Modelling-based identification of factors influencing campylobacters in chicken broiler houses and on carcasses sampled after processing and chilling.

AIMS: To identify production and processing practices that might reduce Campylobacter numbers contaminating chicken broiler carcasses. METHODS AND RESULTS: The numbers of campylobacters were determined on carcass neck skins after processing or in broiler house litter samples. Supplementary information that described farm layouts, farming conditions for individual flocks, the slaughterhouse layouts and operating conditions inside plants was collected, matched with each Campylobacter test result. Statistical models predicting the numbers of campylobacters on neck skins and in litter were constructed. Carcass microbial contamination was more strongly influenced by on-farm production practices compared with slaughterhouse activities. We observed correlations between the chilling, washing and defeathering stages of processing and the numbers of campylobacters on carcasses. There were factors on farm that also correlated with numbers of campylobacters in litter. These included bird gender, the exclusion of dogs from houses, beetle presence in the house litter and the materials used to construct the house frame. CONCLUSIONS: Changes in farming practices have greater potential for reducing chicken carcass microbial contamination compared with processing interventions. SIGNIFICANCE AND IMPACT OF THE STUDY: Routine commercial practices were identified that were correlated with lowered numbers of campylobacters. Consequently, these practices are likely to be both cost-effective and suitable for adoption into established farms and commercial processing.

Quantitative Determination of Campylobacter on Broilers along 22 United Kingdom Processing Lines To Identify Potential Process Control Points and Cross-Contamination from Colonized to Uncolonized Flocks.

ABSTRACT: As part of a program to reduce numbers of the human pathogen Campylobacter on retail chickens, 22 broiler processing lines, representing more than 90% of UK production, were characterized by enumerating Campylobacter on pooled neck skins after exsanguination, scalding, defeathering, evisceration, crop removal, inside-outside washing, and air-chilling stages of processing. Sixteen of the processing lines investigated showed significant (P < 0.05) reductions in Campylobacter numbers because of carcass scalding. However, in all of these lines, the following defeathering stage caused a significant increase in Campylobacter contamination that effectively negated the reductions caused by scalding. On four processing lines, primary chilling also caused a significant reduction in numbers of Campylobacter. On three lines, there was a significant microbiological benefit from inside-outside washing. The stages where Campylobacter numbers were reduced require further investigation to determine the specific mechanisms responsible so that the observed pathogen reductions can be optimized and then more widely implemented. The transfer of up to 4 log CFU Campylobacter per g of neck skin from a colonized flock to a following uncolonized flock was observed. Cross-contamination was substantial and still detectable after 5,000 carcasses from an uncolonized flock had been processed. Numbers of Campylobacter recovered from the uncolonized flocks were highest on the first of the uncolonized birds to pass along the line, and in general, the numbers declined as more uncolonized birds were processed. Air sampling recovered low numbers at the processing stages monitored, indicating that airborne transmission was unlikely to be the primary transfer mechanism operating for cross-contamination between flocks.

Restoring the selectivity of Bolton broth during enrichment for Campylobacter spp. from raw chicken.

AIMS: When isolating Campylobacter spp. from retail raw chicken using BS EN ISO 10272-1:2006, contaminants frequently cause overgrowth on mCCDA plates. Therefore, these organisms proliferate in the enrichment medium, Bolton broth, indicating a lack of selectivity in this medium. This study sought to characterize the contaminant flora and to devise a modified Bolton broth to inhibit their growth. METHODS AND RESULTS: Contaminants (n=30) from separate samples were identified and antibiotic resistances determined. Most (93%) were extended spectrum ß-lactamase (ESBL) producing Escherichia coli, able to hydrolyse the cefoperazone present in Bolton broth and mCCDA. To inhibit these organisms, original formulation Bolton broth was supplemented with potassium clavulanate, at three concentrations, and recoveries of campylobacters from raw chicken were determined. Using standard Bolton broth, only 49% of samples (n=104) yielded campylobacters, but supplementation with 2 mg l(-1) potassium clavulanate increased this significantly (P<0.05), with 91% of samples positive. CONCLUSIONS: Potassium clavulanate can restore the selectivity of Bolton broth when isolating Campylobacter spp. from raw chicken. SIGNIFICANCE AND IMPACT OF THE STUDY: Raw chicken is often contaminated with the pathogen Campylobacter, but the ISO methodology for its detection is becoming compromised by the increasing presence of antibiotic-resistant bacteria. A simple modification ensures effective detection of this pathogen.

