Prevention of intestinal Campylobacter jejuni colonization in broilers by combinations of in‐feed organic acids

Aim: We have tested the effect of various combinations of formic acid and sorbate on Campylobacter jejuni colonization in broiler chickens to reduce the colonization of this zoonotic pathogen in broiler chicken flocks. Methods and Results: Chickens were offered feed supplemented with different concentrations and combinations of formic acid and/or potassium sorbate. We found little or no effect on the Camp. jejuni colonization levels in chickens that were given feed supplemented with formic acid alone. A combination of 1·5% formic acid and 0·1% sorbate reduced the colonization of Camp. jejuni significantly, while a concentration of 2·0% formic acid in combination with 0·1% sorbate prevented Camp. jejuni colonization in chickens. This inhibition was replicated in two independent trials with a combination of three different Camp. jejuni strains. Conclusions: Our results show a novel and promising intervention strategy to reduce the incidence of Camp. jejuni in poultry products and to obtain safer food. Significance and Impact of the Study: To ensure food safety, a reduction of the carcass contamination with Camp. jejuni through reduced colonization of this pathogen in broiler chicken flocks is important. A range of organic acids as additives in feed and drinking water have already been evaluated for this purpose. However, no studies have yet shown a complete inhibition of Camp. jejuni colonization in broiler chickens.     

Ribosomal operon intergenic sequence region (ISR) heterogeneity in Campylobacter coli and Campylobacter jejuni

Aims: The intergenic sequence regions (ISR) between the 16S and 23S genes of Campylobacter jejuni and Campylobacter coli are markedly different for each species. However, in the genomic sequence for Camp. coli RM2228 , two rRNA operons have an ISR that is characteristic of Camp. coli, and the third operon is characteristic of Camp. jejuni. The aim of this study was to determine the prevalence of ISR heterogeneity in these organisms. Methods and Results: PCR primers were designed to yield a 327‐base pair (bp) product for Camp. coli and 166‐bp product for Camp. jejuni. A strain like Camp. coli RM2228 should yield products of both sizes. DNA from a panel of Camp. coli (n = 133) and Camp. jejuni (n = 134) isolates were tested. All of the isolates yielded products of the predicted size for the species. To verify the data for Camp. coli RM2228 , each ribosomal operon from the isolate was individually amplified by PCR and tested with the ISR primer pair. Products of both sizes were produced as predicted. Conclusions: The cross‐species heterogeneity of the ISR seen in Camp. coli RM2228 is uncommon. Significance and Impact of the Study: The heterogeneity must have been caused by horizontal gene transfer at a frequency lower than predicted from housekeeping gene data. Thus, it can be expected that species identification based on the ISR can be confused in rare isolates.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.     

Detection and quantification of Campylobacter jejuni and Campylobacter coli using real-time multiplex PCR

Aims: We describe a real‐time quantitative multiplex polymerase chain reaction (qmPCR) assay to identify and discriminate between isolates of Campylobacter jejuni and Campylobacter coli. Methods and Results: Two novel sets of primers and hydrolysis probes were designed to amplify the unique DNA sequences within the hipO, ccoN and cadF genes that are specific to Camp. jejuni and Camp. coli. Using the designed optimized qmPCR assay conditions, the amplification efficiency is in range from 108 to 116%. These qmPCR assays are highly specific for Camp. jejuni and Camp. coli, as seen through testing of 40 Campylobacter strains and 17 non‐Campylobacter strains. In chicken juice and tap water models spiked with known quantities of Camp. jejuni, qmPCR detected 10²–10³ CFU ml^?1 within 4 h. Conclusions: The qmPCR assays developed in this study provide reliable and simultaneous detection and quantification of Camp. jejuni and Camp. coli, with good amplification reaction parameters. Significance and Impact of the Study: Following further validation, the qmPCR assay reported here has the potential to be applied to various sample types as an alternative and rapid methodology.     

