Microbial Food Cantamination

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MICROBIAL CONTAMINANTS

Food monitoring, 1993-1997. Part 5.

Prepared by:

Sven QvistInstitute of Food Research and Nutrition

Niels Ladefoged NielsenInstitute of Food Safety and Toxicology

Food monitoring, 1993-1997consists of five sub-reports:

Part 1: NutrientsPart 2: Chemical contaminantsPart 3: Production aids (pesticides and veterinary drugs)Part 4: Food additivesPart 5: Microbial contaminants

Ministry of Food, Agriculture and Fisheries

Danish Veterinary and Food Administration


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MICROBIAL CONTAMINANTSFood monitoring, 1993-1997. Part 5.

FdevareRapport 2001:211st Edition, 1st Circulation, September 2003This report is an English version of FdevareRapport 2000:05, Mikrobiologiskeforureninger, Overvgningssystem for levnedsmidler 1993-1997. Del 5Copyright: The Danish Veterinary and Food Administration400 copiesCover: Jeppe HammerichPrinting office: The Danish Veterinary and Food AdministrationPrice: DKK 50.- incl. VATISBN: 87-91189-03-9ISSN: 1399-0829 (FdevareRapport)

Publications costing money can be bought at book shops or:National IT and Telecom AgencyTel +45 35 45 00 00E-mail - [email protected] site: www.netboghandel.dk

The Danish Veterinary and Food AdministrationMrkhj Bygade 19, DK-2860 SborgTel. + 45 33 95 60 00, fax + 45 33 95 60 01Web site: www.foedevaredirektoratet.dkTThe Danish Veterinary and Food Administrationis part of the Danish Ministry ofAgriculture, Food and Fisheries. The Danish Veterinary and Food Administration isresponsible for the administration, research and control within food and veterinaryareas from farm to fork, as well as practical matters relating to animal protection

(otherwise under the Ministry of Justice).

Making of regulations, co-ordination, research and development, take place in theAdministrations center in Moerkhoej. The 11 Regional Authorities handle the practicalinspection of food and veterinary matters, including import/export etc.

The central administration of The Danish Veterinary and Food Administration employa staff of approx. 550 full-time employees, whilst the 11 regional authorities employ afurther approx.1 400 full-time employees.

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http://www.netboghandel.dk/http://www.foedevaredirektoratet.dk/http://www.foedevaredirektoratet.dk/http://www.foedevaredirektoratet.dk/http://www.netboghandel.dk/


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PREFACE

A programme for monitoring nutrients and chemical contaminants in foods was established in

1983, and the Danish Veterinary and Food Administration is now carrying this programmefurther within an expanded field. Results are being reported for periods of five years; thus, the

present report covers the third period, 1993-1997.

The reporting of the third period of the monitoring programme consists of the following sub-

reports:

Part 1: Nutrients

Part 2: Chemical contaminants

Part 3: Production aids (pesticides and veterinary drugs)

Part 4: Food additives

Part 5: Microbial contaminants

The studies are co-ordinated by the Danish Veterinary and Food Administration. The major

part of the chemical analyses was carried out by the regional laboratories in Copenhagen,

Odense, Aalborg, and rhus; however, analyses for veterinary drugs were mainly carried out

by the Danish Veterinary and Food Administration. Microbiological analyses were carried out

by the Danish Veterinary and Food Administration and the municipal environmental and food

control units. The reporting was co-ordinated by Gudrun Hilbert, Institute of Food Research

and Nutrition.

The Danish Veterinary and Food Administrations monitoring programme for foods does not

include analyses of radionucleides; these analyses, as well as the publication of their results,

are being undertaken by Ris National Laboratory.

The text of the report does not take into account the fact that certain activities had a different

organizational placement prior to the re-organization of the Ministry of Food, Agriculture and

Fisheries in 1997, when the Danish Veterinary Service and the National Food Agency of

Denmark were united into the Danish Veterinary and Food Administration. All results from

these two institutions are being referred to as results of work carried out by the Danish

Veterinary and Food Administration.

December, 1999

Danish Veterinary and Food Administration

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TABLE OF CONTENTS

1. MONITORING PROGRAMME FOR FOODS................................................................ 6

2. INTRODUCTION............................................................................................................. 9

3. SALMONELLA.............................................................................................................. 11

3.1 Importance ................................................................................................................ 11

3.2 Ecology and occurrence............................................................................................ 12

3.3 Salmonella in eggs.................................................................................................... 123.4 Salmonella in broilers ............................................................................................... 13

3.5 Salmonella in pigs..................................................................................................... 13

3.6 Salmonella in cattle................................................................................................... 14

4. CAMPYLOBACTER..................................................................................................... 15

4.1 Importance ................................................................................................................ 15


5. YERSINIA ENTEROCOLITICA .................................................................................. 18

5.1 Importance ................................................................................................................ 18


6. ESCHERICHIA COLI O157.......................................................................................... 21

6.1 Importance ................................................................................................................ 21


7. LISTERIA MONOCYTOGENES.................................................................................. 25

7.1 Importance ................................................................................................................ 25


8. STAPHYLOCOCCUS AUREUS................................................................................... 29

8.1 Importance ................................................................................................................ 29


