Campylobacter jejuni: exposure assessment and hazard characterization : growth, survival and infectivity of Campylobacter jejuni

Campylobacter jejuni, a small, curved or spirally shaped highly motile microorganism, is identified as a major cause of bacterial gastroenteritis throughout the world. Serious complications such as the Guillain-Barré syndrome and reactive arthritis might occasionally follow infection. In this thesis data were generated in the fields of hazard characterization and exposure assessment, which were used to identify risk factors for Campylobacter. Although C. jejuni is generally seen as an obligate micro-aerophile, in this thesis it was shown that it can grow aerobically in the presence of pyruvate, and growth might also be possible in the presence of other antioxidants. However, as result of the strict minimal growth temperature (30ºC), C. jejuni is still assumed to be generally unable to grow in foods and therefore growth is not considered as a large risk for campylobacteriosis. C. jejunican, and does, survive effectively for long periods of time under non-growing conditions in various environments. Temperature is the most influencing factor in survival and survival will be optimal at low temperatures (around 4ºC). As many foods are stored chilled, this will prolong the survival of C. jejuni. Furthermore survival is enhanced at low oxygen conditions, as often present in packaged foods. While the effects of environmental conditions on the survival of C. jejuni have been studied extensively, the knowledge of the effect of environmental factors on the infectivity was scarce. Therefore, the effect of environmental conditions (temperature, medium and atmosphere) on both the survival of C. jejuni and the infectivity of the surviving cells was investigated. We revealed that culturability and infectivity are linearly related. Furthermore, our study on the effect on the infectivity of adding nonculturable C. jejuni cells to culturable cell suspensions, showed nonculturable cells not to be infective (in vitro). Therefore, absence of culturable C. jejuni cells indicates that a product can be regarded as representing a very low risk with respect to campylobacteriosis. Food products can be contaminated with Campylobacter during production or afterwards at the consumer’s home. Once contaminated the risk is high that Campylobacter will survive on food products until the moment of consumption, except if the product undergoes an elimination step during preparation, such as heating. The growth requirements for Campylobacter are met in the gastrointestinal tract of warm-blooded animals. As a result especially products of animal origin, like chicken meat and raw milk, are often contaminated by Campylobacter, but also products of non-animal origin, like vegetables and fruits, can be contaminated. Within the exposure assessment cross-contamination in the home, the importance of raw vegetables and fruits as a risk factor for Campylobacter, and the relative importance of chicken meat, raw milk and raw vegetables and fruits were studied. The effect of different cross-contamination routes during the preparation of a chicken fruit salad was investigated. It was shown that the mean transfer rate by cross-contamination was 0.12% of the initial number of C. jejunion the chicken fillet, and that the different tested cross-contamination routes; cutlery, cutting board and hands, were equally important. The high prevalence and concentration of C. jejuni on chicken meat, the high percentage of consumers who exhibit improper hygienic and cleaning behaviour, and the significant transfer of Campylobacter during improper food preparation, indicate cross-contamination from raw foods, such as chicken, to other ready-to-eat foods as a large risk factor for Campylobacter infection. Fresh produce can become contaminated with C. jejuni during cultivation or processing. Especially vegetables and fruits which are eaten raw may present a risk. In this thesis Dutch data on the prevalence of Campylobacter on raw vegetables and fruits were newly analysed. Thirteen of the 5640 vegetable and fruit samples were Campylobacter positive, resulting in a prevalence of 0.23% (95% confidence interval (Cl): 0.12-0.39%). The prevalence on packaged products was significantly higher than on unpackaged products. Finally, the newly acquired data and quantitative literature data on the prevalence and concentration of Campylobacter entering the consumer phase were summarized for three food groups; vegetables and fruit, chicken, and raw milk. These data were used in a quantitative risk assessment model to estimate the exposure and the number of illnesses as result of the consumption of these foods and to compare their relative importance. The prevalence and concentration are low on raw vegetables and fruit, but the quantity of raw vegetables and fruits consumed is very high. In contrast, raw chicken and raw milk show relative high prevalence and concentration levels, but the consumption is low. Despite the differences in parameters, the exposure calculated out of the combination of parameters indicates all these three foods as high risk factors for Campylobacter. So foods which are not heavily contaminated with Campylobacter, but are consumed in large quantities can constitute a similar high risk on Campylobacter infection as foods which are consumed in small quantities but are heavily contaminated with Campylobacter.</p