Multi‐locus sequence types of Campylobacter carried by flies and slugs acquired from local ruminant faeces

Aims: To assess whether flies and slugs acquire strains of Campylobacter jejuni and Campylobacter coli present in local ruminant faeces. Methods and Results: Campylobacter was cultured from flies, slugs and ruminant faeces that were collected from a single farm in Scotland over a 19‐week period. The isolates were typed using multi‐locus sequence typing (MLST) and compared with isolates from cattle and sheep faeces. Campylobacter jejuni and Camp. coli were isolated from 5·8% (n = 155, average of 75 flies per pool) and 13·3% (n = 15, average of 8·5 slugs per pool) of pooled fly and slug samples, respectively. The most common sequence type (ST) in flies was Camp. coli ST‐962 (approx. 40%) regardless of the prevalence in local cattle (2·3%) or sheep (25·0%) faeces. Two positive slug pools generated the same ST that has not been reported elsewhere. Conclusions: Despite their low carriage rate, flies are able to acquire Campylobacter STs that are locally present, although the subset carried may be biased when compared to local source. Slugs were shown to carry a previously unreported Campylobacter ST. Significance and Impact of the Study: This study has demonstrated that flies carry viable Campylobacter and may contribute to the transfer of STs within and between groups of animals on farms. Further, they may therefore present a risk to human health via their contact with ready‐to‐eat foods or surfaces.     

Occurrence of thermotolerant Campylobacter species in surface waters of a Mediterranean area and in its prevailing pollution sources

Aims: To determine the prevalence of Campylobacter in surface waters of a highly populated Mediterranean area. Methods and Results: Surface water and wastewater samples were collected from an area in the north‐east of Spain during a 2‐year study. All the samples were analysed using the MPN method and Multiplex PCR to quantify and identify Campylobacter. It was detected in 82% of the samples from the Llobregat River with a mean of 1·3 MPN 100 ml^?1. The lowest counts were obtained in summer. Campylobacter coli was the predominant species in this river. The bacteria were isolated from marsh water but not from seawater samples. The highest counts of campylobacters were found in poultry wastewater where Camp. jejuni was the predominant species, as in urban sewage. In pig slurry, Camp. coli was the only species detected. Conclusions: Campylobacter jejuni and Camp. coli are present and widely distributed in the surface water of the studied area. The two species co‐exist, with Camp. coli being predominant. In river water, campylobacter counts presented a seasonal distribution. No relationship with faecal indicators was found. Significance and Impact of the Study: This study provides the first data on the occurrence and concentrations of thermotolerant campylobacter species in surface water in a Mediterranean area.     

Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test®

Aims: The adhesion to an inert surface (the first step of biofilm formation) of the two main pathogenic Campylobacter species, Campylobacter jejuni and Campylobacter coli, isolated from diverse origins, was compared. Methods and Results: Adhesion assays were conducted in 96‐well, polystyrene microtiter plates using the BioFilm Ring Test^® method. This new technique, based on magnetic bead entrapment, was shown to be suitable for analysing the adhesion of Campylobacter sp. strains by comparing the adhesion of four C. jejuni strains as revealed by the BioFilm Ring Test^® and immunodetection. Among the 46 strains tested, C. jejuni and C. coli displayed different adhesion capabilities ranging from no adhesion to strong adhesion. However, no strain of C. coli was strongly adherent, and statistically, C. coli adhered less to an inert surface than C. jejuni. In addition, strains isolated from animals or carcasses were less adherent than those isolated from food‐processing and clinical cases. Conclusions: These observations suggest that the food environment and the human body could have selected strains with greater adhesion. Significance and Impact of the Study: The adhesion capability of strains could partly explain the cross‐contamination or re‐contamination of food products by Campylobacter. This property could provide a mode of survival for Campylobacter in the food chain.     