Use of conductimetry to rapidly determine relative stress sensitivity in Salmonella isolates.

AIMS: To compare conventional plate counting and indirect conductimetry as techniques for ranking the resistance of Salmonella spp. to processing stressors. METHODS AND RESULTS: Forty Salmonella isolates were subjected to three separate stressors used in food processing; irradiation, heat and high hydrostatic pressure (HHP). Total viable counts (TVC) using conventional plate counts and time to detection (TTD) using indirect conductimetry were determined. A significant negative correlation between TVC and TTD was seen with irradiation (P < 0.01) and heat (P < 0.05) but not HHP. CONCLUSIONS: For a group of salmonellas, indirect conductimetry can rapidly determine a ranking of isolate sensitivity to irradiation and heat. However, for HHP, the results indicated that conventional plate counting alone cannot be used to determine sensitivity. SIGNIFICANCE AND IMPACT OF THE STUDY: The resistance of micro-organisms to processing systems must be ranked to allow the selection of appropriate isolates for process validation. TTD measurements allow rapid screening of salmonellas to rank isolates for resistance to irradiation and heat stress. However, following HHP, the TVC of survivors is independent of the time required for growth to a set cell density and therefore it cannot be used as the sole measure of relative stress resistance.

Factors affecting the recovery of Campylobacter spp. from retail packs of raw, fresh chicken using ISO 10272-1:2006.

AIMS: This study sought to determine the most effective protocol for the detection of Campylobacter spp. in retail packs of fresh, raw chicken based on ISO 10272-1:2006. METHODS AND RESULTS: Three sample preparation protocols were studied; two based on excision and one combining excision with a rinse of the remaining sample. Enrichment cultures were incubated both in closed bottles and microaerobically, and sub-cultured at 24 and 48 h. Packs of chicken (110) were analysed and only two yielded no Campylobacter spp. Subculturing enrichment broths at 24 h gave the same prevalence as at 48 h, P > 0.4 but microaerobic incubation yielded approximately 50% more positive samples than did incubation in closed bottles. Sampling based on excision plus rinsing gave the highest Campylobacter prevalence (92.7%). CONCLUSIONS: To isolate Campylobacter spp. from retail packs of chicken, enrichment cultures must be incubated in a microaerobic atmosphere and sub-cultured at 24 h and, possibly, 48 h. Sampling packs by excision plus rinsing maximized recoveries. SIGNIFICANCE AND IMPACT OF THE STUDY: ISO 10272-1:2006 permits the use of inefficient protocols which markedly underestimate the true prevalence of Campylobacter spp. in retail, fresh chicken. Equivalent results could be obtained 24 h earlier, with consequent savings. Its revision is essential.

Consequences of Using Two Types of Skin Samples from Chilled Chicken Broiler Carcasses To Measure the Degree of Contamination by Campylobacter spp.

HIGHLIGHTS: Campylobacter levels on chicken neck and breast skin were compared. Neck skin was significantly more contaminated (P < 0.05) than breast skin. No relationship between the two skin types was found for Campylobacter levels. A UK government reduction target for highly contaminated chicken was not achieved.

Use of Hugh and Leifson's medium as a simple screening test to aid in the differentiation of Arcobacter spp. from background flora during their isolation from foodstuffs.

AIMS: To investigate the suitability of Hugh and Leifson's medium (HLM) as the basis of a simple screening test to differentiate between contaminants and Arcobacter spp. during their isolation from foodstuffs. METHODS AND RESULTS: Characterized Arcobacter spp. were obtained from recognized culture collections. Wild-type isolates of Arcobacter spp. and contaminants were obtained using published isolation protocols. Retail packs of red meats were used as the source of the isolates. Eighteen defined Arcobacter spp. gave no reaction on HLM, as did 10 local wild-type isolates. Overall 163 contaminants were studied for oxidative reactions on HLM and 86% of isolates demonstrated this property. CONCLUSIONS: HLM can usefully serve as a simple and effective screening test to differentiate between Arcobacter spp. and contaminants. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacter isolation procedures are still being developed, and no effective diagnostic media currently exist. Rapidly excluding most contaminants can markedly increase the efficiency of isolation procedures by removing the need for extensive biotyping or the requirement to isolate DNA and conduct PCR tests.