De invloed van omgevingsfactoren op de infectiviteit van niet-delende cultures van Campylobacter jejuni

Campylobacter jejuni is de meest frequente bacteriele veroorzaker van voedselinfecties. Onder omstandigheden waarbij C. jejuni niet kan groeien, bijvoorbeeld bij temperaturen lager dan 30 graden C, verliest de bacterie zijn kweekbaarheid, maar lijkt de bacterie niet dood te gaan. In deze studie is onderzocht of een niet-kweekbare C. jejuni een infectie kan veroorzaken, en of de omstandigheden waaronder de kweekbaarheid verloren is gegaan van invloed zijn op een eventuele infectie. Gedurende 40 dagen zijn kweekbaarheid, levensvatbaarheid en vermogen om een infectie te veroorzaken bepaald van C. jejuni die was opgeslagen in twee verschillende media bij drie verschillende temperaturen. Wanneer opgeslagen in een nutrient-arm medium bij lage temperatuur (4 graden C) bleek C. jejuni langer zijn kweekbaarheid te behouden dan bij hogere temperaturen (12 graden C, 25 graden C) of in een rijk medium. Bacterien bleven onder alle condities levensvatbaar. De mate van adhesie en invasie van de opgeslagen cultures nam af in de tijd; bij lage temperatuur in buffer behielden de bacterien het langst hun vermogen om aan darmepitheel- cellen te hechten of om erin binnen te dringen. Terwijl de infectiviteit van de totale cultuur afnam, nam de infectiviteit per kweekbare bacterie in die cultuur toe gedurende de eerste 5 dagen om daarna weer af te nemen. Dit resultaat suggereert dat een niet-kweekbare C. jejuni infectieus kan zijn. Echter, er kan ook een alternatieve verklaring zijn, namelijk dat er slechts een beperkt aantal specifieke bindingsplaatsen voor de hechting van C. jejuni aanwezig zijn. De eventuele aanwezigheid van specifieke bindingsplaatsen voor Campylobacter heeft mogelijk grote gevolgen voor onderzoek aan de invloed van niet-kweekbare cellen. Als niet-kweekbare cellen niet meer infectief zijn, maar wel kunnen hechten aan een bindingsplaats, dan kunnen zij met kweekbare cellen concurreren om een bindingsplaats. Dit zou een geheel nieuw rol betekenen voor niet-kweekbare cellen: niet-kweekbare cellen zouden dan de kans op een infectie verlagen!Campylobacter (C.) jejuni is a causative agent of gastro-enteritis. Growth of this Gram negative bacterium is restricted to a limited number of environmental conditions. Under conditions where growth of C. jejuni is not possible, e.g. at temperatures below 30 degrees C, C. jejuni can remain viable for a certain period of time, but its culturability decreases. In this study we examined the effect of this loss of culturability on the infectivity in cell-lines. We hypothesised that if non-culturable cells affect the process of infection then the infectivity per culturable cell used in the infection assay is not constant, whereas if only culturable cell are infectious then the infectivity per culturable cell is. C. jejuni was stored for 40 days in nutrient poor and nutrient rich medium at three temperatures. During this period we monitored its culturability, viability and infectivity. A decrease in culturability in time was observed. At low temperature (4 degrees C) and in nutrient poor medium cells remained culturable for longer periods than at higher temperatures (12 degrees C, 25 degrees C) or in nutrient rich medium. Non-culturable cells remained viable, as determined by tetrazolium chloride staining. The absolute level of adhesion and invasion showed a decrease in time. At low temperatures and in nutrient poor medium cells retained their capability to adhere and invade longer than at higher temperatures or in nutrient rich medium. While the absolute level of adhesion and invasion decreased, the infectivity per culturable cell added seemed to increase during the first 5 days of storage at 4 degrees C. This result confirmed our hypothesis that non-culturable C. jejuni can affect the process of infection. However, the result can also be explained by the presence of a limited number of bindingsites for C. jejuni in our testmodel. This suggested existence of bindingsites has great importance for the research into the influence of non-culturable cells on the infectivity. If non-culturable cells are not infectious but can block bindingsites, then they can play a protective role by competing with the culturable cells for the limited number of bindingsites.RIV

