Characterization of Campylobacter jejuni biofilms under defined growth conditions

Abstract

Campylobacter jejuni is a major cause of human diarrheal disease in many industrialized countries and is a source of public health and economic burden. C. jejuni, present as normal flora in the intestinal tract of commercial broiler chickens and other livestock, is probably the main source of human infections. The presence of C. jejuni in biofilms found in animal production watering systems may play a role in the colonization of these animals. We have determined that C. jejuni can form biofilms on a variety of abiotic surfaces commonly used in watering systems, such as acrylonitrile butadiene styrene and polyvinyl chloride plastics. Furthermore, C. jejuni biofilm formation was inhibited by growth in nutrient-rich media or high osmolarity, and thermophilic and microaerophilic conditions enhanced biofilm formation. Thus, nutritional and environmental conditions affect the formation of C. jejuni biofilms. Both flagella and quorum sensing appear to be required for maximal biofilm formation, as C. jejuni flaAB and luxS mutants were significantly reduced in their ability to form biofilms compared to the wild-type strain.

FIG. 1.

Effects of growth medium, temperature, and oxygen tension on C. jejuni biofilm formation. (A) C. jejuni M129 biofilm formation in MHB and brucella and Bolton broths at 37°C and 10% CO₂. (B) C. jejuni M129 biofilm formation in MHB in 10% CO₂ or under aerobic conditions at either 37°C or 25°C. Biofilm formation was assessed by CV staining. Experiments were performed in triplicate on three separate occasions, and error bars represent one standard deviation from the mean.

FIG. 2.

Effect of NaCl (A) or sucrose or glucose (B) on the ability of C. jejuni M129 to form biofilms, as measured by CV staining. (B) White bars represent sucrose, and gray bars represent glucose. Experiments were performed three times in triplicate, and error bars represent one standard deviation from the mean.

FIG. 3.

Biofilm formation of C. jejuni M129 on coupons of polystyrene (PS), ABS, PVC, or copper was measured by a viable count following 24 h of incubation at 37°C in 10% CO₂. Experiments were performed three times in triplicate, and the graph depicts data from a representative experiment.

FIG. 4.

Cm inhibits biofilm formation of C. jejuni. C. jejuni strains M129 and F38011 were treated with 0.5 μg/ml Cm for 15 min or left untreated prior to a standard biofilm assay in medium without antibiotic. Experiments were performed three times in triplicate, and error bars represent one standard deviation from the mean.

FIG. 5.

Flagella and quorum sensing positively influence biofilm formation in C. jejuni. C. jejuni M129, M129::flaAB, and M129::luxS strains were subjected to a standard biofilm assay. (A) Biofilm formation at 24, 48, and 72 h postinoculation was measured by CV staining. White bars represent M129, gray bars represent the M129::flaAB mutant, and hatched bars represent the M129::luxS mutant. (B) Representative wells at 24, 48, and 72 h postinoculation showing CV staining prior to decolorization. (C) CSF from wild-type M129 rescued biofilm formation in the M129::luxS mutant. Wild-type M129 CSF was mixed 1:1 with MHB and was used as the growth medium in a standard biofilm assay. Experiments were performed three times in triplicate, and error bars represent one standard deviation from the mean.

FIG. 6.

CSFs from Pseudomonas spp. and A. pyogenes promote C. jejuni biofilm formation. Biofilm assays were performed with C. jejuni M129 in a growth medium consisting of 1:1 mixtures of MHB and CSF from Pseudomonas aeruginosa 9027, Pseudomonas fluorescens PF-5, Chromobacterium violaceum CV206, A. pyogenes BBR1, or C. perfringens strain 13. Trypticase soy broth (TSB) supplemented with 5% newborn calf serum (TSB-5%) and TSB supplemented with 0.5% yeast extract and 0.05% cysteine (TSBYC) were used as controls for A. pyogenes and C. perfringens CSFs, respectively. The source of the CSF or the type of control medium is shown on the x axis. Experiments were performed three times in triplicate, and error bars represent the standard deviation from the mean.

FIG. 7.

Biofilm formation by C. jejuni isolates does not correlate with isolate virulence. Biofilm assays were performed with the C. jejuni type strain NCTC11168, human clinical isolates M129, F38011, and UMC3, and strain S2B. Experiments were performed three times in triplicate, and error bars represent the standard deviation from the mean.