9. CLOSTRIDIUM PERFRINGENS ................................................................................. 31

9.1 Importance ................................................................................................................ 319.2 Ecology and occurrence............................................................................................ 31

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10. BACILLUS CEREUS..................................................................................................... 33

10.1 Importance .............................................................................................................. 33

10.2 Ecology and occurrence.......................................................................................... 33

11. ANTIBIOTIC RESISTANCE ........................................................................................ 3511.1 Monitoring of antibiotic resistance......................................................................... 35

12. SUMMARY AND CONCLUSION ............................................................................... 37

13. REFERENCES ............................................................................................................... 39

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1. MONITORING PROGRAMME FOR FOODS

The object of the monitoring programme is, by means of systematic studies of foods and the

dietary habits of the Danish population,

to ascertain whether our foods are subject to any long-term changes in terms of contents of

desirable and undesirable substances and/or micro-organisms,

to assess the health significance of any such changes in relation to major changes of

dietary habits,

to disclose potential problems within the area and to provide background material and a

basis for decisions to remedy any problems which might have arisen.

The material provided may also serve as a documentation of the health quality of Danish

foods, and be used for updating the food composition data base of the Danish Veterinary and

Food Administration. Monitoring results are used also in other connections; e.g.,

microbiological results are reported to the Danish Zoonosis Centre, and results concerning

residues of pesticides and veterinary drugs are reported to the EU.

The work with the monitoring programme consists of the following:

to monitor, by means of analyses, the contents of desirable and undesirable

substances/micro-organisms in specific foods,

to investigate the dietary habits of the Danish population,

to carry out intake estimates (wherever relevant) by combining contents in foods and data

on the populations diet.

Subsequently, a nutritional and/or toxicological assessment can be made. Such an assessment

will be particularly important whenever changes are found.

Since changes in the contents of foods and changes in our dietary habits usually developslowly, the studies cover a considerable number of years. Every five years, the results are

reviewed, and the analytical results for the foods are compared with the dietary habits over

the period. This permits an assessment of whether the intake of desirable substances is

adequate, and whether the intake of undesirable substances or micro-organisms is acceptably

low.

Content findings and intake estimates are compared with earlier results, thus permitting an

assessment of the development of contents and intakes over time.

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Results are evaluated continuously during the monitoring period, enabling reactions to

violations of existing limits, deviations from contents declarations, or other noteworthy

observations.

The monitoring programme consists of five sub-fields:

Nutrients, including vitamins, minerals, proximates, and dietary fibres.

Chemical contaminants, including trace elements, nitrate, organic environmental

contaminants, and mycotoxins.

Production aids, including residues of pesticides and veterinary drugs.

Food additives.

Microbial contaminants.

Initially, the monitoring programme covered only nutrients and chemical contaminants. The

remaining three subjects are new inclusions under the monitoring concept; these are

production aids (pesticides and veterinary drugs), which have been reported continuously

during several decades and during recent years have attracted increasing attention within the

international co-operation and in the public; food additives which, according to three EU

directives, shall be followed with a view to application and intake; and finally, microbial

contaminants, with an increasing number of reported disease cases which can be attributed to

pathogenic bacteria in foods.

With the merger in 1997 of the National Food Agency of Denmark and the Danish Veterinary

Service into the new Danish Veterinary and Food Administration it has become possible to

compile the data material, especially within the fields concerning microbial contaminants and

veterinary drug residues.

Unlike the first two monitoring periods (1983-1987 and 1988-1992), each of which was

reported as a whole [1,2], the reporting of the third period has been divided into five sub-

reports according to subject. Each sub-report comprises a number of analyses which,

depending on the subject matter, are carried out once or several times during a five-yearperiod. Thus, e.g. vitamins in meat are analysed once, while pesticide residues in fruits and

vegetables are analysed yearly. The difference reflects the fact that empirically, vitamin

contents in meat will not change within a short time, whereas the monitoring of pesticide

residues contains an appreciable element of control, and the use pattern for pesticides is

subject to greater fluctuations.

In 1996, the monitoring programme (nutrients and chemical contaminants) was subjected to

an international evaluation [3]. The main conclusion was that the monitoring programme was

good, but might be improved in some respects. The collection of diet data should be expanded

to include a larger number of methods and be effected continuously, and the use of statisticalexpertise should be optimized, particularly for sampling and processing of results. Further, a

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number of more specific suggestions was mentioned. The experience gathered from the

evaluation has been included in the reporting of the third period as well as in the planning of

the fourth period.

The Ministry of Food, Agriculture and Fisheries has to be informed on the immediate

situation concerning Danish foods, the health significance for Danish consumers, and the

direction in which matters are likely to develop. In this respect, the monitoring programme

can provide background material and a basis for decisions on actions in the form of national

or international regulations.

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2. INTRODUCTION

Within the framework of the Danish Veterinary and Food Administration and with the

assistance of the municipal food control units, a number of microbiological mapping studieshas been carried out during recent years, with the objective of providing information on the

present occurrence of one or more bacteria. In addition, monitoring programmes for bacteria

such as Salmonellaand Campylobacterhave been initiated. The purpose of these monitoring

programmes is to follow the occurrence of particular bacteria in different foods over a period

of time.