Identification of nine sequence types of the 16S rRNA genes of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> subsp. <Emphasis Type="Italic">jejuni</Emphasis> isolated from broilers

Background  

Campylobacter is the most commonly reported bacterial cause of enteritis in humans in the EU Member States and other industrialized countries. One significant source of infection is broilers and consumption of undercooked broiler meat. Campylobacter jejuni is the Campylobacter sp. predominantly found in infected humans and colonized broilers. Sequence analysis of the 16S rRNA gene is very useful for identification of bacteria to genus and species level. The objectives in this study were to determine the degree of intraspecific variation in the 16S rRNA genes of C. jejuni and C. coli and to determine whether the 16S rRNA sequence types correlated with genotypes generated by PFGE analysis of Sma I restricted genomic DNA of the strains.     

High-Resolution Genotyping of Campylobacter Strains Isolated from Poultry and Humans with Amplified Fragment Length Polymorphism Fingerprinting

For epidemiological studies of Campylobacter infections, molecular typing methods that can differentiate campylobacters at the strain level are needed. In this study we used a recently developed genotyping method, amplified fragment length polymorphism (AFLP), which is based on selective amplification of restriction fragments of chromosomal DNA, for genetic typing of Campylobacter jejuni and Campylobacter coli strains derived from humans and poultry. We developed an automated AFLP fingerprinting method in which restriction endonucleases HindIII and HhaI were used in combination with one set of selective PCR primers. This method resulted in evenly distributed band patterns for amplified fragments ranging from 50 to 500 bp long. The discriminatory power of AFLP was assessed with a C. jejuni strain, an isogenic flagellin mutant, and distinct C. jejuni strains having known pulsed-field gel electrophoresis and fla PCR-restriction fragment length polymorphism genotypes. Unrelated C. jejuni strains produced heterogeneous patterns, whereas genetically related strains produced similar AFLP patterns. Twenty-five Campylobacter strains obtained from poultry farms in The Netherlands grouped in three C. jejuni clusters that were separate from a C. coli cluster. The band patterns of 10 C. jejuni strains isolated from humans were heterogeneous, and most of these strains grouped with poultry strains. Our results show that AFLP analysis can distinguish genetically unrelated strains from genetically related strains of Campylobacter species. However, desirable genetically related strains can be differentiated by using other genotyping methods. We concluded that automated AFLP analysis is an attractive tool which can be used as a primary method for subtyping large numbers of Campylobacter strains and is extremely useful for epidemiological investigations.     

Genomic Diversity of Campylobacter coli and Campylobacter jejuni Isolates Recovered from Free-Range Broiler Farms and Comparison with Isolates of Various Origins

In many industrialized countries, the incidence of campylobacteriosis exceeds that of salmonellosis. Campylobacter bacteria are transmitted to humans mainly in food, especially poultry meat products. Total prevention of Campylobacter colonization in broiler flocks is the best way to reduce (or eliminate) the contamination of poultry products. The aim of this study was to establish the sources and routes of contamination of broilers at the farm level. Molecular typing methods (DNA macrorestriction pulsed-field gel electrophoresis and analysis of gene polymorphism by PCR-restriction fragment length polymorphism) were used to characterize isolates collected from seven broiler farms. The relative genomic diversity of Campylobacter coli and Campylobacter jejuni was determined. Analysis of the similarity among 116 defined genotypes was used to determine clusters within the two species. Furthermore, evidence of recombination suggested that there were genomic rearrangements within the Campylobacter populations. Recovery of related clusters from different broiler farms showed that some Campylobacter strains might be specifically adapted to poultry. Analysis of the Campylobacter cluster distribution on three broiler farms showed that soil in the area around the poultry house was a potential source of Campylobacter contamination. The broilers were infected by Campylobacter spp. between days 15 and 36 during rearing, and the type of contamination changed during the rearing period. A study of the effect of sanitary barriers showed that the chickens stayed Campylobacter spp. free until they had access to the open area. They were then rapidly colonized by the Campylobacter strains isolated from the soil.     

