaminated with Salmonella, and in particular, a specific strain of the Newport serovar. This work sought to further investigate the host-microbe interactions between Salmonella Newport and oysters. A procedure was developed to reliably and repeatedly expose oysters to enteric bacteria and quantify the subsequent levels of bacterial survival. The results show that 10 days after an exposure to Salmonella Newport, an average concentration of 3.7 × 10(3)CFU/g remains within the oyster meat, and even after 60 days there still can be more than 10(2)CFU/g remaining. However, the strain of Newport that predominated in the market survey done by Brands et al. does not survive within oysters or the estuarine environment better than any other strains of Salmonella we tested. Using this same methodology, we compared Salmonella Newport's ability to survive within oysters to a non-pathogenic strain of E. coli and found that after 10 days the concentration of Salmonella was 200-times greater than that of E. coli. We also compared those same strains of Salmonella and E. coli in a depuration process to determine if a constant 120 L/h flux of clean seawater could significantly reduce the concentration of bacteria within oysters and found that after 3 days the oysters retained over 10(4)CFU/g of Salmonella while the oysters exposed to the non-pathogenic strain of E. coli contained 100-times less bacteria. Overall, the results of this study demonstrate that any of the clinically relevant serovars of Salmonella can survive within oysters for significant periods of time after just one exposure event. Based on the drastic differences in survivability between Salmonella and a non-pathogenic relative, the results of this study also suggest that unidentified virulence factors may play a role in Salmonella's interactions with oysters.

Prevalence and characterization of Salmonella serovars isolated from oysters served raw in restaurants.

To determine if Salmonella-contaminated oysters are reaching consumer tables, a survey of raw oysters served in eight Tucson restaurants was performed from October 2007 to September 2008. Salmonella spp. were isolated during 7 of the 8 months surveyed and were present in 1.2% of 2,281 oysters tested. This observed prevalence is lower than that seen in a previous study in which U.S. market oysters were purchased from producers at bays where oysters are harvested. To test whether the process of refrigerating oysters in restaurants for several days reduces Salmonella levels, oysters were artificially infected with Salmonella and kept at 4°C for up to 13 days. Direct plate counts of oyster homogenate showed that Salmonella levels within oysters did not decrease during refrigeration. Six different serovars of Salmonella enterica were found in the restaurant oysters, indicating multiple incidences of Salmonella contamination of U.S. oyster stocks. Of the 28 contaminated oysters, 12 (43%) contained a strain of S. enterica serovar Newport that matched by pulsed-field gel electrophoresis a serovar Newport strain seen predominantly in the study of bay oysters performed in 2002. The repeated occurrence of this strain in oyster surveys is concerning, since the strain was resistant to seven antimicrobials tested and thus presents a possible health risk to consumers of raw oysters.

Antimicrobial edible apple films inactivate antibiotic resistant and susceptible Campylobacter jejuni strains on chicken breast.

UNLABELLED: Campylobacter jejuni is the leading cause of bacterial diarrheal illness worldwide. Many strains are now becoming multidrug resistant. Apple-based edible films containing carvacrol and cinnamaldehyde were evaluated for bactericidal activity against antibiotic resistant and susceptible C. jejuni strains on chicken. Retail chicken breast samples inoculated with D28a and H2a (resistant strains) and A24a (a sensitive strain) were wrapped in apple films containing cinnamaldehyde or carvacrol at 0.5%, 1.5%, and 3% concentrations, and then incubated at 4 or 23 °C for 72 h. Immediately after wrapping and at 72 h, samples were plated for enumeration of viable C. jejuni. The antimicrobial films exhibited dose- and temperature-dependent bactericidal activity against all strains. Films with ≥1.5% cinnamaldehyde reduced populations of all strains to below detection at 23 °C at 72 h. At 4 °C with cinnamaldehyde, reductions were variable for all strains, ranging from 0.2 to 2.5 logs and 1.8 to 6.0 logs at 1.5% and 3.0%, respectively. Films with 3% carvacrol reduced populations of A24a and H2a to below detection, and D28a by 2.4 logs at 23 °C and 72 h. A 0.5-log reduction was observed for both A24a and D28a, and 0.9 logs for H2a at 4 °C at 3% carvacrol. Reductions ranged from 1.1 to 1.9 logs and 0.4 to 1.2 logs with 1.5% and 0.5% carvacrol at 23 °C, respectively. The films with cinnamaldehyde were more effective than carvacrol films. Reductions at 23 °C were greater than those at 4 °C. Our results showed that antimicrobial apple films have the potential to reduce C. jejuni on chicken and therefore, the risk of campylobacteriosis. Possible mechanisms of antimicrobial effects are discussed. PRACTICAL APPLICATION: Apple antimicrobial films could potentially be used in retail food packaging to reduce C. jejuni commonly present on food.

