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%.     

Baseline Data from a Belgium-Wide Survey of Campylobacter Species Contamination in Chicken Meat Preparations and Considerations for a Reliable Monitoring Program

From February to November 2007, chicken meat preparations (n = 656) were sampled at 11 processing companies across Belgium. All samples were tested for Campylobacter by enrichment culture and by direct plating according to standard methods. Almost half (48.02%) of the samples were positive for Campylobacter spp. The mean Campylobacter count was 1.68 log₁₀ CFU/g with a standard deviation of ± 0.64 log₁₀ CFU/g. The study revealed a statistically significant variation in Campylobacter contamination levels between companies; processors with a wider frequency distribution range of Campylobacter counts provided chicken meat preparations with higher Campylobacter incidences and concentrations. There was no significant difference between the counts of Campylobacter spp. in various preparation types. However, the Campylobacter counts and incidences in chicken wings were the highest and portioned-form products (legs, wings, and breasts) showed a higher probability of being Campylobacter positive compared to minced-form products (sausages, burgers, and minced meat). The proportion of Campylobacter-positive samples was significantly higher in July than in other months. Recovery of Campylobacter spp. recovery by direct plating was higher (41.0%) compared to detection after enrichment (24.2%). Statistical modeling of the survey data showed that the likelihood of obtaining a positive result by enrichment culture increases with an increase in the Campylobacter concentration in the sample. In the present study, we provide the first enumeration data on Campylobacter contamination in Belgian chicken meat preparations and address proposals for improving Campylobacter monitoring programs.     

Prevalence of Campylobacter spp., Escherichia coli, and Salmonella Serovars in Retail Chicken, Turkey, Pork, and Beef from the Greater Washington, D.C., Area

A total of 825 samples of retail raw meats (chicken, turkey, pork, and beef) were examined for the presence of Escherichia coli and Salmonella serovars, and 719 of these samples were also tested for Campylobacter spp. The samples were randomly obtained from 59 stores of four supermarket chains during 107 sampling visits in the Greater Washington, D.C., area from June 1999 to July 2000. The majority (70.7%) of chicken samples (n = 184) were contaminated with Campylobacter, and a large percentage of the stores visited (91%) had Campylobacter-contaminated chickens. Approximately 14% of the 172 turkey samples yielded Campylobacter, whereas fewer pork (1.7%) and beef (0.5%) samples were positive for this pathogen. A total of 722 Campylobacter isolates were obtained from 159 meat samples; 53.6% of these isolates were Campylobacter jejuni, 41.3% were Campylobacter coli, and 5.1% were other species. Of the 212 chicken samples, 82 (38.7%) yielded E. coli, while 19.0% of the beef samples, 16.3% of the pork samples, and 11.9% of the turkey samples were positive for E. coli. However, only 25 (3.0%) of the retail meat samples tested were positive for Salmonella. Significant differences in the bacterial contamination rates were observed for the four supermarket chains. This study revealed that retail raw meats are often contaminated with food-borne pathogens; however, there are marked differences in the prevalence of such pathogens in different meats. Raw retail meats are potential vehicles for transmitting food-borne diseases, and our findings stress the need for increased implementation of hazard analysis of critical control point (HACCP) and consumer food safety education efforts.     

Clonal Population Structure and Antimicrobial Resistance of Campylobacter jejuni in Chicken Meat from Belgium