Comparison of genotypes and antibiotic resistance of Campylobacter jejuni isolated from humans and slaughtered chickens in Switzerland

Aims: To get an overview of genotypes and antibiotic resistances in Swiss Campylobacter jejuni implicated in human gastroenteritis and to examine the association with isolates from chickens. Methods and Results: Multilocus sequence typing (MLST) and flaB typing were applied to 136 human clinical isolates. Phenotypic resistance to 12 antimicrobials and genotypic resistance to macrolides and quinolones were determined. MLST resulted in 35 known and six new sequence types (ST). The flaB analysis revealed 35 different types, which – in combination with MLST – increased the resolution of the typing approach. Resistance to quinolones, tetracycline and ampicillin was found in 37·5, 33·1 and 8·1% of the isolates, respectively, whereas macrolide resistance was found only once. Genotypic and phenotypic resistance correlated in all cases. A comparison to Camp. jejuni isolated from slaughtered chickens was performed. While 86% of the quinolone‐sensitive human isolates showed overlapping MLST‐flaB types with those of chicken origin, resistant strains showed only 39% of matching types. Conclusion: Mainly quinolone‐sensitive Camp. jejuni strains implicated in human campylobacteriosis showed matching genotypes with isolates originating from chickens. Significance and Impact of the Study: A large proportion of human cases in Switzerland are likely to originate from domestic chickens, confirming that prevention measures in the poultry production are important.     

Campylobacter jejuni as a secondary colonizer of poultry biofilms

Aims: The objective of this study was to determine if survival of culturable Campylobacter jejuni outside the host was increased by entrapment in pre-established biofilms. Methods and Results: Campylobacter jejuni was inoculated into four biofilm populations isolated from poultry environments and cultured at three temperatures. Survival of culturable Camp. jejuni in some pre-established biofilms was extended vs survival of culturable Camp. jejuni in broth. But some biofilms were detrimental to survival of culturable Camp. jejuni. Denaturing gradient gel electrophoresis analysis indicated differences in bacterial profiles depending on initial source and temperature of culturing, which may have had impacts on survival of culturable Camp. jejuni. Further investigation showed no evidence of interspecies cell signalling indicating that secondary colonization was only physical. Conclusions: The results of this study show Camp. jejuni’s attachment to surfaces is facilitated by pre-established biofilms and survival of culturable Camp. jejuni may be extended in some pre-established biofilms, but these biofilms do not fully explain long-term survival of culturable Camp. jejuni outside hosts. Significance and Impact of the Study: This study provides new information concerning survival of culturable Camp. jejuni outside the host and shows biofilms may be important in transmission and prevalence of Camp. jejuni.     

Expression and characterization of cell-signalling molecules in Campylobacter jejuni

Aims: This study investigated the production and effects of cell‐signalling compounds on selected survival and virulence mechanisms of Campylobacter jejuni. Methods and Results: The production of Autoinducer 1 (AI‐1) compounds by Camp. jejuni was investigated in‐vitro using a variety of available AI‐1 bioassays. We further examined the role of a range of commercially available homoserine lactones (HSL) and a novel compound (cjA) isolated from Camp. jejuni. The selected attributes included the transformation to a viable but nonculturable (VBNC) state, biofilm formation, interleukin 8 (IL‐8) stimulation in INT‐407 cells and virulence gene expression as determined by qRT‐PCR. This study is the first to report an HSL or HSL mimic produced by Camp. jejuni. Short chained HSLs and the novel compound cjA prolonged the delay to a VBNC state as well as inhibiting biofilm formation and the majority of HSLs examined and the HSL mimic cjA significantly affected virulence gene expression as well as increasing the production of IL‐8 in challenged INT‐407 cells. Conclusions: Despite the lack of a homologous HSL kinase or sensor, Camp. jejuni appears to produce, as well as detect, exogenous signalling molecules and respond accordingly to aid in the survival and virulence capabilities of this micro‐organism. Significance and Impact of the Study: This study suggests that Camp. jejuni is able to detect and utilize as well as possibly produce cell‐signalling molecules that enhance both survival and virulence attributes. This possibility opens a new field in the search for Camp. jejuni reduction and elimination strategies.     