Complete genome sequence of Campylobacter jejuni strain S3.

Campylobacter jejuni is one of the leading causes of bacterial gastroenteritis in the world; however, there is only one complete genome sequence of a poultry strain to date. Here we report the complete genome sequence and annotation of the second poultry strain, C. jejuni strain S3. This strain has been shown to be nonmotile, to be a poor invader in vitro, and to be a poor colonizer of poultry after minimal in vitro passage.

Analysis of the pan genome of Campylobacter jejuni isolates recovered from poultry by pulsed-field gel electrophoresis, multilocus sequence typing (MLST), and repetitive sequence polymerase chain reaction (rep-PCR) reveals different discriminatory capabilities.

Campylobacter jejuni is one of the leading bacterial causes of food-borne illness in the USA. Molecular typing methods are often used in food safety for identifying sources of infection and pathways of transmission. Moreover, the identification of genetically related isolates (i.e., clades) may facilitate the development of intervention strategies for control and prevention of food-borne diseases. We analyzed the pan genome (i.e., core and variable genes) of 63 C. jejuni isolates recovered from chickens raised in conventional, organic, and free-range poultry flocks to gain insight into the genetic diversity of C. jejuni isolates recovered from different environments. We assessed the discriminatory power of three genotyping methods [i.e., pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and repetitive extragenic palindromic polymerase chain reaction (rep-PCR)]. The rep-PCR fingerprint was generated by determining the presence of repetitive sequences that are interspersed throughout the genome via repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and BOX element PCR (BOX-PCR) and combining the data to form a composite fingerprint. The genetic fingerprints were subjected to computer-assisted pattern analysis. Comparison of the three genotypic methods revealed that repREB-PCR showed greater discriminatory power than PFGE and MLST. ERIC-PCR and BOX-PCR yielded the highest number of PCR products and greatest reproducibility. Regardless of the genotyping method, C. jejuni isolates recovered from chickens reared in conventional, organic, and free-range environments all exhibit a high level of genotypic diversity.

Comparison of in vitro virulence factors of Campylobacter jejuni to in vivo lesion production.

Campylobacter jejuni is recognized as one of the most common agents of food-borne bacterial gastroenteritis in humans. Previous work has shown that C. jejuni isolates vary in their ability to invade and survive in laboratory grown cells. The correlation of these assays to actual lesion development in the hosts has not been determined. Therefore, this study aims to define the relationship between in vitro and in vivo assays for determining the virulence of C. jejuni isolates. Fifty-nine C. jejuni poultry isolates were analyzed in invasion and macrophage assays, and five isolates showing different invasion and survival abilities were examined for pathogenicity in the piglet model. All five isolates examined in the piglet model induced diarrhea without the presence of blood. Four of the five isolates produced microscopic lesions in piglets consisting of mucosal congestion, villous degeneration, and epithelial cell erosion. These studies imply that invasion or macrophage survival had little effect on the production of lesions typical of those noted in patients with campylobacteriosis. The most surprising finding was that isolates that produced a fluid exudate in piglets failed to invade epithelial cells. It is not known if the production of this fluid exudate is related to the production of a toxin(s) by C. jejuni. More work on the identification of the gene expressing this virulence factor is needed to confirm that this is indeed a putative toxin produced by C. jejuni.

Comparative metagenomics reveals host specific metavirulomes and horizontal gene transfer elements in the chicken cecum microbiome.

