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United States Department of Agriculture

Agricultural Research Service


Location: Produce Safety and Microbiology Research

2010 Annual Report

1a.Objectives (from AD-416)
Objective 1: Genome sequencing, annotation, and gene-indexing, of Campylobacter species, Salmonella Enteritidis (SE) and pathogenic E. coli to identify targets for rapid detection and differentiation, and fitness and virulence factors. Objective 2: Develop DNA microarrays, and sequence-based typing methods to detect and analyze multiple critical food-borne pathogens; validate assays with food samples. Objective 3: Develop new and/or improved multi-locus sequence typing (MLST) and multi-locus variable tandem repreat analysis (MLVA) methods for human pathogens with emphasis on enterohemorrhagic E. coli. Combine MLST, MLVA and microarray analysis to identify markers associated with pathogen source and fitness, and relate to epidemiology and culture method bias. Objective 4: Develop specific capture and mass spectrometry (MS) methods to detect and fingerprint foodborne pathogens and threat agents. Objective 5: Evaluate methods for inactivating protein toxins. Problem to be Addressed: Through the use of genomics and proteomics develop multiplex assays to detect, identify and differentiate foodborne pathogens on fresh produce (leafy vegetables) to derive fundamental data to increase the safety and security of this commodity. FY07 Objectives of Research: Genome sequencing, annotation, and gene-indexing, of pathogenic E. coli to identify targets for rapid detection and differentiation, and fitness and virulence factors, with special emphasis on E. coli in the environment of produce production. Use fundamental genomic and proteomic information produced to develop microarray or other multiplex immunoreagent methods to identify and analyze genera, species and strains of critical food-borne pathogens. Identify single nucleotide polymorphism hot-spots in "clonal" pathogens for high resolution fingerprinting. Characterize E. coli O157:H7 strains associated with outbreaks and to identify potential virulence factors and other factors that may enhance fitness in produce production environments (plants, animal hosts, environment).

1b.Approach (from AD-416)
Our objectives address fundamental research to develop high-resolution genotyping methods for characterizing and tracking multiple pathogens related to food. Multiple approaches to methods development are described as contingencies to ensure success. The recent sequence data we have collaborated in producing for Campylobacter and Arcobacter species and collaborations on S. enterica, Ec O157:H7 and Lm genotyping will be invaluable for this work. Recent PSMRU involvement in two outbreak investigations of pre-harvest produce and tree-nuts contaminated with Ec O157:H7 (letter, J. Farrar) and S. Enteritidis (letter, J. Adams), respectively, has confirmed the need for improved methods for tracking pathogens in complex environments, determining their relatedness, and the relevance of these studies also to addressing potential intentional contamination events. The objectives we describe have been organized with the following strategy in mind: (i) Emphasize Campylobacter species, especially emerging species, because they remain underappreciated as food pathogens and causes of serious illness. Recent progress in sequencing and MS analysis facilitate comparative genomics and proteomics, and the expertise gained will be beneficial for development of similar approaches for other pathogens; (ii) Develop microarrays specific for genotyping, to learn as much as possible about virulence factors and fitness characteristics that might be beneficial to interventions during production or processing; (iii) Expand, as appropriate, the microarray approach to more comprehensive DNA microarrays for detection of many pathogens simultaneously; (iv) Develop methods useful for addressing objectives of PSMRU CRIS-040 (“Biology and Control of Human Pathogens on Fresh produce”) to leverage methods and discoveries and increase productivity; (v) Collaborate with other groups who have access to productions systems and/or strains for assessing the robustness of genotyping or protein typing; (vi) Use the novel methods developed to address whether culture bias is affecting the ability to obtain meaningful data about reservoirs, food sources and epidemiology. Replacing 5325-42000-041-00D (4/06).

