MOLECULAR BIOLOGY AND GENOMICS OF FOODBORNE PATHOGENS
Location: Produce Safety and Microbiology Research
Title: Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 27, 2007
Publication Date: May 10, 2007
Citation: Quinones, B., Parker, C., Janda Jr, J.M., Miller, W.G., Mandrell, R.E. 2007. Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays. Applied and Environmental Microbiology. 73(11)3645-3655
Interpretive Summary: Bacterial contamination of food supplies during food harvesting, processing and preparation is an area of increasing concern in food safety. Infections caused by bacterial human pathogens continue to be a serious health issue, and the prevalence of foodborne illnesses is still substantial in the United States. The bacterial agent in the family Campylobacteraceae that is the most significant contributor to human gastrointestinal infections is Campylobacter; in laboratory-confirmed cases of infection in 2005, Campylobacter was second only to Salmonella. Although, the causative agent of these human infections is determined often only at the genus level, the Campylobacter spp. that are associated primarily with human gastrointestinal disease are Campylobacter jejuni and Campylobacter coli. These species are often referred to as thermotolerant Campylobacters given their optimal growth conditions at 42°C and are prominently associated also with poultry by preferentially colonizing the avian gastrointestinal tract. However, other important infection sources identified are pigs for C. coli and untreated water and raw milk for C. jejuni. Among cases of campylobacteriosis, C. jejuni is predominant, and in rare instances, these infections may be followed by autoimmune neuropathies such as Guillain Barré syndrome and Miller Fisher syndrome. It has been postulated that lipooligosaccharides (LOS) on the cell surface of C. jejuni exhibit molecular mimicry with gangliosides on peripheral nerves, resulting in the development of these autoimmune neuropathies.
Arcobacter, another genus in the family Campylobacteraceae, is of importance also to human health and food safety and is phenotypically related to Campylobacter. Although four Arcobacter species have been associated with animals and humans, Arcobacter butzleri is the species most frequently isolated from human infections. Similar to Campylobacter, Arcobacter has been isolated more frequently from poultry than from red meats, suggesting that poultry may be a major reservoir for this pathogen. However, Arcobacter species grow at temperatures much lower (15-37°C) than those required for Campylobacter growth and lower than the normal avian body temperature (~42°C) (29). These observations suggest that the presence of Arcobacter in poultry may be due to contamination from the skin of the birds or through processing and not due to contamination from the bird’s gastrointestinal contents. Given that contaminated food products are a potential source of Campylobacter and Arcobacter, a key issue in food safety is the development of efficient detection and identification methods for these pathogens as a first step in the control of human infections.
Traditionally, foodborne pathogens have been identified by microbiological culture followed by immunological methods, which can be laborious and time consuming. Methods for the isolation and identification of Campylobacters in foods require an enrichment culture for days followed by subculture on selective media for further phenotypic identification. Recently, nucleic acid amplification methods have been developed for the specific and rapid detection of these foodborne pathogens. For example, multiplex PCR assays have been applied to simultaneously identify and discriminate, in a single step reaction, C. coli and C. jejuni or Arcobacter spp. Although, these multiplex PCR assays are more time efficient than traditional culturing methods, they are limited by the number of genes that can be detected in a single reaction (3). In addition, certain contaminants that may be present in crude DNA preparations can inhibit the PCR reactions, resulting in non-specificity.
The advent of whole-genome based methods, as is DNA microarrays, offers another means to enhance the detection capabilities and to overcome the limitations of established procedur
To explore the use of DNA microarrays for pathogen detection in food, we have produced DNA oligonucleotide arrays to identify the presence of Arcobacter and Campylobacter in retail chicken. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and thermotolerant Campylobacter jejuni and Campylobacter coli. These microarrays showed a high level of probe specificity; signal intensities detected for A. butzleri, C. coli or C. jejuni probes were at least 10-fold higher than background levels. The specific identification of A. butzleri, C. coli and C. jejuni was achieved without including a PCR amplification step as the first step in the detection of these pathogens. By adapting an isolation method that employed membrane filtration and selective media, C. jejuni isolates were recovered from package liquid from whole chicken carcass prior to enrichment. Increasing the time of enrichment resulted in the isolation of A. butzleri and also allowed a higher recovery of C. jejuni. C. jejuni isolates were further classified by using an additional subset of probes targeting the lipooligosaccharide (LOS) biosynthesis locus. Our results demonstrated that most of these C. jejuni isolates likely possess LOS classes B, C or H. Validation experiments demonstrated that this DNA microarray had a detection sensitivity threshold of approximately 100 to 1000 C. jejuni cells. Interestingly, the use of C. jejuni-sequence specific primers to label genomic DNA increased the sensitivity of this DNA microarray for detecting C. jejuni in a complex sample. C. jejuni was efficiently detected directly in both package liquid from whole chicken carcasses and also in enrichment broths.