Campylobacter jejuni is one of the most important causes of human diarrhea worldwide. In the present work, multilocus sequence typing was used to study the genotypic diversity of 145 C. jejuni isolates from 135 chicken meat preparations sampled across Belgium. Isolates were further typed by pulsed-field gel electrophoresis, and their susceptibilities to six antimicrobials were determined. Fifty-seven sequence types (STs) were identified; 26.8% of the total typed isolates were ST-50, ST-45, or ST-257, belonging to clonal complex CC-21, CC-45, or CC-257, respectively. One clonal group comprised 22% (32/145) of all isolates, originating from five different companies and isolated over seven sampling months. Additionally, 53.1% of C. jejuni isolates were resistant to ciprofloxacin, and 48.2% were resistant to tetracycline; 28.9% (42/145) of all isolates were resistant to both ciprofloxacin and tetracycline. The correlation between certain C. jejuni clonal groups and resistance to ciprofloxacin and tetracycline was notable. C. jejuni isolates assigned to CC-21 (n = 35) were frequently resistant to ciprofloxacin (65.7%) and tetracycline (40%); however, 90% (18/20) of the isolates assigned to CC-45 were pansusceptible. The present study demonstrates that certain C. jejuni genotypes recur frequently in the chicken meat supply. The results of molecular typing, combined with data on sample sources, indicate a possible dissemination of C. jejuni clones with high resistance to ciprofloxacin and/or tetracycline. Whether certain clonal groups are common in the environment and repeatedly infect Belgian broiler flocks or whether they have the potential to persist on farms or in slaughterhouses needs further investigation.Campylobacter jejuni is among the most common bacterial causes of human gastroenteritis worldwide (4, 23). Infected humans exhibit a range of clinical symptoms from mild, watery diarrhea to severe inflammatory diarrhea (14). In addition, C. jejuni has been identified as an important infectious trigger for Guillain-Barré syndrome, the most common cause of acute flaccid paralysis in polio-free regions (16). Another issue of concern regarding Campylobacter is the increase in antimicrobial resistance appearing in various regions around the world (1). Infection with an antimicrobial-resistant Campylobacter strain may lead to a suboptimal outcome of antimicrobial treatment or even to treatment failure (11).Consumption of contaminated water and raw milk has been implicated in campylobacteriosis outbreaks (23). However, the majority of human cases are sporadic, and consumption or mishandling of contaminated raw or undercooked poultry meat is believed to be an important source of infection. Risk assessment studies, outbreak investigations, and case-control reports all incriminate chicken meat as a major source, perhaps the major source, of food-borne transmission (14, 17, 32, 48). In Belgium in 1999, a controlled withdrawal of poultry products from sale due to alleged dioxin contamination resulted in a 40% reduction in the frequency of human campylobacteriosis (44). Thereafter and since the year 2000, the Campylobacter contamination of Belgian poultry carcasses and meat has been monitored by the Federal Agency for the Safety of the Food Chain, and the rate of positive samples is regarded as high. In 2006, 55.5% of cecal samples (n = 6,443) from Belgian broilers at slaughter tested positive for Campylobacter (3). In 2007, an industry-focused survey reported that 48% of Belgian chicken meat preparations (n = 656) were contaminated with Campylobacter (19).Molecular typing is an important tool in elucidating the diversity and transmission routes of Campylobacter isolates contaminating the food chain. In the United States, molecular analysis of Campylobacter spp. from poultry production and processing environments showed that many of the clones found within a flock are present in the final products, although the diversity of Campylobacter isolates in the final product was lower than that observed in the flock (22). Furthermore, numerous molecular epidemiological studies indicate that the genotypes of C. jejuni isolated from human cases overlap those of poultry origin (17, 47). Various molecular typing methods for the study of the population structure of Campylobacter are currently available (46). Among these, the multilocus sequence typing (MLST) approach is an emerging tool for research on the population structure and molecular epidemiology of Campylobacter. The technique is highly reproducible, portable, and easy to interpret, and results can be shared through a publicly accessible online database (31, 34). As such, MLST is becoming an important tool for studying the molecular epidemiology of Campylobacter in a global context. The accumulation of sequence typing data generated from different countries and settings could allow the creation of more-sophisticated models of the epidemiology and evolution of bacterial pathogens and the development of improved approaches for combating their spread (41).In Belgium, there is a paucity of information regarding the population structure of Campylobacter in the chicken meat supply. No population-based surveys have been conducted to investigate the molecular epidemiology of C. jejuni in chicken meat at points close to human consumption. In this study, MLST and pulsed-field gel electrophoresis (PFGE) were used to characterize the diversity of, and clonal relationships among, 145 C. jejuni isolates from Belgian chicken meat preparations. In addition, we characterized the antimicrobial resistance in this collection and correlated it with C. jejuni genotypes.     

Detection of Cytolethal Distending Toxin Activity and cdt Genes in Campylobacter spp. Isolated from Chicken Carcasses

This study was designed to determine whether isolates from chicken carcasses, the primary source of Campylobacter jejuni and Campylobacter coli in human infections, commonly carry the cdt genes and also whether active cytolethal distending toxin (CDT) is produced by these isolates. Campylobacter spp. were isolated from all 91 fresh chicken carcasses purchased from local supermarkets. Campylobacter spp. were identified on the basis of both biochemical and PCR tests. Of the 105 isolates, 70 (67%) were identified as C. jejuni, and 35 (33%) were identified as C. coli. PCR tests amplified portions of the cdt genes from all 105 isolates. Restriction analysis of PCR products indicated that there appeared to be species-specific differences between the C. jejuni and C. coli cdt genes, but that the restriction patterns of the cdt genes within strains of the same species were almost invariant. Quantitation of active CDT levels produced by the isolates indicated that all C. jejuni strains except four (94%) had mean CDT titers greater than 100. Only one C. jejuni strain appeared to produce no active CDT. C. coli isolates produced little or no toxin. These results confirm the high rate of Campylobacter sp. contamination of fresh chicken carcasses and indicate that cdt genes may be universally present in C. jejuni and C. coli isolates from chicken carcasses.     

Has Retail Chicken Played a Role in the Decline of Human Campylobacteriosis?

Between 2001 and 2006, the incidence of human Campylobacter infections decreased by 10 and 27% in Scotland and the Grampian region of Scotland, respectively. Contemporaneous collection and analyses of human and retail-chicken isolates from Grampian were carried out over a 10-week period in 2001 and again in 2006 in order to determine whether the fall in the incidence of human infections was related to the retail-chicken exposure route. Rates of carriage of Campylobacter on chicken carcasses from retail outlets in Grampian in 2001 and 2006 were estimated. Chicken-derived Campylobacter isolates from 2001 (n = 84) and 2006 (n = 105) and human-derived isolates from patients with clinical cases of infection in 2001 (n = 172) and 2006 (n = 119) were typed by multilocus sequence typing. We found no evidence for statistically significant changes in prevalence and counts per carcass. We found by rarefaction that although the degree of diversity in humans tended to be higher than that in chickens, these differences were not significant. The genetic distance between chicken and human isolates from 2001 according to sequence type, clonal complex (CC), or allele composition was not significant, whereas the distances between 2006 isolates at the CC and allele levels were significant. This difference was attributable to a lower proportion of CC-21's being found in retail-chicken isolates from 2006 than in chicken isolates from 2001. We conclude that human exposure to Campylobacter via retail chicken is important and that changes in the population structure of campylobacters in this reservoir need to be taken into account in investigating human infection.     

