Friederike Hilbert

Survival of Campylobacter jejuni under Conditions of Atmospheric Oxygen Tension with the Support of Pseudomonas spp.

ABSTRACT Campylobacter jejuni is a major food-borne pathogen. Despite causing enteritis in humans, it is a well-adapted intestinal microorganism in animals, hardly ever generating disease symptoms. Nevertheless, as a true microaerophilic microorganism it is still puzzling how Campylobacter cells can survive on chicken meat, the main source of human infection. In this study, we demonstrate that C. jejuni is able to withstand conditions of atmospheric oxygen tension when cocultured with Pseudomonas species, major food-spoiling bacteria that are frequently found on chicken meat in rather high numbers. Using an in vitro survival assay, interactions of 145 C. jejuni wild-type strains and field isolates from chicken meat, broiler feces, and human clinical samples with type strains and food isolates of Pseudomonas spp., Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, and Enterococcus faecalis were studied. When inoculated alone or in coculture with Proteus mirabilis, Citrobacter freundii, Micrococcus luteus, or Enterococcus faecalis type strains, Campylobacter cells were able to survive ambient oxygen levels for no more than 18 h. In contrast, Campylobacter bacteria inoculated with type strains or wild-type isolates of Pseudomonas showed a prolonged aerobic survival of up to >48 h. This microbial commensalism was diverse in C. jejuni isolates from different sources; isolates from chicken meat and humans in coculture with Pseudomonasputida were able to use this survival support better than fecal isolates from broilers. Scanning electron microscopy revealed the development of fiberlike structures braiding P. putida and C. jejuni cells. Hence, it seems that microaerophilic C. jejuni is able to survive ambient atmospheric oxygen tension by metabolic commensalism with Pseudomonas spp. This bacterium-bacterium interaction might set the basis for survival of C. jejuni on chicken meat and thus be the prerequisite step in the pathway toward human infection.

Bacteriophages isolated from chicken meat and the horizontal transfer of antimicrobial resistance genes

ABSTRACT Antimicrobial resistance in microbes poses a global and increasing threat to public health. The horizontal transfer of antimicrobial resistance genes was thought to be due largely to conjugative plasmids or transposons, with only a minor part being played by transduction through bacteriophages. However, whole-genome sequencing has recently shown that the latter mechanism could be highly important in the exchange of antimicrobial resistance genes between microorganisms and environments. The transfer of antimicrobial resistance genes by phages could underlie the origin of resistant bacteria found in food. We show that chicken meat carries a number of phages capable of transferring antimicrobial resistance. Of 243 phages randomly isolated from chicken meat, about a quarter (24.7%) were able to transduce resistance to one or more of the five antimicrobials tested into Escherichia coli ATCC 13706 (DSM 12242). Resistance to kanamycin was transduced the most often, followed by that to chloramphenicol, with four phages transducing tetracycline resistance and three transducing ampicillin resistance. Phages able to transduce antimicrobial resistance were isolated from 44% of the samples of chicken meat that we tested. The statistically significant (P = 0.01) relationship between the presence of phages transducing kanamycin resistance and E. coli isolates resistant to this antibiotic suggests that transduction may be an important mechanism for transferring kanamycin resistance to E. coli. It appears that the transduction of resistance to certain antimicrobials, e.g., kanamycin, not only is widely distributed in E. coli isolates found on meat but also could represent a major mechanism for resistance transfer. The result is of high importance for animal and human health.