In the following, a description is given of the pathogenic bacteria which are most important

under Danish conditions, as well as a list of results from study programmes carried out during

the period 1993-1997. It should be noted that such monitorings are not undertaken every year,

with the exception of Salmonellaand Campylobacter.

A common feature of Salmonella, Campylobacter, Yersinia enterocolitica, and verotoxin-

producingEscherichia coliO157 is their reservoir in the gastro-intestinal tract of production

and meat animals. From this reservoir, the bacteria may contaminate the foods during the

slaughtering process and further processing, thereby constituting a risk of disease in humans.

These pathogenic bacteria are responsible for a very large proportion of the total incidence of

food-borne diseases. During recent years, such bacteria have caused a yearly total of 7,000-

8,000 registered disease cases in Denmark. However, only a part of the food-borne diseasecases are diagnosed and registered. The actual incidence of such diseases is assumed to be at

least 10-20 times higher than the number of registered cases.

The prevention and control of these pathogenic bacteria are issues of very high priority.

Control must take place first and foremost in the primary production, but also the subsequent

steps from stable to table must be involved. Development of methods for measuring

antibodies in e.g. blood, meat juice, and eggs make it possible to establish the infection status

of herds with respect to bacteria such as Salmonellaand Y. enterocolitica.

In addition to this, the spread of bacteria to foods must be prevented and controlled at thefood manufacturing as well as the wholesale and retail stages. At these stages, the prevention

is based on self-imposed control according to the Hazard Analysis Critical Control Point

(HACCP) principle. With the widespread occurrence of pathogenic bacteria in meat animals,

carcasses may very easily be contaminated. Modern industrialized slaughter methods tend to

increase the infection levels. Therefore, an effort is necessary in order to improve the hygiene

in critical processes on the slaughter line, such as removal of intestines, removal and hooking

of chest organs, lymph node incisions at meat inspection, and trimming of head meat.

In addition to improved hygiene on the slaughter line, separate slaughterings of infected and

non-infected animals may contribute to lower contamination levels in meat for consumption.

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At the retail stages, prevention of cross-contamination from raw meat to other food items,

especially to consumption-ready products, is essential. Adequate heat treatment is another

essential factor for preventing the transmission of pathogenic bacteria to humans. Correct

refrigeration and application of other growth-inhibiting principles, including acidity and water

activity, are also important critical points in the preventive efforts.

Finally, in order to prevent disease, consumers must be advised about the correct handling

and storage of foods.

The identification of primary infection sources and their importance permits a targeted and

efficient prevention and control. Essential tools for this purpose are the collection and typing

of bacteria from humans, animals, and foods, case-control studies, and monitoring of the

occurrence of bacteria in various food groups.

Table 1 gives a list of the number of samples analysed in relation to the bacteria that arediscussed in the present report. Samples analysed in connection with routine control,

consumer complaints, and the like, are not included.

Table 1.Number of samples analysed in microbiological mapping and monitoringprogrammes, 1993-1997.

Bacteria Number of samples analysed

Salmonella 50,918

Campylobacter 5,696

Escherichia coli O157 4,012

Yersinia enterocolitica 1,326

Listeria monocytogenes 2,810

Staphylococcus aureus 1,309

Clostridium perfringens 1,262

Bacillus cereus 1,309

The decision to include the subject of microbiology in the monitoring programme was madequite late in the monitoring period. Consequently, the reporting has not yet assumed its final

form; the report covering the next monitoring period is expected to contain, among other

things, literature references.

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3. SALMONELLA

3.1 Importance

Salmonellosis has been one of the most frequently occurring food-borne diseases in many

countries for the last 20 years.

In the early 1980es, the number of salmonellosis cases in Denmark was approx. 1,000,

increasing to around 3,000 per year in the late 1980es. 4,300 cases were registered in 1994,

and in 1995 and 1996 the number decreased to 3,600 and 3,200, respectively. In 1997, 5,000

cases were registered, which is the highest number till now.

The majority of registered cases is assumed to be sporadic or minor outbreaks associated withfamilies or parties. Only a minor proportion is associated with more widespread outbreaks.

The genus Salmonella (S.) consists of two species: S.enterica, comprising more than 2,400

serotypes, and S. bongori, comprising approx. 20 serotypes. Generally speaking, all

Salmonella serotypes must be considered as pathogenic. However, 80-90% of all cases of

human salmonellosis are caused by the serotypes S.Typhimuriumand S.Enteritidis. This is

due to the widespread occurrence of especially these serotypes in the primary production

systems. S.Typhimuriumis found in all types of production animals, whereas S.Enteritidisis

found mainly in market egg-producing hens and in broilers. Also other serotypes may be

transmitted from production animals, but only occasionally cause serious problems. One suchcase was the S. Infantis outbreak in 1993, where more than 500 people were infected after

having eaten pork from a minor slaughterhouse in Jutland.

In recent years, many countries have seen an increase in the number of registered cases as

well as outbreaks caused by S.Enteritidis. Thus, S.Enteritidis is now by far the most

frequently isolated serotype, whereas S.Typhimuriumis the source of less than 20% of cases.

In the early 1980es, on the other hand, S. Typhimurium was the most frequently isolated

serotype in foods in Europe.