Epidemiology, Relative Invasive Ability, Molecular Characterization, and Competitive Performance of Campylobacter jejuni Strains in the Chicken Gut

One hundred forty-one Campylobacter jejuni isolates from humans with diarrhea and 100 isolates from retailed poultry meat were differentiated by flaA typing. The bacteria were isolated in a specific geographical area (Dunedin) in New Zealand over a common time period. Twenty nine flaA types were detected, one of which (flaA restriction fragment length polymorphism type 15 [flaA-15]) predominated among isolates from humans (~30% of isolates). This strain was of low prevalence (5% of isolates) among poultry isolates. flaA-15 strains were five to six times more invasive of HEp2 cells in an in vitro assay than a flaA type (flaA-3) that was commonly encountered on poultry meat (23% of isolates) but was seldom associated with human illness (5%). Competitive-exclusion experiments with chickens, utilizing real-time quantitative PCR to measure the population sizes of specific strains representing flaA-15 (T1016) and flaA-3 (Pstau) in digesta, were carried out. These experiments showed that T1016 always outcompeted Pstau in the chicken intestine. Genomic comparisons of T1016 and Pstau were made using DNA microarrays representing the genome of C. jejuni NCTC 11168. These comparisons revealed differences between the strains in the gene content of the Cj1417c-to-Cj1442c region of the genome, which is associated with the formation of capsular polysaccharide. The strains differed in Penner type (T1016, O42; Pstau, O53). It was concluded that poultry meat was at least one source of human infection with C. jejuni, that some Campylobacter strains detected in poultry meat are of higher virulence for humans than others, and that bacterial attributes affecting strain virulence and commensal colonization ability may be linked.     

Monitoring chicken flock behaviour provides early warning of infection by human pathogen Campylobacter

Campylobacter is the commonest bacterial cause of gastrointestinal infection in humans, and chicken meat is the major source of infection throughout the world. Strict and expensive on-farm biosecurity measures have been largely unsuccessful in controlling infection and are hampered by the time needed to analyse faecal samples, with the result that Campylobacter status is often known only after a flock has been processed. Our data demonstrate an alternative approach that monitors the behaviour of live chickens with cameras and analyses the ‘optical flow’ patterns made by flock movements. Campylobacter-free chicken flocks have higher mean and lower kurtosis of optical flow than those testing positive for Campylobacter by microbiological methods. We show that by monitoring behaviour in this way, flocks likely to become positive can be identified within the first 7–10 days of life, much earlier than conventional on-farm microbiological methods. This early warning has the potential to lead to a more targeted approach to Campylobacter control and also provides new insights into possible sources of infection that could transform the control of this globally important food-borne pathogen.     

Flock Health Indicators and Campylobacter spp. in Commercial Housed Broilers Reared in Great Britain

This study investigated the relationship between flock health and Campylobacter infection of housed commercial broilers in Great Britain. Thirty ceca were collected at slaughter from batches of broilers from 789 flocks, at either full or partial depopulation, between December 2003 and March 2006 and examined individually for Campylobacter by direct plating onto selective media. Management and health data were collected from each flock and included information on mortality or culling during rearing, the number of birds rejected for infectious or noninfectious causes at slaughter, the proportion of birds with digital dermatitis (also termed hock burn), and other general characteristics of the flock. Campylobacter spp. were isolated from 280 (35%) flocks. The relationship between bird health and welfare and Campylobacter status of flocks was assessed using random-effects logistic regression models, adjusting for region, month, year, and rearing regime. Campylobacter-positive batches of ceca were associated with higher levels of rejection due to infection (odds ratio [OR], 1.5; 95% confidence interval [CI_95%], 0.98 to 2.30) and digital dermatitis (OR, 2.08; CI_95%, 1.20 to 3.61). Furthermore, higher levels of these conditions were also associated with the highest-level category of within-flock Campylobacter prevalence (70 to 100%). These results could indicate that improving health and welfare may also reduce Campylobacter in broilers.     

