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).     

Quantification of Campylobacter Species Cross-Contamination during Handling of Contaminated Fresh Chicken Parts in Kitchens

Numerous outbreak investigations and case-control studies for campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a risk factor for infection and illness. There is currently extremely limited quantitative data on the levels of Campylobacter cross-contamination in the kitchen, hindering risk assessments for the pathogen commodity combination of Campylobacter and chicken meat. An exposure assessment needs to quantify the transfer of the bacteria from chicken to hands and the kitchen environment and from there onto ready-to-eat foods. We simulated some typical situations in kitchens and quantified the Campylobacter transfer from naturally contaminated chicken parts most commonly used in Germany. One scenario simulated the seasoning of five chicken legs and the reuse of the same plate for cooked meat. In another, five chicken breast filets were cut into small slices on a wooden board where, without intermediate cleaning, a cucumber was sliced. We also investigated the transfer of the pathogen from chicken via hands to a bread roll. The numbers of Campylobacter present on the surfaces of the chicken parts, hands, utensils, and ready-to-eat foods were detected by using Preston enrichment and colony counting after surface plating on Karmali agar. The mean transfer rates from legs and filets to hands were 2.9 and 3.8%. The transfer from legs to the plate (0.3%) was significantly smaller (P < 0.01) than the percentage transferred from filets to the cutting board and knife (1.1%). Average transfer rates from hands or kitchen utensils to ready-to-eat foods ranged from 2.9 to 27.5%.     

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.     

Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants

Aims: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC‐A. Methods and Results: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD–PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage‐resistant mutants were tested against phages PL‐1, J‐1, A2 and MLC‐A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC‐A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Conclusions: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC‐A but also to other Lact. casei and Lact. paracasei phages. Significance and Impact of the Study: This study highlights isolation of spontaneous bacteriophage‐resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.     

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.     

Quantification of campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens

Numerous outbreak investigations and case-control studies for campylobacteriosis have provided evidence that handling Campylobacter-contaminated chicken products is a risk factor for infection and illness. There is currently extremely limited quantitative data on the levels of Campylobacter cross-contamination in the kitchen, hindering risk assessments for the pathogen commodity combination of Campylobacter and chicken meat. An exposure assessment needs to quantify the transfer of the bacteria from chicken to hands and the kitchen environment and from there onto ready-to-eat foods. We simulated some typical situations in kitchens and quantified the Campylobacter transfer from naturally contaminated chicken parts most commonly used in Germany. One scenario simulated the seasoning of five chicken legs and the reuse of the same plate for cooked meat. In another, five chicken breast filets were cut into small slices on a wooden board where, without intermediate cleaning, a cucumber was sliced. We also investigated the transfer of the pathogen from chicken via hands to a bread roll. The numbers of Campylobacter present on the surfaces of the chicken parts, hands, utensils, and ready-to-eat foods were detected by using Preston enrichment and colony counting after surface plating on Karmali agar. The mean transfer rates from legs and filets to hands were 2.9 and 3.8%. The transfer from legs to the plate (0.3%) was significantly smaller (P < 0.01) than the percentage transferred from filets to the cutting board and knife (1.1%). Average transfer rates from hands or kitchen utensils to ready-to-eat foods ranged from 2.9 to 27.5%.     

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.     

LuxS distribution and AI-2 activity of Campylobacter spp

Aims: This study investigates the distribution of LuxS within Campylobacter (Camp.) species and Autoinducer (AI)‐2 activity of Camp. jejuni NCTC 11168 in food matrices. Methods and Results: LuxS (S‐ribosylhomocysteinase) sequences of different Campylobacter spp. were compared, and AI‐2 activity was measured with an AI‐2 reporter assay. Highest LuxS homologies were shared by Camp. jejuni, Camp. coli and Camp. upsaliensis, and their LuxS sequences had more similarities to the analysed Arcobacter and Vibrio harveyi strains than to all other analysed Campylobacter species. Of 15 analysed species only Camp. lari, Camp. peloridis and Camp. insulaenigrae did not produce AI‐2 molecules. Cultivation of Camp. jejuni NCTC 11168 in chicken juice reduced AI‐2 activity, and this reduction is not because of lower luxS expression or functionality. Conclusion: Not all Campylobacter species encode luxS. Food matrices can reduce AI‐2 activity in a LuxS‐independent manner. Significance and Impact of the Study: Besides, Camp. lari, Camp. peloridis and Camp. insulaenigrae do not show AI‐2 activity. Further investigations should clarify the function of AI‐2 in Campylobacter spp. and how species lacking luxS could overcome this alteration. Furthermore, the impact of food matrices on these functions needs to be determined as we could show that chicken juice reduced AI‐2 activity.     