Evaluation of the use of conductimetry for the rapid and precise measurement of Salmonella spp. growth rates.

The growth rates of 14 Salmonella serovars in tryptone soy broth plus yeast extract (TSBYE) were estimated using conventional plating techniques and indirect conductimetry using a Don Whitley RABIT system. Both methods gave identical results for the maximum specific growth rate (mumax) P>0.05. However, using the conductimetric method, mumax for a single serovar was determined in less than 7 h, whereas the conventional method required an additional 24 h. In addition, the conductimetric method was considerably more precise, much less labour-intensive and required the use of considerably less consumables. Using conductimetry, a trained operator could accurately determine mumax for 24 serovars in 3 working days, but only one serovar using the conventional plate counting technique. Hence, the use of conductimetry can markedly increase the precision and accuracy of mumax determinations by allowing a very significant increase in the number of results obtained and in their precision. The data generated will allow the development of better mathematical growth models. The method can also be used to compare growth media and conditions and hence rapidly optimise detection protocols for this pathogen.

Optimising recovery of Campylobacter spp. from the lower porcine gastrointestinal tract.

To determine the incidence of campylobacters in Northern Ireland pigs, ileal contents and anal swabs were taken shortly after death. Direct streaking onto Preston agar, and modified charcoal cefoperazone desoxycholate agar (mCCDA), were compared, as was enrichment in selective broths prior to streaking onto the corresponding solid medium. For anal swabs direct plating on mCCDA was most efficient, with 100% of samples positive, whilst for ileal contents enrichment in mCCD broth was best with 86% of samples positive. Although only 34% of ileal samples enriched in Preston broths were positive they yielded three species not isolated from mCCD broth, and hence indicated that some pigs were infected by at least two species of Campylobacter. Overall, the number of samples found to contain campylobacters, and the range of species isolated, was seen to be markedly affected by both the choice of selective medium and the isolation procedures.

Detection of Salmonella in animal protein by Rappaport-Vassiliadis broth using indirect impediometry.

Indirect impedance methodology for the detection of Salmonella was investigated using a rapid automated bacterial impedance technique (RABIT) system. Four commercially available Rappaport-Vassiliadis (RV) enrichment broths were evaluated for their sensitivity and selectivity in detecting Salmonella using this technique. The RV from Lab M and Oxoid (new) gave the shortest detection times and showed good correlation between Salmonella numbers and detection times. Using Lab M medium, the indirect impedance technique could distinguish between Salmonella spp. and the closely related genera, Proteus and Citrobacter. The impedance technique showed recoveries of Salmonella from processed animal protein and raw meats equivalent to, or better than, those obtained with RV used in a conventional Salmonella isolation procedure.

Impedance detection of Salmonella in processed animal protein and meat.

The impedance technique showed a detection rate (95%) equal to that of conventional enrichment for raw meat contaminated with Salmonella. For processed animal protein samples impedance was less sensitive. A commercially available Easter and Gibson impedance medium used for the selective enrichment of salmonellae proved superior to the laboratory prepared equivalent for the detection of Salmonella in processed animal protein. The rate of false-positive results with the impedance technique was high.

Benefits of indirect impediometry, using Rappaport-Vassiliadis broth, for the detection of Salmonella in processed animal protein.

Four hundred samples of processed animal protein were analysed for the presence of Salmonella using two impediometric methods. The direct method using both Easter and Gibson's, and Ogden's media, as defined in legislation applying throughout the UK, was less selective and sensitive, detecting 30% fewer positive samples than the indirect method using Rappaport-Vassiliadis broth. The indirect method detected 63 positive samples with a false positive rate of less than 10% and about 87% of positive samples were detected less than 12 h after being loaded onto the impedance equipment. Thus the indirect method was superior in terms of performance as well as being a cheaper and simpler methodology for the detection of Salmonella.

Occurrence of Campylobacter in retail foods in Ireland.

