Comparison of different sampling techniques and enumeration methods for the isolation and quantification of Campylobacter spp. in raw retail chicken legs

Comparable quantitative data of Campylobacter spp. on chicken products are a major data lack for quantitative risk assessment approaches. The objective of this study was to compare two different sampling techniques for the isolation and enumeration of Campylobacter spp. in chicken and to evaluate a suitable enumeration method comparing the most probable number (MPN) technique to the direct plating method. For this, 90 packages containing at least two raw chicken legs were examined for the comparison of sampling techniques, rinsing one leg and homogenizing 25 g of skin of the other leg of each package; both sample preparation types were examined by direct plating method and MPN technique in 40 out of 90 packages. Of the skin samples, 70% (63/90), and of the rinse samples, 77% (69/90), were Campylobacter-positive. Enumeration of Campylobacter spp. by direct plating revealed a median of log 4 cfu/leg surface in skin samples (S.D.=0.6) and a median of log 4.3 cfu/leg surface in rinse samples (S.D.=0.9) of the rinse samples; 73% (37/51) had higher numbers of Campylobacter spp. than the skin samples although the difference was not significant (p=0.08). The correlation coefficient of Campylobacter counts in skin and rinse samples was 0.43. The prevalence of Campylobacter spp. in rinse samples was 58% (23/40). In 5% (2/40) of the rinse samples, numbers of Campylobacter spp. could be detected only by the MPN technique due to the lower detection limit compared to the direct plating method. The MPN technique turned out to be unsuitable for the enumeration of Campylobacter spp. in skin samples because a layer formation on the top of the incubated MPN-tubes leads to irregular MPN results. Out of 80% (16/20) of the compared rinse samples, the direct plating detected higher numbers of Campylobacter spp., with a median count of log 4.2 cfu/leg surface (S.D.=1) compared the MPN technique where a median of log 4 cfu/leg surface (S.D.=1.1) was obtained. The difference was not significant (p=0.05). A highly positive correlation coefficient of 0.9 was observed between the direct plating and the MPN technique. Both sampling methods, rinsing the chicken leg and homogenizing the skin, are suitable for the detection and quantification of Campylobacter spp.; the direct plating method was superior to the MPN technique for enumerating Campylobacter spp. in raw chicken legs at retail level because enumeration is more rapid and less laborious.     

Salmonella and Campylobacter in United Kingdom retail raw chicken in 2005

The United Kingdom Food Standards Agency commissioned a survey of Salmonella and Campylobacter in raw, whole chickens at retail in Wales and Northern Ireland between March and December 2005 to measure the baseline prevalence rates of these two significant pathogens. In total, 877 retail samples were examined for Campylobacter and Salmonella by enrichment methods. Overall contamination rates of 70.2% for Campylobacter and 4.0% for Salmonella were found. There was a statistically significant difference in Campylobacter rates between fresh and frozen samples, with fresh samples having a higher rate. There was no statistically significant difference between samples taken from retailers and butchers. Campylobacter was significantly more common in Northern Ireland than in Wales. Salmonella was significantly more common in Wales. The findings indicate the need for further investigation to explore why measures that have been successful in reducing Salmonella in the United Kingdom in recent years have failed to contribute to the control of Campylobacter. Identifying the factors responsible could lead to the introduction of more effective controls throughout the industry.     

Ciprofloxacin-resistant Campylobacter persists in raw retail chicken after the fluoroquinolone ban

In 2005, the FDA withdrew approval for the use of fluoroquinolones in live poultry production. To assess any changes in countable numbers of ciprofloxacin-resistant Campylobacter before and after the fluoroquinolone withdrawal, retail whole raw chicken carcasses (RTCC) purchased in Northwest Arkansas from 2004 to 2006 were sampled for this purpose. Using a previously published direct-plating method developed in our laboratory, we quantified trends of Campylobacter and ciprofloxacin-resistant Campylobacter loads by direct plating whole chicken carcass rinses on Campylobacter agar (CA) or Campylobacter agar containing 8.6 µg/ml ciprofloxacin (CCA). Countable populations of Campylobacter were recovered from 74, 96, and 63% of carcasses sampled in 2004, 2005, and 2006 respectively. The percentages of carcasses with minimum detectable levels of ciprofloxacin-resistant Campylobacter were 20, 42 and 33%, sampled in 2004, 2005 and 2006, respectively. Our 3 year analysis in one geographical area indicated a persistence of Campylobacter and ciprofloxacin-resistant Campylobacter on retail raw chicken carcasses despite the cessation of fluoroquinolone use in poultry production.     