Probiotic Characteristics of <Emphasis Type="Italic">Lactobacillus curvatus</Emphasis> DN317, a Strain Isolated from Chicken Ceca

The probiotic characteristics of Lactobacillus curvatus DN317, a strain isolated from chicken ceca, were evaluated. This strain was selected for study from the isolated Lactobacillus strains because it has specific anti-microbial activity against Campylobacter jejuni ATCC 33560, Camp. jejuni NCTC 11168, Listeria monocytogenes ATCC 7644, and Bacillus subtilis ATCC 8633. Lact. curvatus DN317 showed an auto-aggregation percentage of 72% and presented the highest co-aggregation with Lact. monocytogenes ATCC 7644 (68%) compared to B. subtilis ATCC 8633 (45%), Camp. jejuni ATCC 33560 (36%), and Camp. jejuni NCTC 11168 (35%). The data revealed that Lact. curvatus DN317 could survive at 0.25% bile, maintain viability at pH 2.5 for 30 min, produce biosurfactants, and adhere to Caco-2 cells. Quantification of IL-6, IL-8, IL-10, and β-defensin 2 levels shows that Lact. curvatus DN317 induces an increase in IL-8 and β-defensin 2 secretion, while the levels of IL-6 and IL-10 do not change. Lact. curvatus DN317 showed high levels of esterase and cysteine arylamidase activities (5); moderate levels of esterase lipase, β-galactosidase, and α-galactosidase activities (4, 3); and weak levels of leucine arylamidase, valine arylamidase, and acid phosphatase activity (1). Various activities were obtained of α-chymotrypsin, β-glucuronidase, β-glucosidase, and N-acetyl-β-glucosaminidase, which have been associated with intestinal diseases. Lact. curvatus DN317 lowered the cholesterol level in MRS with and without bile. Antibiotic susceptibility tests indicated that DN317 was sensitive to ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, clindamycin, erythromycin, and vancomycin but was resistant to chloramphenicol and ciprofloxacin. These results suggest that Lact. curvatus DN317 could potentially function as a probiotic.     

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.     

Demonstration of persistent strains of Campylobacter jejuni within broiler farms over a 1‐year period in Lithuania

Aims: The aim of the study was to investigate the flock prevalence of Campylobacter jejuni and Campylobacter coli in broiler farms in Lithuania and to identify possible persistent strains of Camp. jejuni using amplified fragment length polymorphism (AFLP) typing method. Methods and Results: During 1 year, 42 broiler flocks from 9 broiler farms were examined to determine the prevalence of Campylobacter‐positive broiler flocks in Lithuania. Among 42 broiler flocks examined, 31 flocks (73·8%) were positive for Camp. jejuni and 17 flocks (40·48%) for Camp. coli. Campylobacter jejuni isolates were genotyped by AFLP method using BspDI and BglII restriction enzymes. Typing of 190 isolates generated 50 AFLP genotypes with the highest diversity of strains found in the summer season. Each farm showed one or more predominant AFLP types, and one AFLP type (A32) was found in five broiler farms over a 1‐year period. Conclusions: Campylobacter jejuni and Camp. coli are highly prevalent in broiler farms in Lithuania. Farm‐specific genotypes were identified in all farms examined. Type A32 was present and persisted in different broiler farms, and a common source of transmission of Camp. jejuni was suspected. Significance and Impact of the Study: For the first time, Camp. jejuni in broiler flocks has been genetically characterized in Lithuania. Persistent strains of Camp. jejuni were detected over one period at the beginning of broiler meat production chain and, therefore, the identification of contamination source of such strains and the mechanism of their particular ability to persist are crucial to establish effective control measures against Camp. jejuni infection in broiler farms.     

Response of Lactobacillus casei BL23 to phenolic compounds

Aims: To determine the inhibitory effect of phenolic compounds on Lactobacillus casei BL23, the role of two component signal transduction systems (TCS) and the response of Lact. casei BL23 to p‐coumaric acid. Methods and Results: Growth of Lact. casei BL23 and 17 derivative strains defective in each TCS harboured by this strain in the presence of p‐coumaric acid, ferulic acid, caffeic acid or methyl gallate was monitored. Furthermore, changes in the protein content of Lact. casei BL23 when exposed to p‐coumaric acid were evaluated by 2D‐SDS‐PAGE. Eleven proteins differentially expressed in the presence of p‐coumaric acid were detected. Six of them could be identified: ClpP and HtrA, involved in protein turnover and folding, acetyl‐CoA carboxylase, involved in lipid metabolism, and an arginyl‐tRNA synthetase were more abundant, whereas PurL and PurN, involved in purine biosynthesis, were less abundant. Conclusions: No significant differences were observed between the parental strain and the TCS‐defective mutants. p‐Coumaric acid elicited a response against membrane and cytoplasmic damages. Significance and Impact of the Study: The inhibitory effect of phenolic compounds on Lact. casei BL23 has been determined. For the first time, cytoplasmic proteins presumably involved in the response of Lact. casei BL23 against p‐coumaric acid have been identified.     