Decreasing Trend of Overlapping Multilocus Sequence Types between Human and Chicken Campylobacter jejuni Isolates over a Decade in Finland

We describe the long-term multilocus sequence typing (MLST) analysis of the population structure and dynamics of 454 Finnish human Campylobacter jejuni isolates, as well as 208 chicken isolates, collected during the mid-1990s to 2007. The sequence type clonal complexes (ST CC) ST-45 CC, ST-21 CC, and ST-677 CC were the most common ones found among all isolates, and they covered 73.9% of all isolates. The ST-283 CC also was found frequently among chicken isolates (8.2%). The predominant STs among all isolates were ST-45, ST-50, and ST-677. ST-137 and ST-230 were common among human isolates, and ST-267 was found more frequently among chicken isolates than human isolates. The ST-45 CC was significantly associated with chicken isolates (P < 0.01), whereas the ST-21 CC was associated with human isolates (P < 0.001). The ST-677 CC was not associated with any host (P = 0.5), and an opposite temporary trend of this complex was seen among chicken and human isolates, with an increase in the former and a decrease in the latter during the study period. Furthermore, the ST-22 and ST-48 CCs were significantly associated with human isolates (P < 0.01), but neither of the CCs was found in chicken isolates. The annual overlap between STs from human and chicken isolates decreased from 76% at the beginning of the study to 58% at the end. Our results suggest that the importance of chicken as a reservoir for strains associated with human infections has declined despite the consumption of domestic chicken meat increasing during the follow-up period by 83%.Campylobacter jejuni is the most common bacterial cause of gastroenteritis worldwide. In Finland, registered human Campylobacter infections have increased from 2,629 cases in 1996 to 4,107 cases in 2007 (http://www3.ktl.fi/stat/). Of the 4,107 cases in 2007, 45% were registered in the Helsinki-Uusimaa region, where the annual incidence was highest (122/100,000; the national average is 78/100,000) as well. Most of the cases were associated with traveling to other countries. In 2007, approximately 57% of the patients had traveled outside of Finland prior to their illness, 19% had not traveled abroad, and for 24% information was unavailable (http://www.ktl.fi/attachments/suomi/julkaisut/julkaisusarja_b/2008/2008b09.pdf).In Finland, most of the Campylobacter infections are sporadic and appear during the summer months (http://www.efsa.europa.eu/en/scdocs/doc/130r.pdf). Between 1996 and 2007, approximately 45 to 55% of all registered infections were reported between June and September (http://www3.ktl.fi/stat/). In contrast to sporadic Campylobacter infections, outbreaks of campylobacteriosis are uncommon, usually occurring in spring or autumn, and are associated with drinking contaminated water (36).Epidemiological studies performed in many countries indicate that handling or eating chicken meat is an important risk factor for the acquisition of campylobacteriosis (32, 46). A recent Finnish case-control study (39) identified swimming in lakes and rivers and drinking water from private wells as additional risk factors for acquiring the illness from domestic sources during the summer months. Contact with pets (19) and farm animals (21) and the consumption of raw milk (35) also have been identified as risk factors for Campylobacter infections.The annual consumption of chicken meat in Finland increased by 83% (from 53 million to 95 million kg) from 1997 to 2007 but remained stable, at around 83.5 million kg, between 2002 and 2006. However, from 2006 to 2007, the consumption of chicken meat increased by a further 8% (from 88 million to 95 million kg). Most of the chicken meat consumed in Finland comes from domestic production (https://portal.mtt.fi/portal/page/portal/mtt/mtt/julkaisut/suomenmaatalousjamaaseutuelinkeinot/jul108_SM2008.pdf). Finnish chicken flocks have been monitored for Campylobacter spp. according to the regulations of the European Union since 2004 (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2003:325:0031:0040:EN:PDF). Seasonality similar to that observed in human infections can be found in the prevalence of Campylobacter-positive chicken slaughter batches, i.e., 7.7% of all chicken slaughter batches were Campylobacter positive from June to October in 2007; however, during the rest of the year no positive chicken flocks were detected (http://www.efsa.europa.eu/en/scdocs/doc/130r.pdf).To assess the relevance of a particular source and potential routes of transmission from animals to humans (6), the overlap between genotypes of Campylobacter isolates from patients and potential sources of infection has been studied using molecular typing techniques such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Of the two typing methods, PFGE is more discriminatory and therefore is considered more suitable for short-term epidemiological investigations and for the determination of the source of infection in outbreak situations (24, 26, 28).In our previous studies, we have shown that during a seasonal peak, overlapping serotypes and PFGE genotypes exist between samples from patients suffering from campylobacteriosis and fecal samples from chicken (13, 17). Several other studies also have reported a 34 to 60% overlap between serotypes, genotypes, and/or sequence types (STs) of patient and poultry isolates using various typing techniques (20, 31, 37, 42).Due to the high diversity of types and the lack of standardized nomenclature, PFGE is not a useful technique in long-term epidemiological studies. Unlike PFGE, MLST has been successfully used in long-term epidemiological studies (27, 45) and in deciphering the population structure (2, 20, 28, 45) of Campylobacter on a global scale. MLST has high discriminatory power (23) and standardized nomenclature for STs and clonal complexes (CCs) (3, 26).The aim of our study was to analyze by MLST the overlap and dynamics of C. jejuni types among isolates collected from domestically acquired sporadic human infections from the Helsinki-Uusimaa region from 1996 to 2006 and isolates collected from domestic chicken production from 1999 to 2007.     