Survival with a Helping Hand: Campylobacter and Microbiota

Campylobacteriosis is the most important bacterial food-borne disease in the developed world. Consumption of chicken meat, beef or raw milk, direct contact with ruminants and exposure to contaminated surface water or even consumption of tap water have been identified as risk factors for human disease. However, the most important risk factor is consumption of and/or handling contaminated chicken. Campylobacter spp. are fastidious microorganisms but must somehow survive outside the host, especially in food and agricultural environments and also resist the innate and humoral immune responses inside the host. In this paper we hypothesize that other microorganisms in mixed populations with Campylobacter may act to improve survival outside the host and may also protect the pathogen against the intestinal immune system. Our evidence for this hypothesis is based on: 1. newly generated microbial community analysis; 2. the prolonged survival of Campylobacter in mixed species biofilms and in co-culture with environmental bacteria; 3. improved survival in amoebae and rumen fluid; 4. sulfur release and iron uptake systems within the intestinal lumen. This would make Campylobacter an exceptional food-borne pathogen. With this in mind, new strategies are necessary to combat Campylobacter along the total food chain.

Prevalence and risk factors for shedding of thermophilic Campylobacter in calves with and without diarrhea in Austrian dairy herds

The objectives of this study were to evaluate the presence of thermophilic Campylobacter in feces of calves with and without diarrhea on dairy farms and to survey farm characteristics and management practices to define risk factors for the presence of Campylobacter. Fifty dairy farms were chosen based on the presence of calf diarrhea, and 50 farms in which calves were free from diarrhea served as a standard of comparison. In total, fecal samples were taken from 382 calves. Farm data and management practices were surveyed using a questionnaire on farm. Campylobacter were isolated from fecal samples and colonies were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Campylobacter spp., mainly Campylobacter jejuni (93% of isolated species), were detected on 33% of the farms and in 14.9% of the calves. Detection of Campylobacter did not differ between farms or between calves with and without diarrhea, although we found a tendency for calves suffering from diarrhea to shed Campylobacter more often. Calves may act as a reservoir of Campylobacter and may therefore lead to infections of other animals and humans. To define control strategies to reduce Campylobacter in calves, we identified on-farm risk factors. The presence of poultry on the farm, the time of cow-calf separation following birth, the use of an individual bucket for each calf, the feeding of waste milk, and the duration of individual housing were variables significantly associated with the appearance or absence of Campylobacter.

Multilocus Sequence Typing and Antimicrobial Resistance of Campylobacter jejuni Isolated from Dairy Calves in Austria.

Human campylobacteriosis is primarily associated with poultry but also cattle. In this study, 55 Campylobacter jejuni strains isolated from 382 dairy calves’ feces were differentiated by multilocus sequence typing and tested for antimicrobial resistance. The most prevalent sequence type (ST) was ST883 (20.0%), followed by ST48 (14.5%), and ST50 (9.1%). In contrast to ST48 and ST50, ST883 has rarely been described in cattle previously. Furthermore, risk factor analysis was performed for the presence of the most prevalent STs in these calves. Multiple regression analysis revealed that the type of farm (organic vs. conventional) and calf housing (place, and individual vs. group) were identified as significantly (p < 0.05) associated with the presence of ST883 in calves, and ST50 was associated with calf diarrhea. Antimicrobial resistance was detected in 58.2% of the isolates. Most of the resistant isolates (81.3%) were resistant to more than one antimicrobial. Most frequently, resistance to ciprofloxacin (49.1%), followed by nalidixic acid (42.8%), and tetracycline (14.5%) was observed. The results of the present study support the hypothesis that dairy calves may serve as a potential reservoir for C. jejuni and pose a risk for transmission, including antimicrobial resistant isolates to the environment and to humans.

Rapid urease screening of Yersinia on CIN agar plates

Yersinia enterocolitica is an important foodborne pathogen, but isolation of virulent Yersinia from food sources is still time consuming and requires skills. In this article, we describe a rapid urease screening on cefsulodin-irgasan-novobiocin (CIN) agar plates with an agar overlay assay. This test is simple to perform, all colonies on a plate can be checked simultaneously, it only takes minutes for detection of urease-positive colonies and the colonies survive for transfer, further characterisation, and storage. Additionally, this method is useful to isolate virulent (urease-positive and pYV harbouring) Y. enterocolitica from foodstuffs.

The risk of transfer of foodborne bacterial hazards in Turkey through the consumption of meat; risk ranking of muscle foods with the potential to transfer Campylobacter spp.

