|Harden, Leslie - Les|
|Miller, William - Bill|
|Fagerquist, Clifton - Keith|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2005
Publication Date: 10/1/2005
Citation: Mandrell, R.E., Harden, L.A., Bates, A.H., Miller, W.G., Haddon, W.F., Fagerquist, C.K. 2005. Speciation of campylobacter coli, c. jejuni, c. helveticus, c. lari, c. sputorum, and c. upsaliensis by matrix-assisted laser desorption ionization-time of flight (maldi-tof) mass spectrometry. Applied and Environmental Microbiology. 71:10 p. 6292-6307 Interpretive Summary: Strains of 6 different species of Campylobacter have been analyzed by mass spectrometry (MS) to identify markers that are specific for each species. These species identifying ions are useful for rapid analysis of the many different strains that can be encountered in human, food, animal and environmental samples. The MS method is very rapid and relayively high-throuhgput so it is an advantage over other genetic techniques if MS is available. In addition, useful information regarding the molecules identifed, usually proteins, is obtained and can be exploited by searching databases and identifying the gene sequence encoding the protein and the protein sequence. This analysis identified certain types of cytosolic proteins as those most useful for identification of species and also some intra-species differences can be discerned. In this study, different combinations of media and growth conditions were tested to determine how robust were the data obtained for identifying Campylobacter species. All indications are that the markers identified are very stable under different conditions and thus are useful for identification. Mixed species of campylobacter were identified in some samples thought to be a single species and also strains obtained from different investigators were incorrectly speciated as determined by MS. This approach is being used currently for identifying similar types of ions in other enteric pathogens that cause foodborne illness.
Technical Abstract: Multiple strains of Campylobacter coli (Cc), C. jejuni (Cj), C. helveticus, C. lari (Cl), C. sputorum biovar faecalis (Cspf) and C. upsaliensis (Cu) isolated from clinical, animal, or food samples have been analyzed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar, transferred directly into solvent, then to a spot of dried ferulic acid (matrix). Multiple ions in the 5,000 to 15,000 dalton (Da) mass range were evident in spectra for each strain, including at least one or two ions of consistently high intensity in the 9,500-11,000 Da range. Analysis of ion spectra of multiple reference strains for each of the six species, including the genome strains Cj NCTC11168 and Cj RM1221, yielded identification of 'species-identifying' biomarker ions (SIBIs). The highest intensity Cj SIBIs were cytosolic proteins (e.g. GroES, RplL HU/HCj). Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within ±5 Da. Multiple intra-species SIBIs corresponding probably to non-synonomous single nucleotide polymorphisms (SNPs), also provided some intra-species strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs or cats revealed: (1) some associations of SIBI type with source, (2) strains speciated incorrectly, and (3) 'strains' composed of more than one species. MALDI-TOF MS provides an accurate, sensitive and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.