Ciprofloxacin-resistant Campylobacter persists in raw retail chicken after the fluoroquinolone ban

In 2005, the FDA withdrew approval for the use of fluoroquinolones in live poultry production. To assess any changes in countable numbers of ciprofloxacin-resistant Campylobacter before and after the fluoroquinolone withdrawal, retail whole raw chicken carcasses (RTCC) purchased in Northwest Arkansas from 2004 to 2006 were sampled for this purpose. Using a previously published direct-plating method developed in our laboratory, we quantified trends of Campylobacter and ciprofloxacin-resistant Campylobacter loads by direct plating whole chicken carcass rinses on Campylobacter agar (CA) or Campylobacter agar containing 8.6 mg/ml ciprofloxacin (CCA). Countable populations of Campylobacter were recovered from 74, 96, and 63% of carcasses sampled in 2004, 2005, and 2006 respectively. The percentages of carcasses with minimum detectable levels of ciprofloxacin-resistant Campylobacter were 20, 42 and 33%, sampled in 2004, 2005 and 2006, respectively. Our 3 year analysis in one geographical area indicated a persistence of Campylobacter and ciprofloxacin-resistant Campylobacter on retail raw chicken carcasses despite the cessation of fluoroquinolone use in poultry production.     

Quantification of the contamination of chicken and chicken products in the netherlands with Salmonella and Campylobacter

The research described in this contribution provides quantitative data on contamination levels with Salmonella and Campylobacter in chicken and chicken products in The Netherlands at retail level using the most probable number method and direct counting. Most samples contained <10 Salmonella per carcass, both in fresh (89%) and frozen (68%) products, contamination levels with Campylobacter varied from <10 (18%) to more than 5,500 (18%) per fresh carcass. Most frozen samples (57%) contained < 10 Campylobacter per carcass.     

Prevalence of Campylobacter and Salmonella in raw chicken on retail sale in the republic of Ireland

To assess the current risks to consumers from Campylobacter and Salmonella in raw chicken products sold in the Republic of Ireland, a retail survey was undertaken to define their prevalence. Samples (n = 510) were analyzed using protocols based on ISO 10272-1:2006 and ISO 6579:2002. Processor codes on pack labels showed that 67% of samples were produced in the Republic of Ireland and 25% in the United Kingdom. Salmonella was present in 5.1% of samples, but the eight serovars found caused less than 7% of human salmonellosis reported in the Republic of Ireland. The results suggest that on-farm controls to limit Salmonella infection of broilers have been successful and that in Ireland raw chicken is not a significant cause of salmonellosis in humans. The overall prevalence of Campylobacter spp. was 84.3%. Isolation by the ISO method found 52.7% of samples to be positive, but overgrowth by contaminants was frequently evident. Therefore, in addition to enrichment, an homogenized sample was plated directly onto modified charcoal cefoperazone deoxycholate agar, and this detected a further 31.6%. Speciation of isolates (n = 426) determined that 67% were Campylobacter jejuni and 32% were Campylobacter coli. These species are the most common cause of campylobacteriosis in man. The results indicate that there is a need for poultry producers to introduce interventions to minimize the exposure of consumers in the Republic of Ireland to Campylobacter spp., as has been successfully done for Salmonella.     

Three-year surveillance program examining the prevalence of Campylobacter and Salmonella in whole retail raw chicken

A 36-month study of Campylobacter and Salmonella in retail raw whole chicken was carried out to measure baseline rates at the retail level, establish seasonality, and observe changes in rates over time. In total, 2,228 samples were taken between November 2001 and December 2004. The Campylobacter rate was unchanged over the 3 years of the study, but the Salmonella rates declined significantly between 2001 and 2004. There was also some seasonality in Campylobacter rates in fresh samples. The overall conclusion from the study was that the Salmonella rate in raw chicken available to consumers in Wales fell significantly between 2001 and 2004, while the Campylobacter rate remained unchanged and is still by far the greater problem.     