This paper presents a simple approach to semi-quantitatively estimating the risk for human foodborne campylobacteriosis in Turkey, with a focus on typical/traditional meat and meat dishes. The following factors are considered: prevalence/concentration of the pathogen in raw meat according to species, changes in the pathogen’s titre as a result of heat treatment and cross-contamination, serving frequency and size, and demographic data. Regional differences as well as differences between social groups are considered in particular. The biological hazard “Campylobacter” is characterized as associated with conditions prevailing in the meat chain and this information is linked to food consumption and other data thus allowing an estimate of consumers’ exposure to Campylobacter via typical Turkish meat dishes. The latter are ranked on the basis of the presence of meat components and the associated risks.

Demographic/socio-economic factors and dietary habits determining consumer exposure to foodborne bacterial hazards in Turkey through the consumption of meat

Meat is considered a major source of foodborne bacterial pathogens. Assessing the risks for public health of meat consumption is achieved by “Risk Analysis”- a concept firmly established on a global level - so as to ensure the level of consumer protection as stipulated by international trade agreements. Risk analysis identifies and characterizes potential hazards, which allows estimating the associated risks, provided reliable data on consumer exposure are available. The latter varies by geographical region and associated differences in culture and socio-economic position of the consumer. This paper is dedicated to assessing the exposure of the Turkish population to bacterial hazards resulting from consumption of typical traditional meat dishes. It reviews current data on meat consumption in different regions of Turkey, on the basis of which seven typical preparations and consumption scenarios are identified. Major demographic and socio-economic differences along Turkey`s east–west axis were identified, such as the degree of urbanization, consumers’ age, household size, preferred meat animal species, frequency and volumes of meat consumption, and preparation techniques. Considering all these variables allowed a differentiated and reliable assessment of consumers’ exposure to biological hazards. This information is essential for greater accuracy in estimating the public health risks associated with various meat-borne hazards in this country.

Simple media and conditions for inter-laboratory transport of Campylobacter jejuni isolates

Background: Campylobacter jejuni is one of the most important agents of zoonotic disease. Production as well as companion animals can be the infectious source for Campylobacteriosis in humans. Hence, epidemiological research on animal colonization, survival in food of animal origin, and human Campylobacteriosis is of high priority. As such studies involve worldwide co-operations and should include further typing of isolates in reference centers, using a reliable method for transportation is essential. In the case of C. jejuni, a pathogenic and microaerophilic bacterium, special safety precautions as well as particular transport conditions that guarantee survival of isolates are required. Objective: The purpose of this study was to test various media and temperatures for the transportation of C. jejuni under aerobic conditions and to identify a cheap, effective and easy method that is appropriate for long distance transportation and can be applied by most veterinary/medical laboratories with a basic infrastructure. Materials and methods: We examined Mueller–Hinton (MH) agar with and w/o 2% horse blood and m-CCDA at room temperature and 2 ± 2 (SD)°C under atmospheric conditions for survival of Campylobacter strains. Results: MH agar with 2% horse blood, suitable transport vials, and an optimum temperature of 2 ± 2°C provided survival of three Campylobacter type strains for at least one month under atmospheric conditions. This was validated by a transport test in which 101 isolates were shipped from Turkey to Austria. All isolates could be recultured and 97% survived more than one month in the transport medium. Conclusion: These findings indicate that the described approach is suitable for inter-laboratory transport of C. jejuni isolates.

Safety of Food and Beverages: Poultry and Eggs

Broiler meat and eggs constitute an important part of the human diet. The mode of production ranges from backyard farming to high level of integration from feed/farm to fork. Pathogenic bacteria commonly associated with poultry meat are Campylobacter sp. and Salmonella sp. These pathogens can be harbored in the intestines of otherwise healthy animals, and spread on the carcass surface during slaughter. Effective control is possible for Salmonella sp. Multiresistance against antibiotics is an emerging issue. Eggs may be contaminated in utero before the shell is formed or when cuticula/shell of the laid egg is damaged.