A surveillance study was carried out to determine the prevalence of Campylobacter in a range of retail foods purchased in three Irish cities over a 20-month period between March 2001 and October 2002. In total 2391 food samples were analysed during this period. Campylobacter was isolated from 444 raw chicken (49.9%), 33 turkey (37.5%) and 11 duck samples (45.8%). Lower isolation rates of 7/221 (3.2%), 10/197 (5.1%) and 31/262 (11.8%) were observed for raw beef, pork and lamb, respectively. One sample of pork paté from 120 samples analysed (0.8%) was Campylobacter-positive. A total of three shellfish samples (oysters) from 129 raw specimens examined (2.3%) were found to contain Campylobacter. Low prevalences of the organism (0.9%) were also isolated from fresh mushrooms. Of 62 raw bulk tank milk samples analysed, Campylobacter was recovered in a single sample (1.6%). Campylobacter was not detected in any of the comminuted pork puddings, prepared vegetables and salads, retail sandwiches or cheeses made from unpasteurised milk. In total, 543 Campylobacter were isolated from all of the food samples analysed, of which 453 (83.4%) were confirmed as Campylobacter jejuni and the remaining 90 (16.6%) as Campylobacter coli.

Occurrence of thermophilic Campylobacter spp. in porcine liver in Northern Ireland.

Pork liver (400) from bacon pigs (37 herds) obtained at six pork-processing in Northern Ireland were studied to assess the rate of contamination with Campylobacter spp. These animals average 95 to 100 kg live weight. Deep tissue areas were sampled immediately postevisceration and revealed that ca. 6% of livers were infected with Campylobacter spp., consisting of C. coli (67%), C. jejuni (30%) and C. lari (3%). Mean log10 CFU g(-1) for aerobic plate count and coliforms were 3.60 and 2.94 respectively, indicating reasonable maintenance of slaughter-house hygiene procedures. A combination of direct swabbing of liver coupled with plating on both Skirrow and Blaser-Wang selective media was the most efficient combination of selective media employed. These data confirm the presence of Campylobacter spp. in porcine liver, thereby emphasizing the need to define safe processing parameters in the manufacture of liver-based products that are subjected to mild thermal processes, in order to eliminate the risk of disease to man.

Microbiological quality of chilled beef carcasses in Northern Ireland: a baseline survey.

To standardize the assessment of the hygienic quality of beef carcasses in Northern Ireland (NI) abattoirs, swabbing techniques were evaluated. Six materials, including two commercially produced swabs, were compared for their ability to recover spoilage and pathogenic bacteria and for their ease of use as carcass swabs. A sponge retailed for domestic use was selected on the basis of efficiency of recovery of microorganisms, ease of use, and cost. On sample carcasses, 1,000 cm2 of the brisket was swabbed, since this site is normally readily contaminated. For 9 months, 420 carcasses in seven of the nine European Union-approved abattoirs in NI were sampled while in the chiller (24 to 48 h after kill). Total viable count (TVC), yeasts and molds, and Enterobacteriaceae were enumerated after incubation at 22 (48 h) and 37 degrees C (48 h), and the results were expressed as log CFU/cm2. The mean TVC results at 22 and 37 degrees C were 2.80+/-0.70 and 2.75+/-0.64, respectively. Although 63% of samples had yeasts that grew at 22 degrees C, only 35% were positive at 37 degrees C. The respective mean yeast counts were 1.12+/-0.59 and 0.46+/-0.51. Enterobacteriaceae were present in 15% of samples at 22 degrees C and 21% of samples at 37 degrees C. The mean counts for positive samples were 0.41+/-0.37 and 0.40+/-0.30, respectively. Molds were found in less than 4% of samples. Given that the brisket is normally one of the most heavily contaminated parts of the carcass, these results suggest that good hygienic practices are in operation in NI abattoirs. The results also enabled the abattoirs with the cleanest carcasses to be identified, hence permitting best practices to be found.

Occurrence of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter spp. on beef carcasses in Northern Ireland.

A survey of beef carcasses was conducted in all 10 European community approved abattoirs in Northern Ireland to determine the incidence of Escherichia coli O157:H7. Analyses were based on excised samples of neck meat taken less than 48 h post-kill. Overall, 780 carcasses were sampled and all were negative for E. coli O157:H7. A sub-set of samples was analysed for the presence of Listeria monocytogenes (n=200), Salmonella (n=200) and Campylobacter spp.(n=100). L. monocytogenes was not detected but Listeria innocua was found on five carcasses and Listeria seeligeri on one. Three carcasses carried salmonellas; Salmonella Mbandaka was found on two and Salmonella Thompson on one. Campylobacter spp. were not detected on any carcasses. The results indicate that very few beef carcasses in Northern Ireland appear to carry any of the four pathogens sought, and this may help explain the low incidence of E. coli O157:H7 in the Northern Ireland human population, relative to the rest of the UK.