Quantification and differentiation of Campylobacter jejuni and Campylobacter coli in raw chicken meats using a real-time PCR method

Campylobacter species are one of the most common causes of bacterial diarrhea in humans worldwide. The consumption of foods contaminated with two Campylobacter species, C. jejuni and C. coli, is usually associated with most of the infections in humans. In this study, a rapid, reliable, and sensitive multiplex real-time quantitative PCR was developed for the simultaneous detection, identification, and quantification of C. jejuni and C. coli. In addition, the developed method was applied to the 50 samples of raw chicken meat collected from retail stores in Korea. C. jejuni and C. coli were detected in 88 and 86% of the samples by real-time quantitative PCR and the conventional microbiological method, respectively. The specificity of the primer and probe sets was confirmed with 30 C. jejuni, 20 C. coli, and 35 strains of other microbial species. C. jejuni and C. coli could be detected with high specificity in less than 4 h, with a detection limit of 1 log CFU/ml by the developed real-time PCR. The average counts (log CFU per milliliter) of C. jejuni or C. coli obtained by the conventional methods and by the real-time PCR assay were statistically correlated with a correlation coefficient (R2) between 0.73 and 0.78. The real-time PCR assay developed in this study is useful for screening for the presence and simultaneous differential quantification of C. jejuni and C. coli.     

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.     

Prevalence and antimicrobial resistance of Campylobacter isolated from retail raw chicken and beef meat, Tehran, Iran

Campylobacter spp. is a leading cause of human diarrhea. The common source of infection is contaminated food, particularly poultry. The veterinary use of antimicrobial drugs has been suggested to be largely responsible for resistance in human isolates of this zoonotic pathogen. From April to October 2004, 241 samples of chicken and beef meat for sale in retail outlets in Tehran (Iran) were analyzed for the presence of Campylobacter. Totally, 88 (36.5%) Campylobacter strains were isolated. Campylobacter was isolated from a significantly larger number of chicken (63%) than beef (10%) meat (P < 0.0001). Susceptibilities of 72 strains were determined for eight antimicrobial drugs using the disk diffusion assay. Resistance to nalidixic acid was the most common finding (75%), followed by resistance to ciprofloxacin (69.4%), tetracycline (45.8%), amoxicillin (11.1%), streptomycin (4.2%), chloramphenicol (2.8%) and gentamicin (1.4%). None of the isolates was resistant to erythromycin. Multidrug resistance was seen in 75% of the Campylobacter strains.     

烤鸡腿的研制

以鸡腿为主要原料,添加适量的食盐,辣椒粉和白砂糖等作为辅料,采用单因素试验,正交试验和感官评定的方法对烤鸡腿的配方进行筛选和研究,确定了该产品的最佳工艺配方:辣椒粉0.2%,白砂糖0.5%,孜然粉0.15%,料酒0.2%。最佳烤制工艺条件:烤制温度250℃,烤制时间20～30min。     

Antimicrobial resistance and molecular subtyping of Campylobacter jejuni and Campylobacter coli from retail meats

Campylobacter isolates (n = 297; 202 C. jejuni and 95 C. coli isolates) recovered from 2,513 retail meat samples (chicken breasts, ground turkey, ground beef, and pork chops) were examined for antimicrobial susceptibility. The isolates were further analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE) using SmaI and KpnI restriction enzymes, and a subset of isolates (n = 174) were subtyped by multilocus sequence typing (MLST). The resistance most frequently observed was that to doxycycline (27.6%), followed by ciprofloxacin (13.8%) and erythromycin (6.4%). All isolates were susceptible to gentamicin and meropenem. C. coli showed higher resistance to doxycycline than did C. jejuni (42.1 versus 20.8%) and lower resistance to ciprofloxacin than did C. jejuni (10.5 versus 15.3%). Erythromycin resistance was only observed in C. coli. PFGE using SmaI plus KpnI digestion generated 168 clusters from 297 isolates: 115 from C. jejuni and 53 from C. coli. MLST revealed 44 sequence types (STs) under 10 clonal complexes from 120 C. jejuni and 27 STs under two clonal complexes from 54 C. coli. There was a positive association between PFGE and STs; however, PFGE showed greater discriminatory power than MLST. Subtyping data did not correlate with antimicrobial resistance phenotypes.     