Influx of Enterococci and Associated Antibiotic Resistance and Virulence Genes from Ready-To-Eat Food to the Human Digestive Tract

The influx of enterococcal antibiotic resistance (AR) and virulence genes from ready-to-eat food (RTEF) to the human digestive tract was assessed. Three RTEFs (chicken salad, chicken burger, and carrot cake) were sampled from five fast-food restaurants five times in summer (SU) and winter (WI). The prevalence of enterococci was significantly higher in SU (92.0% of salad samples and 64.0% of burger samples) than in WI (64.0% of salad samples and 24.0% of burger samples). The overall concentrations of enterococci during the two seasons were similar (~10³ CFU/g); the most prevalent were Enterococcus casseliflavus (41.5% of isolates) and Enterococcus hirae (41.5%) in WI and Enterococcus faecium (36.8%), E. casseliflavus (27.6%), and Enterococcus faecalis (22.4%) in SU. Resistance in WI was detected primarily to tetracycline (50.8%), ciprofloxacin (13.8%), and erythromycin (4.6%). SU isolates were resistant mainly to tetracycline (22.8%), erythromycin (22.1%), and kanamycin (13.0%). The most common tet gene was tet(M) (35.4% of WI isolates and 11.9% of SU isolates). The prevalence of virulence genes (gelE, asa1, cylA, and esp) and marker genes for clinical isolates (EF_0573, EF_0592, EF_0605, EF_1420, EF_2144, and pathogenicity island EF_0050) was low (≤12.3%). Genotyping of E. faecalis and E. faecium using pulsed-field gel electrophoresis revealed that the food contamination likely originated from various sources and that it was not clonal. Our conservative estimate (single AR gene copy per cell) for the influx of tet genes alone to the human digestive tract is 3.8 × 10⁵ per meal (chicken salad). This AR gene influx is frequent because RTEFs are commonly consumed and that may play a role in the acquisition of AR determinants in the human digestive tract.     

Cross-contamination in the kitchen: estimation of transfer rates for cutting boards, hands and knives

Aims: To quantify cross-contamination in the home from chicken to ready-to-eat salad. Methods and Results: Based on laboratory scenarios performed by de Jong et al. (2008) , transfer rates were estimated for Campylobacter jejuni and Lactobacillus casei as a tracer organism. This study showed that transfer characteristics for both micro-organisms were comparable when washing regimes and transfer via items (cutting board, hands and knives) were compared. Furthermore, the study showed that the use of separate transfer rates for transfer from chicken to items and from items to salad will lead to an overestimation of campylobacteriosis risk. Applying good hygienic practices resulted in final levels of bacteria in the salad below the detection limit. Our study showed that it is important to include these data points in model fitting. Conclusions: Results obtained in observational studies with Lact. casei can be translated to Camp. jejuni using the transfer rates obtained in this study. Cross-contamination by hands, cutting boards and knives was equally important. Significance and Impact of the Study: Cross-contamination should be incorporated in microbiological risk assessments. The present study contributes to this by quantifying transfer of Camp. jejuni and Lact. casei from raw chicken via various contact surfaces into the ready-to-eat product.     

Isolation and Characterization of Campylobacter Bacteriophages from Retail Poultry

The ability of phages to survive processing is an important aspect of their potential use in the biocontrol of Campylobacter in poultry production. To this end, we have developed a procedure to recover Campylobacter bacteriophages from chilled and frozen retail poultry and have validated the sensitivity of the method by using a characterized Campylobacter phage (i.e., NCTC 12674). By using this method, we have shown that Campylobacter phages can survive on retail chicken under commercial storage conditions. Retail chicken portions purchased in the United Kingdom were screened for the presence of endogenous Campylobacter phages. Thirty-four Campylobacter bacteriophages were isolated from 300 chilled retail chicken portions, but none could be recovered from 150 frozen chicken portions. The phage isolates were characterized according to their lytic profiles, morphology, and genome size. The free-range products were significantly more likely to harbor phages (P < 0.001 by single-factor analysis of variance) than were standard or economy products. This study demonstrates that Campylobacter bacteriophages, along with their hosts, can survive commercial poultry processing procedures and that the phages exhibited a wide range of recovery rates from chicken skin stored at 4°C.     

Contamination of Foods by Food Handlers: Experiments on Hepatitis A Virus Transfer to Food and Its Interruption

Hepatitis A virus (HAV) is an important pathogen which has been responsible for many food-borne outbreaks. HAV-excreting food handlers, especially those with poor hygienic practices, can contaminate the foods which they handle. Consumption of such foods without further processing has been known to result in cases of infectious hepatitis. Since quantitative data on virus transfer during contact of hands with foods is not available, we investigated the transfer of HAV from artificially contaminated fingerpads of adult volunteers to pieces of fresh lettuce. Touching the lettuce with artificially contaminated fingerpads for 10 s at a pressure of 0.2 to 0.4 kg/cm² resulted in transfer of 9.2% ± 0.9% of the infectious virus. The pretreatments tested to interrupt virus transfer from contaminated fingerpads included (i) hard-water rinsing and towel drying, (ii) application of a domestic or commercial topical agent followed by water rinsing and towel drying, and (iii) exposure to a hand gel containing 62% ethanol or 75% liquid ethanol without water rinsing or towel drying. When the fingerpads were treated with the topical agents or alcohol before the lettuce was touched, the amount of infectious virus transferred to lettuce was reduced from 9.2% to between 0.3 and 0.6% (depending on the topical agent used), which was a reduction in virus transfer of up to 30-fold. Surprisingly, no virus transfer to lettuce was detected when the fingerpads were rinsed with water alone before the lettuce was touched. However, additional experiments with water rinsing in which smaller volumes of water were used (1 ml instead of 15 ml) showed that the rate of virus transfer to lettuce was 0.3% ± 0.1%. The variability in virus transfer rates following water rinsing may indicate that the volume of water at least in part influences virus removal from the fingerpads differently, a possibility which should be investigated further. This study provided novel information concerning the rate of virus transfer to foods and a model for investigating the transfer of viral and other food-borne pathogens from contaminated hands to foods, as well as techniques for interrupting such transfer to improve food safety.     

