Dissemination of fluoroquinolone-resistant Campylobacter spp. within an integrated commercial poultry production system

Abstract

While characterizing the intestinal bacterial community of broiler chickens, we detected epsilon-proteobacterial DNA in the ilea of 3-day-old commercial broiler chicks (J. Lu, U. Idris, B. Harmon, C. Hofacre, J. J. Maurer, and M. D. Lee, Appl. Environ. Microbiol. 69:6816-6824, 2003). The sequences exhibited high levels of similarity to Campylobacter jejuni and Campylobacter coli sequences, suggesting that chickens can carry Campylobacter at a very young age. Campylobacter sp. was detected by PCR in all samples collected from the ilea of chicks that were 3 to 49 days old; however, it was detected only in the cecal contents of chickens that were at least 21 days old. In order to determine whether the presence of Campylobacter DNA in young chicks was due to ingestion of the bacteria in food or water, we obtained commercial broiler hatching eggs, which were incubated in a research facility until the chicks hatched. DNA sequencing of the amplicons resulting from Campylobacter-specific 16S PCR performed with the ileal, cecal, and yolk contents of the day-of-hatching chicks revealed that Campylobacter DNA was present before the chicks consumed food or water. The 16S rRNA sequences exhibited 99% similarity to C. jejuni and C. coli sequences and 95 to 98% similarity to sequences of other thermophilic Campylobacter species, such as C. lari and C. upsaliensis. The presence of C. coli DNA was detected by specific PCR in the samples from chicks obtained from a commercial hatchery; however, no Campylobacter was detected by culturing. In order to determine whether the same strains of bacteria were present in multiple levels of the integrator, we cultured Campylobacter sp. from a flock of broiler breeders and their 6-week-old progeny that resided on a commercial broiler farm. The broiler breeders had been given fluoroquinolone antibiotics, and we sought to determine whether the same fluoroquinolone-resistant strain was present in their progeny. The isolates were typed by pulsed-field gel electrophoresis, which confirmed that the parental and progeny flocks contained the same strain of fluoroquinolone-resistant C. coli. These data indicate that resistant C. coli can be present in multiple levels of an integrated poultry system and demonstrated that molecular techniques or more sensitive culture methods may be necessary to detect early colonization by Campylobacter in broiler chicks.

FIG. 1.

Strain typing of C. jejuni isolates by PFGE. (A) Isolates from two different commercial broiler farms. Lanes 1 to 3 contained isolates from farm CF-3, and lanes 4 to 6 contained isolates from farm CF-1. Genomic DNA was digested with SmaI. (B and C) SmaI (B) and KpnI (C) typing of C. coli isolated from a broiler breeder flock and the commercial broiler progeny of this flock. Lanes 1 and 2 contained C. coli isolated from breeder parental chickens, lanes 3 and 4 contained C. coli isolated from their 6-week-old progeny, lanes 5 and 6 contained C. jejuni isolated from the progeny broilers, and lane 7 contained S. cerevisiae molecular weight markers.

FIG. 2.

PCR detection of Campylobacter species present in the intestinal and yolk contents of chicks on the day of hatching. Ten embryonating eggs were obtained from a sarafloxacin-treated broiler breeder flock and hatched in a research hatching cabinet. In addition, 10 chicks from this flock were obtained from the commercial hatchery. On the day of hatching, the ileal, cecal, and yolk contents were aseptically removed, and the DNA was extracted. PCR targeting Campylobacter was performed with pooled samples, and the amplicons were separated on a 1.5% agarose gel. (A) 16S rRNA PCR results for chicks hatched in the research cabinet. Pooled yolk, ileum, and cecum samples contained Campylobacter DNA. Lane 1 contained DNA molecular weight markers, lane 2 contained C. jejuni ATCC 33560 as the positive control, lane 3 contained ileal contents, lane 4 contained cecal contents, lane 5 contained yolk contents, and lane 6 contained a control (no DNA template). (B and C) PCR results for chicks hatched in the research facility (B) and for chicks obtained from the commercial hatchery (C), obtained by using primers specific for the ceuE gene of C. coli. Pooled yolk, ileum, and cecum samples from chicks hatched in the research facility contained C. coli DNA, while only the pooled yolk samples from the commercial hatchery were positive. Lane 1, DNA molecular weight markers; lane 2, C. coli M1-19 (positive control); lane 3, ileal contents; lane 4, cecal contents; lane 5, yolk contents; lane 6, control containing no DNA template.

FIG. 3.

Phylogenetic tree showing the relatedness of chick intestinal 16S rRNA sequences to sequences of Campylobacter species. The 16S rRNA clone libraries were produced by amplification of DNA extracted from the ileum, cecum, and yolk contents of day-of-hatching chicks and from the ileal and cecal contents of chickens that were 3 to 49 days old (23). The tree was constructed by neighbor-joining analysis of a distance matrix obtained from a multiple-sequence alignment of clone library sequences and GenBank ɛ-proteobacterial 16S rRNA sequences. Bootstrap values (expressed as percentages of 100 replications) are indicated at branch points. Branches labeled with DOH represent sequences obtained from the clone library from day-of-hatching chicks; branches labeled ileum or cecum (but without DOH) represent sequences obtained from clone libraries of older chickens. Branches labeled with a specific organism's name and accession number represent sequences obtained from the GenBank database.

FIG. 4.

(Top panel) Integrated structure of the commercial poultry production system. Production companies acquire breeder flocks from the pedigree flocks owned by three to five companies worldwide. These breeder flocks produce hatching eggs that become meat birds and layers for human consumption. (Lower panel) Potential sources of antibiotic-resistant bacteria for poultry. While antibiotic use may select for resistant populations (left box), young chicks lacking a stable intestinal bacterial community may be more susceptible to colonization with resistant strains present in the hatchery or on the farm (middle box). Exposure of hatchlings to resistant bacteria present on the surface of the egg can potentially result in dissemination of resistant strains from the breeder flock to progeny (right box).