Genotyping of Campylobacter coli and C. jejuni from retail chicken meat and humans with campylobacteriosis in Slovenia and Bosnia and Herzegovina

Thermotolerant Campylobacter jejuni and C. coli are one of the major causes of bacterial foodborne enteric infection. Consuming and/or handling poultry meat is the most consistent risk factor, linked to the high prevalence of campylobacters in retail poultry meat. The aim of the present study was to ascertain the genetic diversity and/or possible specificity of thermotolerant Campylobacter isolates according to species (C. coli, C. jejuni), isolation source (retail chicken meat and human clinical samples) and geographic origin (Goriska in Slovenia and Zenica-Doboj Canton in Bosnia and Herzegovina (BIH)). With the pulsed-field gel electrophoresis (PFGE) after SmaI macrorestriction we distinguished 80 PFGE types among 118 strains and CfoI restriction fragment length polymorphism of the amplified flagellin gene (fla-RFLP) gave 12 fla-RFLP types. Beside the higher discriminatory power and strain typeability, PFGE discriminated the C. jejuni and C. coli groups of isolates. A high proportion of C. coli strains was isolated, especially from poultry samples. Identical or very similar PFGE types among the isolates from animal, food and human samples indicate the transmission of C. jejuni and C. coli from the chickens on the farm to the retail chicken meat, as well as possible cross-contamination of retail meat and transmission to humans. However, the identity of the isolates from non-related samples but with identical PFGE and fla-RFLP types should be confirmed with additional typing. Reliable tracing of the source of Campylobacter strains by molecular typing of the chicken meat isolates is therefore very difficult. The reasons include contamination of meat samples with multiple strains, possible cross-contamination and extreme heterogeneity of the isolates (mainly for C. jejuni) on one side and a limited power of the genotyping methods used to distinguish non-related strains on the other side (mainly for C. coli).     

Seasonal influence on the prevalence of thermotolerant Campylobacter in retail broiler meat in Denmark

In Denmark, the incidence of human campylobacteriosis cases, as well as the Campylobacter prevalence in broiler flocks, is strongly influenced by season with a summer peak in July-August. Therefore, it was considered that the prevalence of Campylobacter in broiler meat sold at retail in Denmark might also be influenced by season. A retrospective survey analysis was performed on 2001-2007 national surveillance data of the prevalence of thermotolerant Campylobacter in all conventional broiler flocks at slaughter, and in randomly sampled broiler meat at retail. There was a significant effect of season on the occurrence of Campylobacter in meat at retail; the largest effect was found for domestic chilled meat. Thus, the Campylobacter prevalence in Danish broiler flocks, which fluctuated with season, was found to be a strong predictor for the occurrence of Campylobacter in fresh, chilled, Danish broiler meat. However, besides flock prevalence, there was also a direct effect of season on the occurrence of Campylobacter in Danish broiler meat at retail.     

泡椒凤爪防腐保藏及栅栏因子调控研究

通过研究乳酸添加量、巴氏灭菌温度与灭菌时间对泡椒凤爪菌落总数和口感的影响,采用响应面法对泡椒凤爪加工工艺进行优化,确定泡椒凤爪的最佳加工工艺参数为乳酸添加量0.4mL,灭菌温度83℃,灭菌时间18min。在巴氏灭菌基础上,研究调控防腐剂、辐照及冷藏三种栅栏因子对产品保藏性的影响。三种保藏技术均能有效提高产品保藏性。在产品的加工、贮运及销售过程中,应结合不同栅栏因子的优势对产品保藏进行综合调控,以提高产品品质及营养安全性。      