3.Progress Report
A: Campylobacter genomics. Using next-generation sequencing, the genomes of 18 Campylobacter strains were sequenced to draft level. These supplement the 12 Campylobacter genomes completed already in collaboration with JCVI (formerly TIGR), providing genomic sequence data for all 30 accepted taxa within the genus Campylobacter. Using these genomic data, novel typing methods were developed for emerging Campylobacter species (e.g. C. hyointestinalis and C. lanienae) and used to type campylobacters isolated as part of an environmental survey. B: Survey of central California coast produce production region for Campylobacter. More than 2000 samples of livestock and wildlife feces and water were tested for Campylobacter. C. jejuni, C. coli C. hyointesinalis, C. lanienae, C. upsaliensis, C. fetus and other species were isolated from a variety of samples, with some species isolated frequently from particular species of wildlife. Selected strains representing specific species are being processed for typing for source tracking reservoirs and transport. C: Rapid identification of Shiga toxin 2 (Stx2) from E. coli O157:H7 by tandem mass spectrometry and top-down proteomics. A simple and elegant preparation was developed for rapid identification of the alpha- and beta-subunits of Stx2 from E. coli O157:H7 by top-down proteomics analysis using in-house developed software. This provides a new approach for analyzing Stx. D: Discriminating Salmonella serovars by on-target digestion and MALDI-MS. Four serovars of Salmonella enterica were analyzed by mass spectrometry analysis of on-target digestion of cell lysates; differences were identified in some peptide ions corresponding to serovar. Collaboration with MassTech/Science & Engineering Services, Inc. (Columbia, MD) tested their atmospheric pressure (AP) MALDI technology for analysis of bacterial cell lysates of foodborne pathogens. Preliminary analysis of Salmonella enterica serovars (Enteritidis, Kentucky, Newport, Thompson) identified some potential serovar-specific peptide ions. E: Rapid genotyping of foodborne pathogens with DNA microarrays. Shiga toxin-producing O157 and non-O157 E. coli (STEC) strains were genotyped accurately in 2-4 hours by a DNA microarray with innovative colorimetric detection. E. coli strains recovered from a variety of sources in produce production regions were characterized, facilitating rapid identification of virulence genes. F: Relative toxicities of Shiga toxin 2 variants from Escherichia coli. The relative activity of Shiga toxin 2 variants from E. coli strains recovered from produce production regions in California were evaluated with a cell-based fluorescence assay. STEC strains expressing Stx2 variants were determined to have different toxicities in cultured mammalian cells. These findings demonstrated that the differential expression and induction of Stx2 variants contributes to the increased virulence of STEC strains.

1. Sequencing of Campylobacter upsaliensis genomes. Campylobacter upsaliensis can cause human bacterial gastroenteritis and is associated also with domestic dogs and cats. Previous characterization of C. upsaliensis strains by multilocus sequence typing identified two distinct genotypic clusters. ARS researchers in Albany, CA, completed the genome sequences of two human clinical strains of C. upsaliensis, representatives of each genotypic cluster. Comparative genomics of these two strains and subsequent typing indicated that each cluster is characterized by a unique outer surface structure, one containing phosphorylcholine and the other similar to surface structures in C. jejuni associated with Guillain-Barré syndrome. The presence of these structures in campylobacters other than C. jejuni suggests that other, emerging Campylobacter species may be associated with human food-borne illness.

2. Global gene expression of Campylobacter jejuni regulatory mutant. C. jeuni causes a significant amount of illness in the US and is prevalent in chickens. ARS Scientists in Albany, CA collaborated with Utrecht University to examine a regulatory system important for C. jejuni colonization of chickens. A variety of molecular methods were used to reveal a system controlling genes encoding a pump that transports certain antibiotics, and additional genes encoding periplasmic and membrane proteins. The system enhances early in vivo growth of C. jejuni in 7-day old chicken. This mechanism could provide novel methods for controlling colonization of chickens and minimize contamination of the food supply.

3. Mutants and phenotypic variants of Campylobacter jejuni. C. jejuni is considered naturally competent for DNA uptake, a mechanism important in acquiring new traits for increased fitness. However, not all strains are equally competent. ARS scientists in Albany, CA collaborated with Central Vet. Inst. of Wageningen in monitoring gene content differences resulting in different phenotypes. We compared all of the genes among different strains by microarray profiling to show that C. jejuni incapable of transformation possess genes encoding DNases. This work has revealed a factor that may enhance the fitness and persistence of strains in certain environments, including animals and food.