Effects of Polyphosphate Additives on Campylobacter Survival in Processed Chicken Exudates

Campylobacter spp. are responsible for a large number of the bacterial food poisoning cases worldwide. Despite being sensitive to oxygen and nutritionally fastidious, Campylobacter spp. are able to survive in food processing environments and reach consumers in sufficient numbers to cause disease. To investigate Campylobacter persistence on processed chicken, exudates from chickens produced for consumer sale were collected and sterilized. Two types of exudates from chicken products were collected: enhanced, where a marinade was added to the chickens during processing, and nonenhanced, where no additives were added during processing. Exudates from enhanced chicken products examined in this study contained a mixture of polyphosphates. Exudate samples were inoculated with Campylobacter jejuni or Campylobacter coli strains and incubated under a range of environmental conditions, and viable bacteria present in the resultant cultures were enumerated. When incubated at 42°C in a microaerobic environment, exudates from enhanced chicken products resulted in increased survival of C. jejuni and C. coli compared with that in nonenhanced exudates in the range of <1 to >4 log CFU/ml. Under more relevant food storage conditions (4°C and normal atmosphere), the exudates from enhanced chicken products also demonstrated improved Campylobacter survival compared with that in nonenhanced exudates. Polyphosphates present in the enhanced exudates were determined to be largely responsible for the improved survival observed when the two types of exudates were compared. Therefore, polyphosphates used to enhance chicken quality aid in sustaining the numbers of Campylobacter bacteria, increasing the opportunity for disease via cross-contamination or improperly cooked poultry.Campylobacter species are the major causative agent of food-borne gastrointestinal bacterial infections in the developed world (6, 11, 21). Poultry products are a major source for the introduction of Campylobacter into the food supply (15, 16). Improperly cooked poultry and cross-contamination of other foods by raw poultry are common methods for transmission of Campylobacter to humans (5). However, Campylobacter spp. are nutritionally fastidious organisms that are sensitive to the oxygen levels present in a normal environment (O₂ = 20.9%) (21). Therefore, Campylobacter appears an unlikely candidate to persist within poultry processing and storage environments at levels sufficient to cause human disease. This conundrum directly leads to a question: what then are the elements that contribute to the ability of Campylobacter to survive through poultry processing and cold storage?To investigate this question, a food-relevant environment consisting of chicken weepage or exudate can be used to perform survival experiments on Campylobacter species. Strains of Campylobacter jejuni and Campylobacter coli were used for the survival studies since these two species are responsible for the vast majority of human cases of campylobacteriosis (20, 28). Chicken exudate is the fluid that seeps out from processed poultry carcasses and is often found to be contaminated with considerable numbers of Campylobacter bacteria. It is comprised of water, blood, fats, and other materials added to the poultry during processing. Sterilized poultry exudates make for a convenient experimental material that is also relevant to the conditions which Campylobacter will experience as a contaminant of processed poultry (2, 3). Two different types of chicken exudates were collected from commercial producers, one from chickens processed without additives (nonenhanced) and the other from chickens that were treated with a commercial marinade to increase the quality and appeal of the meat at market (enhanced). The commercial poultry marinades contain a significant amount of polyphosphate additives. Polyphosphates comprise a group of food additives that are utilized within poultry processing to enhance the moisture absorbance, color, and flavor and to reduce product shrinkage of poultry (24, 29-32). Polyphosphates have also been shown to have an antimicrobial effect on several different bacterial species (8, 10, 12). The goal of the research was to determine if polyphosphates contribute to the ability of Campylobacter to survive and persist through the supply chain, thus directly increasing the opportunity for Campylobacter-mediated food poisoning of consumers.     

Campylobacter Infection as a Trigger for Guillain-Barré Syndrome in Egypt

Background

Most studies of Campylobacter infection triggering Guillain-Barré Syndrome (GBS) are conducted in western nations were Campylobacter infection and immunity is relatively rare. In this study, we explored Campylobacter infections, Campylobacter serotypes, autoantibodies to gangliosides, and GBS in Egypt, a country where Campylobacter exposure is common.

Methods

GBS cases (n = 133) were compared to age- and hospital-matched patient controls (n = 374). A nerve conduction study was performed on cases and a clinical history, serum sample, and stool specimen obtained for all subjects.