The incubation period (the period from infection to manifest symptoms) for food-bornesalmonella infections is reported to be -3 days in most cases. Salmonellacauses enteritis in

95-99% of cases, while septicaemia is seen in approx. 1-5% of cases. The risk of developing

septicaemia depends on the serotype that is responsible for the disease. S. Dublin, for

example, typically associated with cattle, causes septicaemia 10-20 times more often than

other serotypes.

The symptoms of salmonellosis are diarrhoea, abdominal pains, fever, and headache. Other

symptoms may be nausea and vomiting. The duration of the symptoms may range from a few

days to several weeks. In cases of septicaemia, the course of the disease may be of longerduration. Late complications in the form of arthralgia are seen in approx. 7% of patients,

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especially in patients having a particular tissue type. The mortality rate of salmonellosis is

estimated to be approx. 0.1% of registered cases.

The infective dose depends on several factors such as age and immunological status. For

healthy persons, an infectious dose of 103-106 bacteria has been indicated. However,

infections caused by much lower numbers have been reported, especially in connection with

intakes of fatty products. Thus, an outbreak in the USA, caused by pasteurized ice cream

produced with raw egg yolks, showed that people had become ill after ingestion of less than

0.003 Salmonellaper g of ice cream (corresponding to one bacterium per serving).

3.2 Ecology and occurrence

Salmonellahas its growth optimum at 37C and growth interval from 5 to 46C. Salmonella

does not survive pasteurization, but is relatively resistant to freezing. The heat resistance of

Salmonella is highly dependent on environmental factors such as water activity and acidity.

Salmonella is facultatively anaerobic (i.e. capable of growth in the presence as well as

absence of oxygen) and is therefore not inhibited by vacuum packing or modified atmosphere

packing. Salmonella has its pH optimum at 6.5-7.5 with a growth range of 4.5-9.0. At water

activities (aw) below approx. 0.94, growth of Salmonella is not possible. The aw limit of

growth depends on factors such as pH and temperature.

The normal habitat ofSalmonellais the intestinal tract of warm-blooded animals. Salmonella

is spread from production animals to raw meat, unpasteurized milk, and eggs, which are themain causes of human infection. In Denmark, outbreaks caused by bean sprouts have also

occurred.

In Denmark, comprehensive monitoring programmes, including sampling of salmonella

bacteria from animals, foods, and humans, have permitted a specification of the relative

importance of individual animal species for disease in humans. The Danish Zoonosis Center

issues annual reports of sources of human salmonella infections.

3.3 Salmonella in eggs

In two studies carried out in 1994 and 1995-96, the occurrence of Salmonella in eggs was

found to be approx. 0.1%. 9 of 10 infected eggs were contaminated exclusively on the shells,

while 1 in 10 was contaminated internally. Eggs may be contaminated with all salmonella

types, but in cases of contaminated egg contents, S.Enteritidisis regarded as by far the most

important, if not the only one. In poultry, S.Enteritidis has a special ability to infect the

oviducts of laying hens, thus inoculating the egg before the shell is formed.

In 1997, a marked increase in egg-borne salmonellosis was observed, which might indicate an

increased occurrence in eggs. The large proportion of the total number of salmonellosis casesaccounted for by egg-borne salmonellosis, is explained by a high consumption of eggs. Thus,

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every Danish person consumes approx. four eggs per week on average. On a yearly basis, this

totals more than one thousand millions of eggs which are used in Danish households.

The occurrence of S.Enteritidisin yolks and whites is believed to be the cause of the great

majority of human infections caused by this salmonella type. Non heat-treated fatty foods

containing raw eggs are assumed to be the main cause of egg-borne salmonella infections.

Salmonellawill often be present in low numbers when the egg is laid, and any increase of the

numbers will depend on how and for how long the egg is stored .How great a risk an infected

egg may present, will depend on conditions of storage, handling, and preparation.

Action plans for the control of Salmonellain the market egg production have been initiated,

including, among other things, monitoring of Salmonellain breeding flocks and in production

flocks. If Salmonellais found in production flocks, or if a flock is suspected of being infected

with Salmonella, the eggs are sent to heat treatment.

3.4 Salmonella in broilers

Salmonella in broilers used to be an important source of human salmonellosis, but this

importance has been on the decrease in recent years. A voluntary action plan from 1989, and

the official action plan from 1994 for the control of Salmonellain poultry, has resulted in a

reduction in the number of salmonella positive broiler flocks sent to slaughter, from approx.

70% to approx. 10-15% by the end of 1997.

In 1997, the municipal food control units routine inspections of poultry meat at the retailstages revealed that 5.7% of non heat-treated poultry products were contaminated with Salmo-

nella (see Table 2).

Broilers are assumed to be the cause of only 1-3% of all salmonella infections. Among the

contributing causes to this may be mentioned consumer information campaigns pointing out

correct handling and heat treatment as the most important preventive measures.

3.5 Salmonella in pigs

An increasing occurrence of S.Typhimurium in pig herds has contributed noticeably to the

number of human salmonellosis cases since the beginning of the 1990es.

In 1995, a four-year action plan was initiated for the control of Salmonella in pig farming,

based on a monitoring of salmonella in finished produce that was started in 1992. The action

plan has brought about a reduction in the occurrence of S.Typhimuriumin pig herds. Thus,

serological monitoring of all Danish pig herds has shown the number of salmonella infected

herds to be lower than 5% (spring, 1997).