De invloed van omgevingsfactoren op de infectiviteit van niet-delende cultures van Campylobacter jejuni

Campylobacter (C.) jejuni is a causative agent of gastro-enteritis. Growth of this Gram negative bacterium is restricted to a limited number of environmental conditions. Under conditions where growth of C. jejuni is not possible, e.g. at temperatures below 30 degrees C, C. jejuni can remain viable for a certain period of time, but its culturability decreases. In this study we examined the effect of this loss of culturability on the infectivity in cell-lines. We hypothesised that if non-culturable cells affect the process of infection then the infectivity per culturable cell used in the infection assay is not constant, whereas if only culturable cell are infectious then the infectivity per culturable cell is. C. jejuni was stored for 40 days in nutrient poor and nutrient rich medium at three temperatures. During this period we monitored its culturability, viability and infectivity. A decrease in culturability in time was observed. At low temperature (4 degrees C) and in nutrient poor medium cells remained culturable for longer periods than at higher temperatures (12 degrees C, 25 degrees C) or in nutrient rich medium. Non-culturable cells remained viable, as determined by tetrazolium chloride staining. The absolute level of adhesion and invasion showed a decrease in time. At low temperatures and in nutrient poor medium cells retained their capability to adhere and invade longer than at higher temperatures or in nutrient rich medium. While the absolute level of adhesion and invasion decreased, the infectivity per culturable cell added seemed to increase during the first 5 days of storage at 4 degrees C. This result confirmed our hypothesis that non-culturable C. jejuni can affect the process of infection. However, the result can also be explained by the presence of a limited number of bindingsites for C. jejuni in our testmodel. This suggested existence of bindingsites has great importance for the research into the influence of non-culturable cells on the infectivity. If non-culturable cells are not infectious but can block bindingsites, then they can play a protective role by competing with the culturable cells for the limited number of bindingsites.Campylobacter jejuni is de meest frequente bacteriele veroorzaker van voedselinfecties. Onder omstandigheden waarbij C. jejuni niet kan groeien, bijvoorbeeld bij temperaturen lager dan 30 graden C, verliest de bacterie zijn kweekbaarheid, maar lijkt de bacterie niet dood te gaan. In deze studie is onderzocht of een niet-kweekbare C. jejuni een infectie kan veroorzaken, en of de omstandigheden waaronder de kweekbaarheid verloren is gegaan van invloed zijn op een eventuele infectie. Gedurende 40 dagen zijn kweekbaarheid, levensvatbaarheid en vermogen om een infectie te veroorzaken bepaald van C. jejuni die was opgeslagen in twee verschillende media bij drie verschillende temperaturen. Wanneer opgeslagen in een nutrient-arm medium bij lage temperatuur (4 graden C) bleek C. jejuni langer zijn kweekbaarheid te behouden dan bij hogere temperaturen (12 graden C, 25 graden C) of in een rijk medium. Bacterien bleven onder alle condities levensvatbaar. De mate van adhesie en invasie van de opgeslagen cultures nam af in de tijd; bij lage temperatuur in buffer behielden de bacterien het langst hun vermogen om aan darmepitheel- cellen te hechten of om erin binnen te dringen. Terwijl de infectiviteit van de totale cultuur afnam, nam de infectiviteit per kweekbare bacterie in die cultuur toe gedurende de eerste 5 dagen om daarna weer af te nemen. Dit resultaat suggereert dat een niet-kweekbare C. jejuni infectieus kan zijn. Echter, er kan ook een alternatieve verklaring zijn, namelijk dat er slechts een beperkt aantal specifieke bindingsplaatsen voor de hechting van C. jejuni aanwezig zijn. De eventuele aanwezigheid van specifieke bindingsplaatsen voor Campylobacter heeft mogelijk grote gevolgen voor onderzoek aan de invloed van niet-kweekbare cellen. Als niet-kweekbare cellen niet meer infectief zijn, maar wel kunnen hechten aan een bindingsplaats, dan kunnen zij met kweekbare cellen concurreren om een bindingsplaats. Dit zou een geheel nieuw rol betekenen voor niet-kweekbare cellen: niet-kweekbare cellen zouden dan de kans op een infectie verlagen

