Multilocus Sequence Typing Methods for the Emerging Campylobacter Species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus

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.

Comprehensive Detection and Discrimination of Campylobacter Species by Use of Confocal Micro-Raman Spectroscopy and Multilocus Sequence Typing

“…MLST was performed as previously described under the conditions and with the primer sets of Miller et al (52,53). MLST amplifications were performed on a Tetrad thermocycler (Bio-Rad, Hercules, CA).…”

The Family Campylobacteraceae

“…For each housekeeping gene, the different sequences present within a bacterial species are assigned as distinct alleles, and for each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST) (Dingle et al 2001). This technique has been used successfully to determine the sequence types of C. coli in pig liver (von Altrock et al 2013); C. jejuni in human, poultry, and bovine isolates (de Haan et al 2013); and C. hyointestinalis and other emerging campylobacters (Miller et al 2012). The MLST genotyping approach is less susceptible to factors causing genomic instability than earlier genomic methods using restriction site polymorphisms or single genetic markers (Wassenaar et al 2000).…”

Characterisation of Campylobacter concisus strains from South Africa using Amplified Fragment Length Polymorphism (AFLP) profiling and a Genomospecies-specific Polymerase Chain Reaction (PCR) assay: Identification of novel genomospecies and correlation with clinical data

“…Of the Campylobacter species for which DNA-based subtyping has been undertaken on appreciable numbers of strains, C. concisus appears to be the most polymorphic. Interestingly, despite the apparent lack of clonality as inferred in AFLP (Aabenhus et al, 2005;Kalischuk and Inglis, 2011;this study) or MLST analysis (Miller et al, 2012), the latter has not yet identified substantive recombination events between strains. Additional work is needed to resolve the population genetics of this intriguing species.…”

“…As in previous studies (Aabenhus et al, 2005;Kalischuk and Inglis, 2011), two major groups were revealed, that corresponded to GS1 (that includes CCUG 13144, the type strain of oral origin) and GS2 (that includes reference strains CCUG 19995 and RH 13826 from human gastroenteritis). No appreciable similarity between South African isolates was obtained with reference profiles of GS3 (RH 7656) and GS4 (RH 13570; which was indicated by MLST to be C. curvus : Miller et al (2012). On the other hand, two profiles were observed, that were highly distinctive and occupied well separated positions in the dendrogram, and potentially representing new C. concisus genomo-species (GS 5 and 6 respectively).…”

“…We found an excellent correlation with C. concisus genomospecies identification between these two approaches, although the novel genome-species (GS5 and GS6) we describe would be identified as GS2 according to the PCR assay (Table 1). However, MLST analysis of the GS5 strain places it in a cluster dominated by known GS2 strains (Miller et al, 2012). This may indicate that the PCR assay correlates more accurately with more distant strain phylogenetic relationships than recent evolutionary events detected by those more finely resolved by AFLP.…”