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The action plan has furthermore contributed to a reduction in the occurrence of Salmonellain

fresh pork from approx. 3% to approx. 1% in 1997. The occurrence of Salmonellain minced

pork at the retail stage is around 2%, or about twice that in pieces of fresh meat (see Table 2).

3.6 Salmonella in cattle

In general, beef is less often contaminated with Salmonellathan poultry and pork. This may

be due to the fact that the slaughter process for cattle differs from those for pigs and poultry.

Analyses of beef at slaughterhouses and at the retail stage showed that Salmonella was

present in approx. 0.2-1% of samples in 1997. However, in minced beef the occurrence is

approx. 1%. S.Typhimuriumand S. Dublinare the most frequent serotypes in cattle herds; but

the incidence is not known for a certainty, since Salmonellais not monitored in the primary

production.An action plan for Salmonella in cattle has not been established, but herds are

analysed whenever salmonellosis is indicated.

Table 2 shows findings of Salmonellain poultry and pork during the period 1994-1997.

Table 2.Occurrence of Salmonella in chicken and pork meat from the retail stage, 1994-1997.

Chicken Pork

Non heat-treated Heat-treated Non heat-treated Heat-treated

% positive Number ofsamples % positive Number ofsamples % positive Number ofsamples % positive Number ofsamples

1994 16.1 663 0.2 968 2.5 2,071 0.06 7,583

1995 6.9 492 0.3 1,294 1.2 3,733 0.1 12,090

1996 9.5 462 0.3 1,373 1.8 3,371 0.01 8,411

1997 5.7 404 0 624 1.4 2,235 0.06 5,144

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4. CAMPYLOBACTER

4.1 Importance

Bacteria belonging to the genus Campylobacterare assumed to be one of the most important

causes of diarrhoea in humans all over the world today. In a number of European countries,

the number of registered cases of campylobacter infections exceeds the number of salmonella

infections.

Within the genus Campylobacter, the following species have most frequently been described

as the causes of campylobacteriosis in humans: C. jejunisubsp.jejuni, C. coli, C. lari, C. fetus

subsp.fetus,and C. upsaliensis. C. jejunisubsp.jejuniis reported to be the cause of 80-90%

or more of all registered cases.

Denmark has seen an increase in the number of registered campylobacter infections from

approx. 1,100 in 1992 to approx. 3,000 in 1996. In 1997, the number of registered cases was

approx. 2,700.

The infective dose for food-borne campylobacteriosis has not been established, but analyses

indicate that it may be as low as 500 bacteria.

The incubation period for food-borne campylobacter infections is reported to be 2-5 days in

most cases.

The symptoms are gastro-enteritis with watery diarrhoea, and in some cases, fresh blood in

the faeces is seen after 1-2 days. Concomitant to diarrhoea, affected general condition, fever,

and abdominal pains are often seen. Other symptoms may be nausea, vomiting, stomach

cramps, haemorrhagic diarrhoea, and arthralgia. The normal duration of symptoms is 5-10

days. Complications are relatively uncommon, but approx. 2% of patients develop a reactive

arthritis (incomplete Reiters syndrome). These symptoms may persist for weeks or months.

Reiters syndrome, comprising, in addition to reactive arthritis, conjunctivitis, ureteritis, and

dermatitis, is described as a sequela to acute campylobacteriosis. In rare cases, patients may

develop Guillain-Barrs syndrome (acute polyneuritis).

Only few mortality cases have been associated with campylobacter infections. International

assessments report one mortality case in 20,000 campylobacter infections.

The majority of human campylobacter infections occur as sporadic single cases or as minor,

family-related outbreaks. However, major outbreaks have been reported after ingestion of

unpasteurized milk, contaminated drinking water, or insufficiently heat-treated meat.

Sporadic cases occur most frequently in summer.

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Members of the genus Campylobacter are microaerofilic (i.e. growing best in reduced oxygen

content in relation to atmospheric air). Oxygen tolerance varies within species and strains.Human-pathogenic Campylobacterspecies do not grow at temperatures below 30C, with the

exception of C. fetus subsp. fetus, which is able to grow at 25C. Maximum growth

temperature is 42-43C. This means that Campylobacterdoes not propagate in refrigerated

foods. On the other hand, it has been demonstrated that Campylobacter survives better at 4C

than at room temperature. Campylobacteris more sensitive to heat treatment than most other

bacteria.

Campylobacter is sensitive to desiccation and to salt concentrations above 0.5%. Optimum

pH interval for growth is 6.5-7.5. Growth is inhibited at pH values below 5.1. Campylobacter

may, when exposed to physical or chemical stress factors, transform into so-called viable

non-culturable forms.

The natural habitat of the majority of Campylobacterspecies is the intestinal tract in warm-

blooded animals, including birds. The main reservoir of C. jejuni is probably poultry,

including chickens and turkeys. In 1997, the flock incidence in Danish broilers was 37%. Due

to intensive production methods and the horizontal transmission route of the bacteria, all

animals in infected flocks are likely to carry the bacteria. Poultry flocks may be infected via

drinking water, rodents, insects, dogs, cats, and humans (tools, footwear, work clothes). C.

jejuniis also frequently isolated from cattle, sheep, and goats.