4. Comparative genomics of outbreak strains of Salmonella Enteritidis linked to raw almonds. S. Enteritidis is a major cause of food-borne illness and is associated mostly with eggs and poultry. From 2001 to 2006, S. enteritidis outbreak isolates with rare bacteriophage types (PT), PT30 and PT9c, were associated with almonds. ARS researchers in Albany, CA, differentiated S. enteritidis strains by genotyping methods and confirmed that that the majority of the differences corresponded to bacteriophage-related genes. Although we were unable to discriminate S. enteritidis PT30 isolates related to outbreaks from unrelated clinical isolates, we could demonstrate distinct metabolic fingerprints for S. enteritidis PT30 clinical isolates as compared to PT30 orchard isolates. These data indicate that S. enteritidis PT9c and 30 isolates are highly related genetically, and that PT30 orchard isolates differ from clinical PT30 strains metabolically, possibly as a result of fitness adaptations maintained after isolation.

5. Shiga toxin 2 (Stx2) identification by proteomics. Stx2 is an important virulence factor of Shiga toxin positive E. coli (STEC). ARS researchers in Albany, CA, developed a top-down proteomics method to identify with in-house software the a- and b-subunits of Stx2 from bacterial cell lysates of E. coli O157:H7. This approach provides a rapid method to identify pathogenic E. coli toxins in food.

6. Mass spectrometry (MS) analysis of bacterial proteins. The ability to identify bacterial proteins and toxins rapidly by MS requires recognition of chemical changes in the predicted molecular ions. ARS scientists in Albany, CA discovered that certain chemical compounds used in MALDI MS (such as sinapinic, caffeic, ferullic but not a-cyano-cinnamic acid) react with disulfide bond-containing proteins at low pH. This has implications for pattern recognition analysis of MS data facilitating identification of bacteria. This has been exploited for identification by MS of protein biomarkers and protein toxins, such as the b-subunit of Stx2, which has a single disulfide bond. This provides a rapid and simple method for identifying important bacterial proteins and toxins.

7. Rapid, simple and inexpensive assays for pathogen detection and genotyping. Improved methods for DNA microarray-based pathogen detection are needed with sufficient sensitivity, cost-effectiveness, and suitability for routine testing of ARS researchers in Albany, CA. In collaboration with InDevR, Inc. (Boulder, CO), a novel, colorimetric detection method (ampliPHOX™), based on light-initiated signal amplification through polymerization, was evaluated for rapidly genotyping E. coli isolates recovered from produce production regions in California. This method is a reasonable approach for regulatory lab surveys of food samples for pathogens. FSIS has expressed interest in evaluating the method with chips designed by a researcher in PSMRU. This method has been adapted for detection of other pathogens, including Salmonella enterica, Campylobacter spp., and Vibrio spp.

8. Cell-based assay for measuring toxicities of Stx2 variants from E. coli (STEC). Stx are important virulence factors linked to severe human illness. Current methods that examine Stx activity can be time consuming and often produce poor quantitative data. A sensitive, quantitative, and high-throughput cell-based assay was developed by ARS researchers in Albany, CA, to monitor the Stx2-induced inhibition of protein synthesis. This assay was used to quantify the relative toxicities of Stx2 proteins expressed by different strains. Our results indicate that STEC strains harboring certain Stx2 subtypes have an increased virulence potential that may lead to severe disease outcomes. This addresses a critical need in evaluating whether all STEC should be considered adulterants of food.

9. Apple juice inactivates Shiga toxin 2. Shiga toxins are produced by E. coli O157:H7. A family of related toxins with two major groups, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) are produced. Stx2 is reportedly much more toxic than Stx1. In the present study, ARS researchers in Albany, CA, discovered that freshly prepared juice from locally purchased Red Delicious apples, but not juice from Golden Delicious apples, inactivated the biological activity of the toxin. However, both Golden Delicious juice and water with added 0.3% polyphenol-rich grape pomace, a byproduct of wine production, also inactivated the Shiga toxin. A related study showed that apple juice also inhibited the biological activity of the Staphylococcus enterotoxin A (SEA). These collaborative study results suggest that food-compatible and safe anti-toxin compounds can be used to inactivate Shiga toxins in apple juice and possibly also in other liquid and solid foods.

10. Edible apple antimicrobial films inactivate antibiotic-resistant Campylobacter jejuni strains on poultry. C. jejuni on contaminated poultry is a leading cause of bacterial diarrheal illness worldwide. Many Campylobacter strains are now becoming multi-drug resistant. To help overcome this problem, ARS researchers in Albany, CA, evaluated the effectiveness of apple-based edible films containing carvacrol and cinnamaldehyde against both antibiotic-resistant and susceptible C. jejuni strains on chicken. The films with cinnamaldehyde were more effective than carvacrol-containing films. Reductions of pathogens at 23 °C were greater than at 4 °C. The results of this suggest that edible antimicrobial apple films have the practical potential to reduce both resistant and susceptible C. jejuni populations on chicken and risk of campylobacteriosis.