Evidence that Certain Clones of Campylobacter jejuni Persist during Successive Broiler Flock Rotations

Through the national surveillance program for Campylobacter spp., nine broiler chicken farms that were infected with Campylobacter jejuni in at least five rotations in 1998 were identified. One additional farm, located at the island of Bornholm where divided slaughter is used extensively, was also selected. Twelve broiler houses located on 10 farms were included in the study. The C. jejuni isolates collected from the selected houses during the surveillance were typed using fla typing and macrorestriction profiling (MRP), and a subset of the isolates, representing each of the identified clones, was serotyped according to the Penner scheme. Pulsed-field gel electrophoresis typing using SmaI and KpnI revealed that the majority of houses (11 of 12) carried identical isolates in two or more broiler flocks. Such persistent clones were found in 63% of all flocks (47 of 75). The majority of persistent clones (7 of 13) had fla type 1/1, but MRPs distinguished between isolates from different houses, and fla type 1/1 clones belonged to different serotypes. Seven houses carried persistent clones that covered an interval of at least four broiler flock rotations, or at least one half year. The dominant fla type (1/1) was represented by 44% of isolates, or by at least one isolate from 31 of 62 broiler flocks. This significantly exceeded the prevalence of fla type 1/1 C. jejuni isolates that we have estimated from other studies and suggests that isolates carrying this fla type are overrepresented in flocks with recurrent Campylobacter problems. The MRPs of clones belonging to fla type 1/1 serotype O:2 isolated from persistently infected flocks shared a high percentage of bands compared to the remaining isolates, indicating that some clones that have the ability to cause persistent infections in broiler farms are highly related to each other.     

Niche segregation and genetic structure of Campylobacter jejuni populations from wild and agricultural host species

Bacterial populations can display high levels of genetic structuring but the forces that influence this are incompletely understood. Here, by combining modelling approaches with multilocus sequence data for the zoonotic pathogen Campylobacter, we investigated how ecological factors such as niche (host) separation relate to population structure. We analysed seven housekeeping genes from published C. jejuni and C. coli isolate collections from a range of food and wild animal sources as well as abiotic environments. By reconstructing genetic structure and the patterns of ancestry, we quantified C. jejuni host association, inferred ancestral populations, investigated genetic admixture in different hosts and determined the host origin of recombinant C. jejuni alleles found in hybrid C. coli lineages. Phylogenetically distinct C. jejuni lineages were associated with phylogenetically distinct wild birds. However, in the farm environment, phylogenetically distant host animals shared several C. jejuni lineages that could not be segregated according to host origin using these analyses. Furthermore, of the introgressed C. jejuni alleles found in C. coli lineages, 73% were attributed to genotypes associated with food animals. Our results are consistent with an evolutionary scenario where distinct Campylobacter lineages are associated with different host species but the ecological factors that maintain this are different in domestic animals such that phylogenetically distant animals can harbour closely related strains.     

Transfer of Campylobacter jejuni from raw to cooked chicken via wood and plastic cutting boards