C. coli is mainly associated with pigs, but is also isolated from chickens. C. lari is most

frequently found in the intestinal tract of birds. C. fetussubsp.fetusis mainly found in cattle

and sheep in which it is described as a cause of abortions.

The occurrence of Campylobacterin beef and pork for sale at the retail stages is usually low

(0.5-5%). Thus, in Danish mapping studies carried out in 1995, 1996, and 1997,

Campylobacterwas demonstrated in 1% of samples of both beef and pork (see Table 3).

In similar mapping studies, the occurrence of Campylobacter in poultry meat was found torange between 25 and 50% (see Table 3). The occurrence in poultry was found to be lower

during the winter months than in summer.

Campylobacter can be isolated from surface water as a result of faecal contamination from

wildlife, birds, and humans, or in ditches from fields where manure has been spread. In

countries where the water supply is based to some extent on surface water, outbreaks due to

contaminated water reservoirs have been described. Campylobacter has also been isolated

from seawater, and shellfish have been reported as causes of campylobacteriosis outbreaks.

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Unpasteurized milk has been described as the cause of several major outbreaks of human

campylobacteriosis. The presence of Campylobacter in raw milk is primarily due to faecal

contamination, but a few cases of campylobacter mastitis in dairy cattlehave been described.

Eggs have not been described as a source of campylobacter infections in humans.

The different sources of human campylobacteriosis, and their relative importance, have not

yet been fully clarified. During the period from May 1996 to September 1997, the Danish

Zoonosis Center carried out a case-control study concerning sporadic human

campylobacteriosis. The result of the study indicates a number of factors causing increased

risk of campylobacteriosis. These are: foreign travel within four weeks of the onset of disease;

ingestion of inadequately heat-treated poultry; ingestion of beef or pork which has been

grilled/barbecued or roasted over open fire; ingestion of drinking water having a foul smell or

taste, in combination with private water supplies. The development and implementing of

suitable typing methods would contribute substantially to the assessment of the relativeimportance of individual infection sources of human campylobacteriosis.

Results of mapping studies and monitoring programmes during the period 1995-1997 are

presented in Table 3.

Table 3.Occurrence of Campylobacter in foods, 1995-1997.

Product type 1997 1996 1995

%

positivesamples

Number

ofsamples

%

positivesamples

Number

ofsamples

%

positivesamples

Number

ofsamples

Non heat-treated chicken

25 637 41 186 40 133Danishproduce

Non heat-treated turkey

29 238 24 103 25 191

Other poultry 35 77 22 9 0 2

Imported Non heat-treated chicken

31 320 41 88 68 34

Non heat-

treated turkey

17 140 28 43 25 24

Other poultry 25 299 35 88 50 14

Beef 0.7 573 2 198 1 395

Pork 1 495 2 177 1 408

Venison 3 202 19 144

Vegetables 0 154

Fruits 0 138

Shellfish 0 186

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5. YERSINIA ENTEROCOLITICA

5.1 Importance

Infections caused by Yersinia enterocolitica were largely unknown until the early 1960es,

when the first cases of gastro-enteritis caused by this bacterium were diagnosed.

Since then, yersiniosis has been diagnosed with increasing frequency until a culmination in

1985 with approx. 1,500 cases. Subsequently the number has gradually decreased, and 426

cases were registered in 1997. These figures comprise positive cultivations of faecal samples

from patients suffering from gastro-enteritis. To these should be added an unknown number

of diagnosed cases of increased antibody contents in serum. The true incidence is not known,

as the course of the disease varies from mild cases that are easily overcome, to severe casesrequiring hospitalization. There are indications that the infection is considerably more

widespread than evidenced by the number of diagnoses. Thus, antibodies have been found in

8% of Danish donor blood samples. In a German study, antibodies were found in up to 39%

of donor blood samples.

Yersiniosis in humans usually manifests itself as gastro-enteritis. The disease varies from a

slight stomach trouble to a severe and protracted course. In some cases the intestinal lymph

nodes are affected, which may cause severe pains similar to those of appendicitis.

Furthermore, localized infections elsewhere in the organism are sometimes seen, and in a few

cases septicaemia. The incubation period varies from approx. 3 to 10 days in most cases.

In some patients also a secondary symptom complex is seen, characterized by reactions from

especially skin and connective tissues. Among other things, tumefaction of joints may occur,

so-called reactive arthritis, of such severe nature that patients may be disabled for several

months. Characteristically, persons having the tissue type HLA-B27 are particularly exposed

to these sequelae. Thus, approx. 80% of patients with sequelae have this tissue type, which is

shared by only approx. 10% of the Danish population.

Especially the yersinia types O:3 and O:9 exhibit a tendency to incur late reactions. Thesetypes are widespread in Europe, whereas in the USA, where type O:8 is predominant, late

reactions are rarely seen.

The sequelae, being caused by the reaction of the organism to liberation of endotoxin from

the cell walls of the bacteria, occur relatively frequently after yersinia infections. However,

such reactions may also be seen after other bacterial infections.

In Denmark, virtually all pathogenic strains belong to biotype 4, serotype O:3, while strains

belonging to biotype 1 are regarded as non-pathogenic environmental types.

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The infective dose is believed to be in the order of 105-106bacteria, but considerable variation

must be taken into account, depending on the immunological and overall health status of

exposed persons.