Prevalence of salmonella on retail chicken meat in Russian Federation

The objective of this study was to estimate the prevalence of Salmonella on raw retail chicken meat in Russia. Broiler chicken carcasses (n = 698) were collected from three regions of Russia: central (i.e., Moscow area), northwest (i.e., St. Petersburg area), and southern (i.e., Krasnodar area). In each region, samples were collected to represent various cities and districts, as well as different types of retail stores and carcass storage temperatures (i.e., chilled and frozen). All chicken samples were analyzed for the presence of Salmonella using a whole-carcass rinse method. The overall Salmonella prevalence was 31.5%. There were significant differences (P < 0.05) in Salmonella prevalence by (i) region-29.3% (n = 464) in Moscow, 38.5% (n = 192) in St. Petersburg, and 23.8% (n = 42) in Krasnodar; (ii) retail store type-28.8% (n = 236) in hypermarkets, 31.9% (n = 260) in supermarkets (part of chain stores), 44.3% (n = 61) in independent supermarkets, 42.9% (n = 28) in independent minimarkets, and 26.6% (n = 113) in wet markets; and (iii) poultry company-34.3% (n = 545) on chickens produced by integrated companies compared with 22.9% (n = 118) on chickens produced by nonintegrated companies. Strategies such as good agriculture and management practices should be enhanced to reduce Salmonella prevalence on raw poultry in Russia and therefore increase the safety of chicken products.     

Prevalence, numbers, and subtypes of Campylobacter jejuni and Campylobacter coli in uncooked retail meat samples

A national quantitative survey of Campylobacter jejuni and Campylobacter coli in 1,011 uncooked retail meat samples (beef, unweaned veal, chicken, lamb and mutton, and pork) was undertaken from August 2003 to June 2004 to establish baseline proportionality data. The presence, number, and type of Campylobacter present in each sample was assessed. Prevalences of C. jejuni and C. coli were 89.1% in chicken, 9.1% in pork, 6.9% in lamb and mutton, 3.5% in beef, and 10% in unweaned veal. C. jejuni was identified in the majority of positive samples (246 of 259). In chicken samples positive for C. jejuni, 40.2% had counts of <0.3 most probable number (MPN)/g, 50.5% had 0.3 to 10.0 MPN/g, 8.8% had 10.1 to 50.0 MPN/g, and 0.5% had 110 MPN/g. In other meats (49 samples), Campylobacter counts were < or = 0.3 MPN/g, except for one unweaned veal sample at > 10.9 MPN/g. Penner serotyping and SmaI macrorestriction genotyping using pulsed-field gel electrophoresis with 247 isolates revealed 17 Penner serotypes and 56 electrophoresis profiles. Seven Penner serotypes (HS1 complex, 2, 4 complex, 6, 11, 27, and 42) were represented by 10 or more isolates from chicken. When data from both typing methods were combined, 62 sero-genotypes were generated. In a comparison of these sero-genotypes with historical data for isolates from human cases, 71% of the beef isolates, 50% of the lamb and mutton isolates, 50% of the pork isolates, 41% of the chicken isolates, and 25% of the unweaned veal isolates were common to both sources. These results provide baseline proportionality profiles of Campylobacter in these five meats and will facilitate exposure assessment in combination with other information such as consumption data and subsequent quantitative risk assessment.     

Antimicrobial resistance among Campylobacter jejuni isolated from raw poultry meat at retail level in Denmark

Campylobacter jejuni isolated from raw poultry meat collected at retail shops in Denmark in the period 1996-2003 were tested for susceptibility to seven antimicrobial agents. The food samples consisted of raw chicken meat and other raw poultry meat of domestic or imported origin. The highest levels of resistance among C. jejuni were observed for tetracycline, nalidixic acid and ciprofloxacin, whereas macrolide resistance was rarely detected. C. jejuni originating from other poultry meat (mainly duck and turkey meat) exhibited the highest occurrences of antimicrobial resistance monitored; approximately one third of the isolates were tetracycline resistant (N=100). Among chicken meat isolates, the occurrence of tetracycline resistance was significantly higher (P<0.005) in C. jejuni isolated from imported chicken meat (N=88) than in C. jejuni from Danish chicken meat (N=367). The same tendency was observed for chloramphenicol, nalidixic acid and ciprofloxacin (P<0.05). The trends in resistance in the period 1996-2003 among C. jejuni isolates from chicken meat indicate a decrease in the occurrence of resistance towards fluoroquinolones. This may be due to reduced application of fluoroquinolones for food animals. Monitoring of the occurrence of antimicrobial resistance in C. jejuni isolated from raw uncooked poultry has been performed on a yearly basis since 1996, thus providing useful insight into consumer exposure to antimicrobial-resistant C. jejuni.