Results

Most (63.3%) cases were demyelinating type; median age four years. Cases were more likely than controls to have diarrhea (29.5% vs. 22.5%, Adjusted Odds Ratio (ORa) = 1.69, P = 0.03), to have higher geometric mean IgM anti-Campylobacter antibody titers (8.18 vs. 7.25 P<0.001), and to produce antiganglioside antibodies (e.g., anti-Gd1a, 35.3 vs. 11.5, ORa = 4.39, P<0.0001). Of 26 Penner:Lior Campylobacter serotypes isolated, only one (41:27, C. jejuni, P = 0.02) was associated with GBS.

Conclusions

Unlike results from western nations, data suggested that GBS cases were primarily in the young and cases and many controls had a history of infection to a variety of Campylobacter serotypes. Still, the higher rates of diarrhea and greater antibody production against Campylobacter and gangliosides in GBS patients were consistent with findings from western countries.     

Spatiotemporal Homogeneity of Campylobacter Subtypes from Cattle and Sheep across Northeastern and Southwestern Scotland

Source attribution using molecular subtypes has implicated cattle and sheep as sources of human Campylobacter infection. Whether the Campylobacter subtypes associated with cattle and sheep vary spatiotemporally remains poorly known, especially at national levels. Here we describe spatiotemporal patterns of prevalence, bacterial enumeration, and subtype composition in Campylobacter isolates from cattle and sheep feces from northeastern (63 farms, 414 samples) and southwestern (71 farms, 449 samples) Scotland during 2005 to 2006. Isolates (201) were categorized as sequence type (ST), as clonal complex (CC), and as Campylobacter jejuni or Campylobacter coli using multilocus sequence typing (MLST). No significant difference in average prevalence (cattle, 22%; sheep, 25%) or average enumeration (cattle, 2.7 × 10⁴ CFU/g; sheep, 2.0 × 10⁵ CFU/g) was found between hosts or regions. The four most common STs (C. jejuni ST-19, ST-42, and ST-61 and C. coli ST-827) occurred in both hosts, whereas STs of the C. coli ST-828 clonal complex were more common in sheep. Neither host yielded evidence for regional differences in ST, CC, or MLST allele composition. Isolates from the two hosts combined, categorized as ST or CC, were more similar within than between farms but showed no further spatiotemporal trends up to 330 km and 50 weeks between farm samples. In contrast, both regions yielded evidence for significant differences in ST, CC, and allele composition between hosts, such that 65% of isolates could be attributed to a known host. These results suggest that cattle and sheep within the spatiotemporal scales analyzed are each capable of contributing homogeneous Campylobacter strains to human infections.Campylobacter species are the largest cause of bacterial intestinal infection in the developed and developing world (3). Almost all reported human Campylobacter infections in the United Kingdom are caused by Campylobacter jejuni, which accounts for approximately 92% of cases, and Campylobacter coli, which accounts for most of the rest (8). Campylobacter species are carried asymptomatically in a wide range of host animals and excreted into the environment in feces (23). Humans can be infected by several routes including consumption of contaminated water (14) or food (23); indeed, case control studies indicate that consumption of poultry meat is a risk factor (11, 12, 28), but other foods including unpasteurized milk (33) and meat from cattle and sheep contaminated at the abattoir might be important (30).Cattle and sheep on farms are major hosts of Campylobacter, with up to 89% of cattle herds (31) and up to 55% of sheep flocks (26) being infected. The prevalence of C. jejuni and C. coli combined, estimated at the level of individual animals from fecal specimens, is 23 to 54% in cattle (22, 25) and up to 20% in sheep (37). Campylobacter enumeration in feces shed from individual animals ranges from <10² to 10⁷ CFU/g in both hosts (31), and the concentration shed varies with time. Meat products of cattle and sheep, by contrast, have generally lower levels of Campylobacter contamination. Prevalence values are 0.5 to 4.9% in surveys of retail beef (11a, 17, 36) and 6.9 to 12.6% in surveys of retail lamb and mutton (17, 35).Clinical Campylobacter strains can be attributed to infection sources in animals by comparing subtype frequencies in clinical cases with those in different candidate sources, including cattle, sheep, pigs, and the physical environment. Campylobacter subtype data sets are most transferable when subtypes are defined as sequence type (ST) using multilocus sequence typing (MLST). Three recent MLST-based studies based in northwestern England (34), mainland Scotland (29), northeastern Scotland (32), and New Zealand (24) have used source attribution models to infer the source of human clinical infection. The results suggest that retail chicken is the source with the highest (55 to 80%) attribution while cattle and sheep combined are ranked second (20 to 40%). These attribution models require further empirical validation but appear to be showing broadly similar results.Attribution of human Campylobacter infections to cattle and sheep raises the question of whether Campylobacter subtypes infecting farm cattle and sheep are generally homogeneous or tend to have spatiotemporal structure. Regarding spatial differences, isolates of C. jejuni from a 100-km² study of farmland area with dairy cattle and sheep in northwestern England displayed increased genetic similarity up to 1 km apart but no further trend over distances of 1 to 14 km apart (7), and isolates from three dairy cattle farms 2 or 5 km apart in the same area demonstrated differences in the frequencies of strains of clonal complexes (CCs) ST-42 and ST-61 (15). Regarding temporal differences, isolates of C. jejuni from five dairy cattle farms in the same area demonstrated differences in the frequency of strains of CC ST-61 between the spring and summer of 2003 (15). Lastly, regarding host-associated strains, STs of CCs ST-21, ST-42, and ST-61 are associated with cattle, and the more limited data for STs from sheep also show the presence of ST-21 and ST-61 (7, 15).The larger-scale spatiotemporal structure of Campylobacter strains from cattle and sheep is poorly known. The main aims of the present study were (i) to characterize C. jejuni and C. coli from cattle and sheep from two distinct geographical Scottish regions in terms of Campylobacter prevalence and enumeration and C. jejuni and C. coli ST composition and (ii) to estimate the extent of host association of C. jejuni and C. coli STs from cattle versus sheep.     