Y. enterocolitica is capable of growth at temperatures close to 0C, and may thus grow in

refrigerated foods. Due to its facultatively anaerobic properties (i.e. growing in the presence

as well as absence of oxygen), it is also capable of growing in vacuum packed or modified

atmosphere packed foods.

With respect to its salt and pH tolerance, Y. enterocoliticadoes not differ much from other

intestinal bacteria. It can grow in foods having a salt concentration up to 5-7% salt in the

aqueous phase; i.e. in relatively salty foods such as brine-pickled ham, bacon, or cured saddle

of pork. It grows in the pH interval of approx. 4-9, with its optimum at 7.2-7.4. Y.

enterocoliticais rather heat sensitive and will be inactivated by heat treatment at 60C for a

mere 1-3 minutes. It is also quite sensitive to ionizing radiation. Experiments seem to indicate

that Y. enterocolitica has a poor ability to grow in a mixed flora with other cold-tolerant

bacteria.

Light salting in connection with vacuum packing or modified atmosphere packing will,

provided optimum hygienic conditions of handling and packing, result in a long shelf-life for

refrigerated foods. This involves a risk of improved possibilities for cold-tolerant bacteria

such as Y. enterocolitica to multiply to infective levels even under marginal growthconditions. In a Danish study of vacuum-packed delicatessen goods, carried out in 1990,

pathogenic types of Y. enterocolitica could not be demonstrated. On the other hand, non-

pathogenic types were isolated in some cases, which seems to indicate growth conditions also

for pathogenic types.

In a mapping study of raw and non heat-treated pork products, Y. enterocolitica was

demonstrated in 1.2% of 508 samples of preserved, non heat-treated products such as bacon

and smoked fillet. In raw pork, the bacteria could be demonstrated in 3.5% of 398 samples of

minced meat. In cut-out pieces of meat, the bacteria were found in 1.4% of samples (see

Table 4).

Already in the early 1960es, pigs were associated with Y. enterocolitica, because these

bacteria were found in a study of the natural intestinal flora of pigs. Since then, numerous

studies have shown healthy slaughter pigs to be carriers of pathogenic types of Y.

enterocolitica. Occurrences of 25% in faeces and more than 80% in tonsils have been

demonstrated. In quantitative studies, levels up to 104-105pathogenic Y. enterocoliticaper g

of faeces have been found.

In a Danish study of the incidence in Danish pig herds, 82% were found to be infected. It was

not possible to demonstrate any relation between the occurrence of the bacteria and the

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production system of the farms. Thus, the bacteria are also widespread within the SPF system

(specific pathogen-free pig stocks).

Pigs are symptom-free carriers, and Y. enterocoliticahas been found to be widespread among

pigs in most countries where published studies have been carried out. This applies to

countries such as England, Japan, New Zealand, and the USA. The serotypes, however, vary

appreciably from one country to another. In the different countries, the types found in pigs

and the types found to cause human yersiniosis are coincident.

The transmission routes of Y. enterocoliticahave not yet been fully clarified; but there can

hardly be any doubt that pigs constitute the primary source of human yersiniosis.

An obvious infection risk is cross-contamination from infected raw pork to foods which,

when refrigerated, have sufficient keepability to permit the bacteria to multiply to infective

levels. Studies have shown that this can happen within 4-5 days.

Milk has been the source of several major outbreaks; but in nearly all cases, a relation to pigs

has subsequently been found.

Results of the 1997 mapping study are presented in Table 4.

Table 4.Occurrence of Yersinia enterocolitica in pork from retail stages, 1997.

Product type Number of samples Y. enterocoliticademonstrated

(% positive)

Cut-out pieces

Minced meat

Processed minced meat

279

398

128

1.4

3.5

1.6

Preserved, non heat-treated products(e.g. bacon, fillet)

508 1.2

Total 1,326 2.0

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6. ESCHERICHIA COLI O157

6.1 Importance

Since the early 1980es, verotoxin (VT)-producing Escherichia coli, serotypes O157:H7 and

O157:H-, (VTEC O157) has been known as an important food-borne pathogenic zoonotic

bacterium. It has caused a number of very large outbreaks in various countries; and VTEC

O157 infections are characterized by occasionally concomitant severe complications that

ultimately may be lethal.

VT-producingE. coliserotypes O157:H7 and O157:H-are characterized by producing toxins

which may kill vero cells, which is a renal cell line from the African Vero monkey. The

toxins, designated VT1 and VT2, are closely related to the shigatoxin, which is produced bythe bacterium Shigella dysenteria type 1, and are referred to as verotoxin, verocytotoxin,

Shiga Like Toxin (SLT), or merely shigatoxin (ST).

Verotoxin is formed by several different E. coli serotypes, collectively referred to as VT-

producing E. coli (VTEC). More than 160 different serotypes are known as VT-producing,

but their pathogenic potential has not been clarified for all serotypes. At the international

level, increasing attention is focused on serotypes other than O157:H7 and O157:H-.

Primarily, this goes for the serotypes O26:H11, O26:H-, O104:H21, O111:H-, and O145:H-,

all of which have been reported as causes of food-borne infections.

