Abstract
Multilocus sequence typing (MLST) systems have been reported previously for multiple food- and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.
Highlights
Campylobacters are a major cause of human bacterial gastrointestinal illness in the industrialized world (Mølbak and Havelaar, 2008; Olson et al, 2008); campylobacterioses (12.68 cases per 100,000) were second only to Salmonella infections (16.2/100,000) in the United States in 2008 (Anonymous, 2009)
DESIGN OF THE NOVEL CAMPYLOBACTER Multilocus sequence typing (MLST) METHODS Construction of the novel Campylobacter MLST methods was facilitated by the availability of genome sequences for all of the taxa typed in this study
Primer design based on a sequence from a single strain might not lead to a successful MLST method if the sequence variation within that taxon prevents the design of primer pairs that efficiently amplify all strains
Summary
Campylobacters are a major cause of human bacterial gastrointestinal illness in the industrialized world (Mølbak and Havelaar, 2008; Olson et al, 2008); campylobacterioses (12.68 cases per 100,000) were second only to Salmonella infections (16.2/100,000) in the United States in 2008 (Anonymous, 2009). The majority of Campylobacter strains isolated from human clinical samples have been identified as C. jejuni subsp. Pathogenic campylobacters outside of the C. jejuni/C. coli group, termed here as emerging Campylobacter species, have been isolated more frequently from food and/or food animals. Recovery of these more fastidious, emerging Campylobacter species from food has not been reported often; isolation of such strains is likely limited by the culture conditions employed, conditions that favor Campylobacter species such as C. jejuni and C. coli. Lynch et al (2011) using novel culture conditions, reported the isolation of multiple emerging Campylobacter spp., e.g., C. concisus, C. curvus, and C. sputorum, from chicken, beef, and pork samples. Emerging campylobacters isolated from food animals are often strains of species associated typically with livestock, such as C. hyointestinalis in sheep, cattle, and swine (Hakkinen et al, 2007; Salihu et al, 2009; Oporto and Hurtado, 2011), C. lanienae in cattle and swine (Sasaki et al, 2003; Inglis et al, 2004; Oporto and Hurtado, 2011), and C. sputorum in cattle and sheep (Terzolo, 1988; On et al, 1998)
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