Aims: We quantified Campylobacter jejuni transferred from naturally contaminated raw chicken fillets and skins to similar cooked chicken parts via standard rubberwood (RW) and polyethylene cutting boards (PE). Methods and Results: RW and PE cutting boards (2·5 × 2·5 cm²) were constructed. RW surfaces were smooth and even, whereas PE was uneven. Scoring with scalpel blades produced crevices on RW and flaked patches on the PE boards. Raw chicken breast fillets or skin pieces (10 g) naturally contaminated with Camp. jejuni were used to contaminate the cutting boards (6·25 cm²). These were then briefly covered with pieces of cooked chicken. Campylobacter jejuni on raw chicken, the boards, and cooked chicken pieces were counted using a combined most‐probable‐number (MPN)‐PCR method. The type of cutting board (RW, PE; unscored and scored) and temperature of cooked chicken fillets and skins were examined. Unscored PE and RW boards were not significantly different in regards to the mean transfer of Camp. jejuni from raw samples to the boards. The mean transfer of Camp. jejuni from scored RW was significantly higher than from scored PE. When the chicken fillets were held at room temperature, the mean transfer of Camp. jejuni from scored RW and PE was found to be 44·9 and 40·3%, respectively. Conclusions: RW and PE cutting boards are potential vehicles for Camp. jejuni to contaminate cooked chicken. Although cooked chicken maintained at high temperatures reduced cross‐contamination via contaminated boards, a risk was still present. Significance and Impact of the Study: Contamination of cooked chicken by Camp. jejuni from raw chicken via a cutting board is influenced by features of the board (material, changes caused by scoring) and chicken (types of chicken parts and temperature of the cooked chicken).     

Use of Multilocus Sequence Typing To Investigate the Association between the Presence of Campylobacter spp. in Broiler Drinking Water and Campylobacter Colonization in Broilers

The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration. Campylobacter presence was independent of the water source and whether the water was treated. The genotypes of Campylobacter jejuni isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters.     

Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocols

Aims: To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. Methods and Results: Two experiments were undertaken, in which 14‐day‐old chickens were colonized with 1 × 10⁷–1 × 10⁹ CFU g^?1Camp. jejuni strain 81116P and then treated with enrofloxacin at 12–500 ppm in drinking water for various times. Caecal colonization levels were determined at various time‐points after start‐of‐treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 μg ml^?1 ciprofloxacin, MICs of 16 μg ml^?1 and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12–250 ppm reduced Camp. jejuni colonization over the first 48–72 h after start‐of‐treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re‐established within 4–6 days. Fluoroquinolone‐resistant organisms were recoverable within 48 h of start‐of‐treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post‐treatment withdrawal. Conclusions: In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12–250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone‐resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. Significance and impact of the study: Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre‐colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters.     

Resistance to quinolones in Campylobacter jejuni and Campylobacter coli from Danish broilers at farm level

AIMS: To investigate the prevalence of quinolone resistance among Campylobacter jejuni and Camp. coli isolates from Danish poultry at the farm level, as well as for the whole country. METHODS AND RESULTS: Data and isolates were collected from a national surveillance of Campylobacter in poultry. Quinolone resistance was investigated by determination of minimum inhibitory concentration (MIC) to nalidixic acid and enrofloxacin. Among Camp. jejuni and Camp. coli combined, 7.5% were resistant to nalidixic acid. Quinolone resistance varied considerably from farm to farm, with 0% on some farms and almost 100% on others, but the resistance was evenly distributed geographically. With respect to isolates from farms where resistance was detected, quinolone resistance was higher among Camp. coli (28.7%) than among Camp. jejuni (11.3%). PFGE typing of quinolone-resistant and quinolone-susceptible isolates from four farms indicated that certain resistant isolates belonged to specific clones that were able to persist on the farms during several rotations, even in the absence of selective pressure. Some clones were present and repeatedly isolated in both a quinolone-susceptible and quinolone-resistant variant. CONCLUSIONS: Overall, quinolone resistance among Campylobacter isolates from Danish broilers was 7.5% in 1998 and 1999; it was higher among Camp. coli than Camp. jejuni. Genetic diversity among resistant isolates was lower than among susceptible isolates, and certain clones existed in both a resistant and a susceptible variant. Some resistant clones appeared to persist on the farms and were repeatedly isolated from poultry flocks. SIGNIFICANCE AND IMPACT OF THE STUDY: The study is important for the understanding of persistence and dynamics of Campylobacter in broiler houses. It also highlights the extent, farm-to-farm variation and persistence of quinolone-resistant Campylobacter in broiler houses.