Campylobacter jejuni: a study on environmental conditions affecting culturability and in vitro adhesion/invasion

Aims: Nongrowing cultures of Campylobacter jejuni lose their culturability. It is unclear whether this loss in culturability also affects their ability to interact with host cells. The purpose of this study was to determine the relevance of the number of culturable cells to the ability to adhere/invade in Caco-2 cells. Methods and Results: For C. jejuni C356, culturability and adhesion/invasion were monitored in time (days) under different storage conditions (temperature, medium, atmosphere). Decrease rates of both culturability and adhesion/invasion were dependent on the conditions used, but the number of adhering/invading cells per culturable cell was not affected by the environmental conditions. Furthermore five strains were monitored at one condition. The culturability and adhesion/invasion decrease rates did not significantly differ per strain; however the number of adhering/invading cells per culturable cell was strain dependent. Conclusions: Culturability and adhesion/invasion of C. jejuni are linearly related. The number of adhering/invading cells per culturable C. jejuni cell is strain dependent, but is not affected by environmental conditions. Significance and Impact of the Study: It was shown that the number of culturable cells is a good measure for the in vitro adhesion/invasion of. C. jejuni

The influence of environmental conditions on the infectivity of non-growing Campylobacter jejuni

Campylobacter jejuni is de meest frequente bacteriele veroorzaker van voedselinfecties. Onder omstandigheden waarbij C. jejuni niet kan groeien, bijvoorbeeld bij temperaturen lager dan 30 graden C, verliest de bacterie zijn kweekbaarheid, maar lijkt de bacterie niet dood te gaan. In deze studie is onderzocht of een niet-kweekbare C. jejuni een infectie kan veroorzaken, en of de omstandigheden waaronder de kweekbaarheid verloren is gegaan van invloed zijn op een eventuele infectie. Gedurende 40 dagen zijn kweekbaarheid, levensvatbaarheid en vermogen om een infectie te veroorzaken bepaald van C. jejuni die was opgeslagen in twee verschillende media bij drie verschillende temperaturen. Wanneer opgeslagen in een nutrient-arm medium bij lage temperatuur (4 graden C) bleek C. jejuni langer zijn kweekbaarheid te behouden dan bij hogere temperaturen (12 graden C, 25 graden C) of in een rijk medium. Bacterien bleven onder alle condities levensvatbaar. De mate van adhesie en invasie van de opgeslagen cultures nam af in de tijd; bij lage temperatuur in buffer behielden de bacterien het langst hun vermogen om aan darmepitheel- cellen te hechten of om erin binnen te dringen. Terwijl de infectiviteit van de totale cultuur afnam, nam de infectiviteit per kweekbare bacterie in die cultuur toe gedurende de eerste 5 dagen om daarna weer af te nemen. Dit resultaat suggereert dat een niet-kweekbare C. jejuni infectieus kan zijn. Echter, er kan ook een alternatieve verklaring zijn, namelijk dat er slechts een beperkt aantal specifieke bindingsplaatsen voor de hechting van C. jejuni aanwezig zijn. De eventuele aanwezigheid van specifieke bindingsplaatsen voor Campylobacter heeft mogelijk grote gevolgen voor onderzoek aan de invloed van niet-kweekbare cellen. Als niet-kweekbare cellen niet meer infectief zijn, maar wel kunnen hechten aan een bindingsplaats, dan kunnen zij met kweekbare cellen concurreren om een bindingsplaats. Dit zou een geheel nieuw rol betekenen voor niet-kweekbare cellen: niet-kweekbare cellen zouden dan de kans op een infectie verlagen