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.     

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.     

Cross-contamination in the kitchen: effect of hygiene measures

Aims:  To determine the effect of hygiene measures on cross-contamination of Campylobacter jejuni at home and to select a safe tracer organism for C. jejuni.
Methods and Results:  Comparative tests were conducted with nonpathogenic Escherichia coli and Lactobacillus casei and L. casei was chosen as the safe tracer organism. Salads containing chicken breast fillet contaminated with a known number of C. jejuni and L. casei were prepared according to different cross-contamination scenarios and contamination levels of salads were determined. Cross-contamination could be strongly reduced when cleaning cutting board and cutlery with hot water (68°C), but generally was not prevented using consumer-style cleaning methods for hands and cutting board.
Conclusions:  Dish-washing does not sufficiently prevent cross-contamination, thus different cutting boards for raw meat and other ingredients should be used and meat–hand contact should be avoided or hands should be thoroughly cleaned with soap. Lactobacillus casei can be used as a safe tracer organism for C. jejuni in consumer observational studies.
Significance and Impact of the Study:  Cross-contamination plays an important role in the transmission of food-borne illness, especially for C. jejuni . This study delivers suitable data to quantitatively assess the risk of campylobacteriosis caused by cross-contamination and it shows the effect of different preventive hygiene measures.     

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.     

Effects of thymol and diphenyliodonium chloride against Campylobacter spp. during pure and mixed culture in vitro

Aims: To determine if the purported deaminase inhibitors diphenyliodonium chloride (DIC) and thymol reduce the growth and survivability of Campylobacter. Methods and Results: Growth rates of Campylobacter jejuni and Camp. coli were reduced compared to unsupplemented controls during culture in Muellar–Hinton broth supplemented with 0·25 μmol DIC or thymol ml^?1 but not with 0·01 μmol monensin ml^?1 or 1% ethanol. Recovery of Camp. jejuni and Camp. coli was reduced >5 log₁₀ CFU from controls after 24 h pure culture in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Similarly, each test Campylobacter strain was reduced >3 log₁₀ CFU from controls after 24 h mixed culture with porcine faecal microbes in Bolton broth supplemented with 0·25 or 1·0 μmol DIC ml^?1 or with 1·0 μmol thymol ml^?1. Treatments with 0·25 μmol thymol ml^?1, 0·01 μmol monensin ml^?1 or 1% ethanol were less effective. Ammonia production during culture or incubation of cell lysates was reduced by 0·25 or 1·0 μmol DIC ml^?1 but only intermittently reduced, if at all, by the other treatments. Conclusions: Diphenyliodonium chloride and thymol reduced growth, survivability and ammonia production of Camp. jejuni and Camp. coli. Significance and Impact of the Study: Results suggest a potential physiological characteristic that may be exploited to develop interventions.     

Correlation Between In Vitro Secretion of Virulence-Associated Proteins of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and Colonization of Chickens

The mechanisms used by Campylobacter jejuni to colonize the (chicken) intestinal tract have not been defined. In this study, we obtained evidence that in the presence of chicken serum and mucus, C. jejuni secreted proteins that may play a role in the colonization of chicken gut (Campylobacter invasion antigen = Cia). C. jejuni strains NCTC11168V1 and 81-176, as well as an NCTC11168V1 flaA mutant, were found to colonize intestinal tract and secrete proteins in the presence of chicken mucus, chicken serum, or fetal bovine serum in cell culture–conditioned medium. C. jejuni strain NCTC11168V26, which was observed to be a poor colonizer compared with the other C. jejuni isolates, did not secrete Cia proteins. Secreted proteins were also recognized by Western immunoblot using sera from birds that had been colonized by C. jejuni. These data suggest that C. jejuni secretes Cia proteins during colonization of chicken gut and that these Cia proteins play an important role in colonization.     

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.     

Inhibitory effect of oral Lactobacillus against oral pathogens

Aims: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. Methods and Results: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis‐ and caries‐related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis‐ and caries‐related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1–SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. Conclusions: The results have shown that oral Lactobacillus SD1–SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram‐negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Significance and Impact of the Study: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.