Review Publications
Healy, B., Huynh, S., Mullane, N., O'Brian, S., Iverson, C., Stephen, R., Parker, C., Fanning, S. 2009. Microarray-based Comparative Genomic Indexing of the Cronobacter genus (Enterobacter sakazakii). International Journal of Food Microbiology. 136:159-64

Fagerquist, C.K., Garbus, B.R., Miller, W.G., Williams, K.E., Yee, E., Bates, A.H., Boyle, S., Harden, L.A., Cooley, M.B., Mandrell, R.E. 2010. Rapid Identification of Protein Biomarkers of E. coli O157:H7 by MALDI-TOF-TOF Mass Spectrometry and Top-Down Proteomics. Analytical Chemistry. 82:2717-2725.

Fagerquist, C.K., Garbus, B., Williams, K., Bates, A.H., Harden, L.A. 2010. Covalent attachment and dissociative loss of sinapinic acid to/from cysteine-containing proteins from bacterial cell lysates analyzed by MALDI-TOF-TOF mass spectrometry. Journal of American Society for Mass Spectrometry. 21,819-832.

Gaasbeek, E.J., Wagenaar, J.A., Guilhabert, M., Van Putten, J.P., Parker, C., Vanderwal, F.J. 2010. Nucleases Encoded by Integrated Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter jejuni. Journal of Bacteriology. 192:936-941.

Haff, R.P., Quinones, B., Swimley, M.S., Toyofuku, N. 2010. Automatic image analysis and spot classification for detection of pathogenic Escherichia coli on glass slide DNA microarrays. Computers and Electronics in Agriculture. 71(2):163-169.

Miller, W.G., Wesley, I.V., On, S., Houf, K., Megraud, F., Wang, G., Yee, E., Srijan, A., Mason, C. 2009. First multilocus sequence typing scheme for Arcobacter spp.. BMC Microbiology. doi:10.1186/1471-2180-9-196

Parker, C., Huynh, S., Quinones, B., Harris, L., Mandrell,2010 R.E. Comparative genotypes of Salmonella enterica serovar Enteritidis Phage type 30 and 9c strains isolated from three outbreaks associated with raw almonds. Applied and Environmental Microbiology. 76: 3723-3731

Wilson, M.K., Lane, A.B., Law, B.F., Miller, W.G., Joens, L.A., Konkel, M.E., White, B.A. 2009. Analysis of the Pan Genome of Campylobacter jejuni Isolates Recovered from Poultry by Pulse-Field Gel Electrophoreis, MLST and rep-PCR Reveals Different Discriminatory Capabilities. Microbial Ecology. DOI 10, 1007/s00248-009-9571-3

Wesley, I.V., Miller, W.G. 2010. Arcobacter: An Opportunistic Human Foodborne Pathogen? In: Scheld, W.M., Grayson, M.L., Hughes, J.M., editors. Emerging Infections 9. Washington, DC: American Society of Microbiology Press. p. 185-211.

Wosten, M.M., Van Dijk, L., Parker, C., Guilhabert, M.R., Van Der Meer-Jannsen, Y.P., Wagenaar, J., Van Putten, J.P. 2010. Growth phase-dependent activation of the DccRS regulon of Campylobacter jejuni. Journal of Bacteriology. 192:2729-2736

Du, W., Olsen, C.W., Avena-Bustillos, R.D., Mc Hugh, T.H., Levin, C.E., Mandrell, R.E., Friedman, M. 2009. Antibacterial Effects of Allspice, Garlic, and Oregano Essential Oils in Tomato Films Determined by Overlay and Vapor-Phase Methods. Journal of Food Science. 74(7):M390-M397.

Munyaneza, J.E., Sengoda, V.G., Crosslin, J., De La Rosa-Lozano, G., Sanchez, A. 2009. First Report of Candidatus Liberibacter psyllaurous in Potato Tubers with Zebra Chip Disease in Mexico. Plant Disease. 93:552.

Last Modified: 4/17/2014
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