Essential Oil Microemulsions Inactivate Antibiotic-Resistant Bacteria on Iceberg Lettuce during 28-Day Storage at 4 °C.

The objective of this study was to investigate the antimicrobial activities of essential oil-based microemulsions in the wash water against Escherichia coli O157:H7 and Pseudomonas fluorescens on Iceberg lettuce. Evaluated wash microemulsions included oregano oil, lemongrass oil, and cinnamon oil, along with a plant-based emulsifier for improved solubility. Iceberg lettuce was inoculated for 2 min with E. coli O157:H7 (6.0 log CFU/g) or P. fluorescens (6.0 log CFU/g) and then dip-treated in a phosphate buffered saline (PBS) control, 50 ppm chlorine, 3% hydrogen peroxide treatment or a 0.1%, 0.3%, or 0.5% microemulsion solution. Treated leaves were stored at 4 °C, and analyzed for surviving bacteria on days 0, 3, 7, 10, 14, 21, and 28. Efficacies of the antimicrobials were concentration and storage-time dependent. There was a 1.26−4.86 log CFU/g reduction in E. coli O157:H7 and significant reductions (0.32−2.35 log CFU/g) in P. fluorescens during storage at days 0−28 (p < 0.05). The 0.1% oregano oil microemulsion resulted in the best visual appeal in Iceberg leaves inoculated with E. coli O157:H7 and showed better improvement in the quality of the Iceberg leaves inoculated with spoilage bacteria P. fluorescens. The results suggest that 0.5% cinnamon and 0.3% oregano oil treatments have the potential to provide natural, eco-friendly, and effective alternatives to chemicals for the decontamination of leafy greens, eliminating E. coli O157:H7 and P. fluorescens.

Enumeration of Salmonella and Campylobacter spp. in environmental farm samples and processing plant carcass rinses from commercial broiler chicken flocks.

A prospective cohort study was performed to evaluate the prevalences and loads of Salmonella and Campylobacter spp. in farm and processing plant samples collected from 55 commercial broiler chicken flocks. Environmental samples were collected from broiler houses within 48 h before slaughter, and carcass rinses were performed on birds from the same flocks at 4 different stages of processing. Salmonella was detected in farm samples of 50 (90.9%) flocks and in processing samples of 52 (94.5%) flocks. Campylobacter was detected in farm samples of 35 (63.6%) flocks and in processing samples of 48 (87.3%) flocks. There was a significant positive relationship between environmental farm samples and processing plant carcass rinses with respect to both Salmonella and Campylobacter prevalences and loads. Campylobacter loads were significantly higher than Salmonella loads, and the correlations between samples collected from the same flocks were higher for Campylobacter than they were for Salmonella. Boot socks were the most sensitive sample type for detection of Salmonella on the farm, whereas litter samples had the strongest association with Salmonella loads in pre- and postchill carcass rinses. Boot socks, drag swabs, and fecal samples all had similar sensitivities for detecting Campylobacter on the farm, and all were more strongly associated with Campylobacter loads in carcass rinses than were litter samples. Farm samples explained a greater proportion of the variability in carcass rinse prevalences and loads for Campylobacter than they did for Salmonella. Salmonella and Campylobacter prevalences and loads both decreased significantly as birds progressed through the processing plant.

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.

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.

Carvacrol and cinnamaldehyde inactivate antibiotic-resistant Salmonella enterica in buffer and on celery and oysters.

The emergence of antibiotic-resistant Salmonella is of concern to food processors. The objective of this research was to identify antimicrobial activities of cinnamaldehyde and carvacrol against antibiotic-resistant Salmonella enterica in phosphate-buffered saline (PBS) and on celery and oysters. Twenty-three isolates were screened for resistance to seven antibiotics. Two resistant and two susceptible strains were chosen for the study. S. enterica cultures (10(5) CFU/ml) were added to different concentrations of cinnamaldehyde and carvacrol (0.1, 0.2, 0.3, and 0.4% [vol/vol]) in PBS, mixed, and incubated at 37 degrees C. Samples were taken at 0, 1, 5, and 24 h for enumeration. Celery and oysters were inoculated with S. enterica (10(6-7) CFU/ml), treated with 1% cinnamaldehyde or 1% carvacrol, incubated at 4 degrees C, and then sampled for enumeration on days 0 and 3. Both antimicrobials induced complete inactivation of S. enterica in PBS at 0.3 and 0.4% on exposure, and on 0.2% in 1 h. Exposure to cinnamaldehyde at 0.1% inactivated all pathogens at 1 h, and survivors were observed only for Salmonella Newport with 0.1% carvacrol at 1 h. In celery, 1% carvacrol reduced S. enterica populations to below detection on day 0, while 1% cinnamaldehyde reduced populations by 1 and 2.3 log on day 0 and day 3, respectively. In oysters, both antimicrobials caused about 5-log reductions on day 3. These results show the potential antimicrobial effects of carvacrol and cinnamaldehyde against antibiotic-resistant S. enterica in vitro and in foods.

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.

Plant-derived compounds inactivate antibiotic-resistant Campylobacter jejuni strains.

Sixty-three Campylobacter jejuni isolates were screened for their resistance to the antibiotics ampicillin, cefaclor, ciprofloxacin, erythromycin, gentamycin, tetracycline, and trimethoprim-sulfamethoxazole. Based on this screen, the resistant strains D28a and H2a and the nonresistant strain A24a were selected for evaluation of their resistance and susceptibility to inactivation by cinnamaldehyde and carvacrol, the main constituents of plant-derived cinnamon and oregano oils, respectively. Different concentrations (0.05, 0.1, and 0.2% [vol/vol] in sterile phosphate-buffered saline) of cinnamaldehyde and carvacrol were added to C. jejuni cultures with initial populations of 10(4) CFU/ml. The samples were then mixed thoroughly and incubated at 37 degrees C. Viable bacterial populations were enumerated at incubation periods of 0, 30, 60, and 120 min. The results indicate that the extent of inhibition of microbial survival was related to both the nature and concentration of antimicrobials and the incubation time. Both cinnamaldehyde and carvacrol exhibited rapid antimicrobial activity against both antibiotic-resistant and non-resistant C. jejuni strains, at concentrations of approximately 0.1% and higher. The antimicrobial efficacy of cinnamaldehyde was greater than that of carvacrol. The possible significance of the results for microbiological food safety is discussed.