Campylobacter jejuni: a study on environmental conditions affecting culturability and in vitro adhesion/invasion

Aims: Nongrowing cultures of Campylobacter jejuni lose their culturability. It is unclear whether this loss in culturability also affects their ability to interact with host cells. The purpose of this study was to determine the relevance of the number of culturable cells to the ability to adhere/invade in Caco-2 cells. Methods and Results: For C. jejuni C356, culturability and adhesion/invasion were monitored in time (days) under different storage conditions (temperature, medium, atmosphere). Decrease rates of both culturability and adhesion/invasion were dependent on the conditions used, but the number of adhering/invading cells per culturable cell was not affected by the environmental conditions. Furthermore five strains were monitored at one condition. The culturability and adhesion/invasion decrease rates did not significantly differ per strain; however the number of adhering/invading cells per culturable cell was strain dependent. Conclusions: Culturability and adhesion/invasion of C. jejuni are linearly related. The number of adhering/invading cells per culturable C. jejuni cell is strain dependent, but is not affected by environmental conditions. Significance and Impact of the Study: It was shown that the number of culturable cells is a good measure for the in vitro adhesion/invasion of. C. jejuni

Cross-contamination in the kitchen: effect of hygiene measures

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

Cross-contamination in the kitchen: effect of hygiene measures

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

Pyruvate relieves the necessity of high induction levels of catalase and enables Campylobacter jejuni to grow under fully aerobic conditions

Aims: Several cases of campylobacteriosis reported worldwide seemingly conflict with the strict growth requirements and sensitivity to environmental stress of Campylobacter jejuni. In this study, the need for a micro-aerobic environment [dissolved oxygen tension (DOT): 0·1¿90%; 100% air saturation)] and the adaptive responses to oxygen stress were studied. Methods and Results: The growth of C. jejuni in continuous culture was assessed under different DOT in the presence or absence of pyruvate. In a medium without pyruvate, continuous cultures of C. jejuni showed typically micro-aerobic behaviour and cells were unable to grow under fully aerobic conditions. However in the presence of pyruvate (25 mmol l¿1), continuous cultures of C. jejuni were able to grow in a broad DOT range, varying from 0·1% to at least 90%, and the catalase activity was decreased. Conclusions: Addition of pyruvate results in the decrease in the concentration of hydrogen peroxide, which enables C. jejuni to grow aerobically. Significance and Impact of the Study: New information on the oxidative physiology of C. jejuni and its ability to grow aerobically in media supplemented with pyruvate is presente

Quantification of Campylobacter jejuni cross-contamination via hands, cutlery, and cutting board during preparation of a chicken fruit salad

Using artificially contaminated chicken, the quantitative overall effect of Campylobacter jejuni cross-contamination, either via cutlery, cutting board, or hands, on the microbiological quality of a chicken salad was tested to identify the most critical transfer route. The end contamination level of salads prepared according to different scenarios, with or without cross-contamination, was compared. It was shown that the mean transfer rate calculated for all salads prepared allowing cross-contamination was 0.12% of the initial number of C. jejuni on the chicken fillet (8.8 ± 0.2 log CFU). The difference in calculated transfer rates for the tested cross-contamination routes was not significantly different (P > 0.05). The prevention of cross-contamination by replacing cutlery and cutting board after handling raw chicken and the prevention of hand contact resulted in considerably reduced end contamination levels