BACKGROUND: The complex microbiome of the ceca of chickens plays an important role in nutrient utilization, growth and well-being of these animals. Since we have a very limited understanding of the capabilities of most species present in the cecum, we investigated the role of the microbiome by comparative analyses of both the microbial community structure and functional gene content using random sample pyrosequencing. The overall goal of this study was to characterize the chicken cecal microbiome using a pathogen-free chicken and one that had been challenged with Campylobacter jejuni. METHODOLOGY/PRINCIPAL FINDINGS: Comparative metagenomic pyrosequencing was used to generate 55,364,266 bases of random sampled pyrosequence data from two chicken cecal samples. SSU rDNA gene tags and environmental gene tags (EGTs) were identified using SEED subsystems-based annotations. The distribution of phylotypes and EGTs detected within each cecal sample were primarily from the Firmicutes, Bacteroidetes and Proteobacteria, consistent with previous SSU rDNA libraries of the chicken cecum. Carbohydrate metabolism and virulence genes are major components of the EGT content of both of these microbiomes. A comparison of the twelve major pathways in the SEED Virulence Subsystem (metavirulome) represented in the chicken cecum, mouse cecum and human fecal microbiomes showed that the metavirulomes differed between these microbiomes and the metavirulomes clustered by host environment. The chicken cecum microbiomes had the broadest range of EGTs within the SEED Conjugative Transposon Subsystem, however the mouse cecum microbiomes showed a greater abundance of EGTs in this subsystem. Gene assemblies (32 contigs) from one microbiome sample were predominately from the Bacteroidetes, and seven of these showed sequence similarity to transposases, whereas the remaining sequences were most similar to those from catabolic gene families. CONCLUSION/SIGNIFICANCE: This analysis has demonstrated that mobile DNA elements are a major functional component of cecal microbiomes, thus contributing to horizontal gene transfer and functional microbiome evolution. Moreover, the metavirulomes of these microbiomes appear to associate by host environment. These data have implications for defining core and variable microbiome content in a host species. Furthermore, this suggests that the evolution of host specific metavirulomes is a contributing factor in disease resistance to zoonotic pathogens.

Characterization of genetically matched isolates of Campylobacter jejuni reveals that mutations in genes involved in flagellar biosynthesis alter the organism's virulence potential.

Phenotypic and genotypic evidence suggests that not all Campylobacter jejuni isolates are pathogenic for humans. We hypothesized that differences in gene content or gene expression alter the degree of pathogenicity of C. jejuni isolates. A C. jejuni isolate (Turkey) recovered from a turkey and a second C. jejuni isolate (CS) recovered from a chicken differed in their degrees of in vitro and in vivo virulence. The C. jejuni Turkey isolate invaded INT 407 human epithelial cells and secreted the Cia (Campylobacter invasion antigen) proteins, while the C. jejuni CS isolate was noninvasive for human epithelial cells and did not secrete the Cia proteins. Newborn piglets inoculated with the C. jejuni Turkey isolate developed more severe clinical signs of campylobacteriosis than piglets inoculated with the C. jejuni CS isolate. Additional work revealed that flagellin was not expressed in the C. jejuni CS isolate. Microarray and real-time reverse transcription-PCR analyses revealed that all flagellar class II genes were significantly downregulated in the C. jejuni CS isolate compared to the C. jejuni Turkey isolate. Finally, nucleotide sequencing of the flgR gene revealed the presence of a single residue that was different in the FlgR proteins of the C. jejuni Turkey and CS isolates. Complementation of the C. jejuni CS isolate with a wild-type copy of the flgR gene restored the isolate's motility. Collectively, these findings support the hypothesis that critical differences in gene content or gene expression can alter the pathogenic potential of C. jejuni isolates.

Characterization of Campylobacter jejuni biofilms under defined growth conditions.

Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.

Genotypes and antibiotic resistance of salmonella newport isolates from U.S. market oysters.