Killing of Campylobacter on contaminated plastic and wooden cutting boards by glycerol monocaprate (monocaprin)

Aims: Contamination in the kitchen with foodborne bacteria is a risk factor in human exposure to these pathogens, an important route being transfer of bacteria from contaminated cutting boards and other surfaces to humans. The aim of this study was to test microbicidal emulsions of glycerol monocaprate (monocaprin) against Campylobacter on contaminated cutting boards. Methods and Results: Plastic and wooden cutting boards, soiled with meat juice heavily contaminated with Campylobacter, were treated for 2 min with emulsions of monocaprin (MC) made in water or in buffer at low pH. Viable Campylobacter counts were reduced below the detectable level on plastic board surfaces after treatment with MC emulsions with or without 1·25% washing‐up liquids (WUL). The counts were also greatly reduced on wooden boards (P < 0·05). Conclusions: Monocaprin emulsions and mixtures of MC emulsions and WUL may be useful as sanitizers/disinfectants in kitchens and in other food preparing and processing facilities. Significance and Impact of the Study: Cleaning with MC emulsions with or without WUL may reduce the risk of human exposure to Campylobacter.     

Rapid host switching in generalist Campylobacter strains erodes the signal for tracing human infections

Campylobacter jejuni and Campylobacter coli are the biggest causes of bacterial gastroenteritis in the developed world, with human infections typically arising from zoonotic transmission associated with infected meat. Because Campylobacter is not thought to survive well outside the gut, host-associated populations are genetically isolated to varying degrees. Therefore, the likely origin of most strains can be determined by host-associated variation in the genome. This is instructive for characterizing the source of human infection. However, some common strains, notably isolates belonging to the ST-21, ST-45 and ST-828 clonal complexes, appear to have broad host ranges, hindering source attribution. Here whole-genome sequencing has the potential to reveal fine-scale genetic structure associated with host specificity. We found that rates of zoonotic transmission among animal host species in these clonal complexes were so high that the signal of host association is all but obliterated, estimating one zoonotic transmission event every 1.6, 1.8 and 12 years in the ST-21, ST-45 and ST828 complexes, respectively. We attributed 89% of clinical cases to a chicken source, 10% to cattle and 1% to pig. Our results reveal that common strains of C. jejuni and C. coli infectious to humans are adapted to a generalist lifestyle, permitting rapid transmission between different hosts. Furthermore, they show that the weak signal of host association within these complexes presents a challenge for pinpointing the source of clinical infections, underlining the view that whole-genome sequencing, powerful though it is, cannot substitute for intensive sampling of suspected transmission reservoirs.     

Antimicrobial Resistance in Salmonella enterica Serovar Heidelberg Isolates from Retail Meats, Including Poultry, from 2002 to 2006

Salmonella enterica serovar Heidelberg frequently causes food-borne illness in humans. There are few data on the prevalence, antimicrobial susceptibility, and genetic diversity of Salmonella serovar Heidelberg isolates in retail meats. We compared the prevalences of Salmonella serovar Heidelberg in a sampling of 20,295 meats, including chicken breast (n = 5,075), ground turkey (n = 5,044), ground beef (n = 5,100), and pork chops (n = 5,076), collected during 2002 to 2006. Isolates were analyzed for antimicrobial susceptibility and compared genetically using pulsed-field gel electrophoresis (PFGE) and PCR for the bla_CMY gene. A total of 298 Salmonella serovar Heidelberg isolates were recovered, representing 21.6% of all Salmonella serovars from retail meats. One hundred seventy-eight (59.7%) were from ground turkey, 110 (36.9%) were from chicken breast, and 10 (3.4%) were from pork chops; none was found in ground beef. One hundred ninety-eight isolates (66.4%) were resistant to at least one compound, and 49 (16.4%) were resistant to at least five compounds. Six isolates (2.0%), all from ground turkey, were resistant to at least nine antimicrobials. The highest resistance in poultry isolates was to tetracycline (39.9%), followed by streptomycin (37.8%), sulfamethoxazole (27.7%), gentamicin (25.7%), kanamycin (21.5%), ampicillin (19.8%), amoxicillin-clavulanic acid (10.4%), and ceftiofur (9.0%). All isolates were susceptible to ceftriaxone and ciprofloxacin. All ceftiofur-resistant strains carried bla_CMY. PFGE using XbaI and BlnI showed that certain clones were widely dispersed in different types of meats and meat brands from different store chains in all five sampling years. These data indicate that Salmonella serovar Heidelberg is a common serovar in retail poultry meats and includes widespread clones of multidrug-resistant strains.