Subspecies characterization of porcine Campylobacter coli and Campylobacter jejuni by multilocus enzyme electrophoresis typing.

Enteropathogenic Campylobacterjejuni, C. coli and C. lari are currently the most common causes of acute infectious diarrhoeal illness in the UK. Many domestic animals, including pigs, act as natural reservoirs of these organisms and infection may occur through the ingestion of contaminated foodstuffs. C jejuni and C. coli, isolated from the livers of bacon pigs, were examined at subspecies level by multilocus enzyme electrophoresis (MEE) typing with seven enzymic loci. Polymorphological variation was highest with indophenol oxidase, isocitrate dehydrogenase and L-phenylalanyl-L-leucine peptidase giving 5. 5 and 4 alleles at these loci, respectively. The 35 Campylobacter isolates examined in this study (12 C. jejuni and 23 C coli) represented 30 unique electrophoretic types (ETs). Of these ETs, 8 unique types were detected for the 12 C jejuni isolates and 19 unique ETs were detected for the 23 C coli isolates. In addition, 3 types (ETs 2, 5, 10) were shared in common among C. jejuni and C coli. The average number of alleles per enzyme locus was 3.28. The mean genetic diversity, i.e. arithmetic average over all loci assayed, including monomorphic values, was 0.5573 and 0.5350 for C jejuni and C coli. respectively. Alleles were shared by C jejuni and C coli, suggesting an exchange of genetic material between the species. MEE analyses of isolates showed that there was a wide range of subspecies types within both C. jejuni and C coli in porcine livers. In certain cases, up to four phenotypically different strains of C coli were isolated from one liver, indicating multiple infections.

Comparison of disc diffusion and epsilometer (E-test) testing techniques to determine antimicrobial susceptibility of Campylobacter isolates of food and human clinical origin.

The antibiotic resistance profiles of 75 Campylobacter isolates of food and human clinical origin was determined by two agar diffusion susceptibility methods; disc diffusion and epsilometer-test (E-test). The most common therapeutic antimicrobials, erythromycin, ciprofloxacin and tetracycline were studied, along with chloramphenicol, ampicillin and naladixic acid. The resistance observed for each antimicrobial, as determined by both of methods, were statistically compared using Fisher two-tailed analysis. Of the six antimicrobials studied only two were shown to have statistically different patterns when resistance was compared by disc diffusion and E-test. The percentage of isolates resistant to clinically relevant antimicrobials using both techniques ranged from 6.6 to 21.3% for erythromycin, 25.3-26.6% for tetracycline and 33.3-36.0% for ciprofloxacin. The prevalence of multi-drug resistant (MDR) campylobacters (isolates resistant to 2 or more antimicrobials) for both disc diffusion and E-test was 44%. It can be concluded that, for four of the six antimicrobials assessed, antimicrobial resistance prevalences could be equally determined by either of the methods studied.

A genotyping investigation of the colonization of piglets by Campylobacter coli in the first 10 weeks of life.

AIMS: To examine the Campylobacter genotypes colonizing a litter of piglets during the first 10 weeks of life and compare them with those of the sow. METHODS AND RESULTS: Campylobacters were isolated by direct plating of anal swabs. Piglets (n = 6) were sampled six times and five isolates per piglet obtained each time. The sow was also sampled but 20 isolates per sampling obtained. Isolates were genotyped by random amplification of polymorphic DNA, pulsed field gel electrophoresis and polymerase chain reaction/restriction fragment length polymorphism of the flagellin gene. Initially piglets were colonized by Campylobacter coli genotypes from the mother but after 66 days 33% of piglet isolates (based on a mean of the three-genotyping methods) were from other sources. The sow died after 14 days and the initial Campylobacter flora of the foster sow was subsequently replaced by genotypes from the piglets mother. However these constituted only a minor part of her flora after 52 days. Both foster sow and piglets carried multiple genotypes of Camp. coli: up to four in a single piglet sample and seven in one from the sow. SIGNIFICANCE AND IMPACT OF THE STUDY: Piglets are initially colonized by Camp. coli genotypes from their mother but later other genotypes displace them. Colonization is dynamic with the sow able to acquire genotypes from the piglets. CONCLUSIONS: The large numbers of Camp. coli genotypes carried by pigs, and frequent successions of dominant types, will render epidemiological studies problematic.