The consumption of raw oysters contributes to food-borne diseases such as salmonellosis. Prevalence studies in our laboratory have shown that Salmonella spp. were present in 7.4% of U.S. market oysters, with the majority (78/101) of isolates being of the Salmonella enterica Newport serovar. E-tests were performed to assess the susceptibility of the S. Newport oyster isolates to antibiotics used for treatment of Salmonella infections. The oyster isolates were susceptible to Gentamicin, Trimethoprim/Sulphamethoxazole, and Ciprofloxacin, while for the most part they were resistant to Ampicillin and Tetracycline. Consistent with the uniform antibiotic susceptibility profile of these isolates, only three pulsed-field gel electrophoresis (PFGE) profiles were identified among the isolates. Of these three profiles, one was present in 63 of the 78 isolates, indicating not only the presence of S. Newport in oysters, but the predominance of a single PFGE type.

Prevalence of Salmonella spp. in oysters in the United States.

Food-borne diseases such as salmonellosis can be attributed, in part, to the consumption of raw oysters. To determine the prevalence of Salmonella spp. in oysters, oysters harvested from 36 U.S. bays (12 each from the West, East, and Gulf coasts in the summer of 2002, and 12 bays, four per coast, in the winter of 2002-2003) were tested. Salmonella was isolated from oysters from each coast of the United States, and 7.4% of all oysters tested contained Salmonella. Isolation tended to be bay specific, with some bays having a high prevalence of Salmonella, while other bays had none. Differences in the percentage of oysters from which Salmonella was isolated were observed between the summer and winter months, with winter numbers much lower probably due to a variety of weather-related events. The vast majority (78/101) of Salmonella isolates from oysters were Salmonella enterica serovar Newport, a major human pathogen, confirming the human health hazard of raw oyster consumption. Contrary to previous findings, no relationship was found between the isolation of fecal coliforms and Salmonella from oysters, indicating a necessity for specific monitoring for Salmonella and other pathogens rather than the current reliance on fecal coliform testing.

Potential virulence and antimicrobial susceptibility of Campylobacter jejuni isolates from food and companion animals.

Infection in humans with Campylobacter jejuni is commonly associated with exposure to food animal fecal material. In this study, we report on the recovery, potential for virulence and antimicrobial resistance levels of C. jejuni isolated from food and companion animals. Three hundred and seventy-eight fecal samples from food and companion animals and surface swabs from beef carcasses were tested for the presence of C. jejuni. C. jejuni was isolated from 13.8% (11/80) of dogs, 5% (1/20) of goats, 28.3% (17/60) of dairy cattle, 0% (0/65) of range cattle, 73.5% (36/49) of feedlot cattle, and 94.7% (18/19) of beef carcasses. Beef cattle from a single Arizona herd showed a considerable increase in fecal shedding of C. jejuni from pasture to feedlot and over time on the feedlot. Forty-two isolates were tested for susceptibility to four antimicrobial agents, each representing a class of antimicrobial drug approved for use in both humans and animals. None of the isolates were found to be resistant to erythromycin or gentamicin, whereas 2.4% of isolates were resistant to ciprofloxacin and 28.6% of isolates were resistant to tetracycline. The presence of virulence traits among the 42 isolates was assessed using in vitro macrophage survival and epithelial cell adherence and invasion assays. Of the isolates examined, 17 were able to survive within macrophages through 72 h at viable counts of >/=10(3)/well and 12 were capable of invading epithelial cells at viable counts of >/=10(3)/well. Data from these studies suggests that many of the isolates recovered from the non-poultry animal sources have the capacity to cause disease if transmitted to humans.

FeoB is not required for ferrous iron uptake in Campylobacter jejuni.

Among strains of Campylobacter jejuni, levels of ferrous iron (Fe2+) uptake was comparable. However, C. jejuni showed a lower level of ferrous iron uptake than Escherichia coli. Consistent with studies of E. coli, Fe2+ uptake in C. jejuni was significantly enhanced by low Mg2+ concentration. The C. jejuni genome sequence contains a single known ferrous iron uptake gene, feoB, whose product shares 50% amino acid identity to Helicobacter pylori FeoB and 29% identity to E. coli FeoB. However, Fe2+ uptake could not be attributed to FeoB for several reasons. Site-directed mutations in feoB caused no defect in 55Fe2+ uptake. Among C. jejuni strains, various nucleotide alterations were found in feoB, indicating that some C. jejuni feoB genes are defective. In addition, uptake could not be attributed to the magnesium transporter CorA, since no reduction in 55Fe2+ uptake was observed in the presence of a CorA-specific inhibitor.