In Denmark, serotype O157 was the cause of one-third of all VTEC cases in 1997, while

types other than O157 were responsible for two-thirds of cases. However, international

experience showsE.coliO157:H7 to be by far the most frequent cause of VTEC outbreaks.

E. coliO157 is demonstrated by identification of the O157 antigen. Since serotype O157 is

not inevitably VT-producing, it is necessary always to analyse for toxin production and a

number of other virulence characteristics associated with pathogenic VTEC. Diagnostic

means of demonstrating VTEC serotypes other than O157 in foods are as yet limited, and the

elucidation of infection sources of disease in humans is therefore difficult.Those serotypes of VTEC that are most often associated with disease cases, are also referred

to as enterohaemorrhagicE. coli(EHEC). The term enterohaemorrhagic refers to the typical

symptoms of VTEC O157 infection, haemorrhagic diarrhoea. However, the symptoms may

also be non-complicated watery diarrhoea or asymptomatic excretion. The incubation period

of the infection varies from 1 to 7 days, 3-4 days being typical. In some cases (0-10%) the

infection is attended by potentially lethal complications in the form of acute renal failure and

anaemia: haemolytic uraemic syndrome (HUS). Children seem to be particularly susceptible

to HUS, and the infection may be lethal.

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The first registration of VT-producing E. coliO157:H7 as the cause of food-borne disease

outbreaks was in 1982 in the USA, where contaminated hamburgers from a fast-food

restaurant chain resulted in haemorrhagic diarrhoea. Since then, numerous small and large

outbreaks of VTEC O157 have been reported. The largest outbreak till now was registered in

Japan in the summer of 1996, involving more than 9,000 persons of which approx. 800 werehospitalized, and 11 died.

In many countries, the number of VTEC O157 cases has shown an increasing tendency

through the 1990es. In England and Wales, 411, 792, and 660 cases were reported in the years

1994, 1995, and 1996, respectively, corresponding to between 0.8 and 1.52 cases per 100,000

inhabitants. However, the actual number is assumed to be considerably higher.

Among the Nordic countries, Finland and Sweden have reported outbreaks. In Sweden, a

pattern of outbreaks was observed for the first time in the autumn of 1995, with up to 10-15

registered disease cases within a few weeks. The sources of the outbreaks were never broughtto light, but some of the cases were ascribed to raw milk from a dairy herd infected with

VTEC O157. Until the 1995 outbreak, Sweden had reported 0-3 cases per year; but after the

outbreak, 1-2 cases per week were diagnosed.

Till now, Denmark has been spared outbreaks of VTEC, and only few sporadic disease cases

are registered every year. During the period 1986-1996, the Danish Serum Institute reported

60 cases, of which 23 involved serotype O157. In 1997, 12 sporadic cases of VTEC O157

were diagnosed. This figure, being high in relation to Danish conditions, is primarily ascribed

to improved diagnostics and increased awareness of the bacteria.

The infectivedose of VTEC O157 is low. A few hundred bacteria may cause disease; thus,

propagation in foods is not necessary. There have also been reports of communicative

infection, which fact is of special importance in relation to outbreaks in families with young

children. Infection between siblings and dissemination in kindergartens are well-known

phenomena. Healthy carriers may therefore present a considerable infection risk.


E. coliO157:H7 grows at temperatures between 8 and 45C, and survives refrigeration andfreezing for months with no substantial reduction. By pasteurization or heating of foods to a

core temperature of 75C, elimination is ensured.

The lower pH limit for growth is stated to be around 4-4.5, but the pH tolerance range is

considerably wider. Thus, E. coliO157:H7 may survive in standard culture media at pH 2.

The growth of the bacteria is inhibited by 4% NaCl at 10C, and at a NaCl concentration of

8%, growth at 37C in standard culture media will cease.

E. coli is an intestinal bacterium of normal occurrence in humans and in most warm-blooded

animals. In ruminants it is also usual that a portion of the intestinal flora consists of different

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VTEC serotypes. This fact is illustrated in analyses of beef, where 5-20% of samples being

contaminated with different VTEC serotypes is not an unusual occurrence.

VTEC serotype O157 is often demonstrated in cattle, sheep, and red deer. In foreign studies, a

VTEC O157 incidence of 0-4.9% in cattle is reported; and studies in Denmark seem to

indicate an essentially similar incidence in Danish cattle. The figures do not reveal the facts

that there are significant seasonal fluctuations, and that the incidence in cattle also appears to

decrease with increasing age. In studies of one high-level infected English cattle herd it was

reported that up to 68% of heifers and 14% of lactating cows may excreteE. coliO157.

E. coliO157 may be demonstrated in foods; but in general, available studies indicate low

occurrences, often less than one per cent. A Danish mapping study in 1996 showed that

among 2,112 samples of minced beef and pork from the retail stage, 7 samples were

contaminated with E. coli O157, including 4 samples contaminated with VTEC O157. In

1997, one of 1,100 beef samples was found to be contaminated with VTEC O157, and 4samples with non-VT-producingE. coliO157 (see Table 5).

VTEC O157 has been isolated from pigs in a few cases; but pigs are not normally regarded as

a reservoir of VTEC O157, and findings in pork may quite well be due to cross-contamination

from other meat types.

At the international level, the majority of outbreaks are associated with foods containing beef,

and insufficiently heat-treated minced beef is often the most exposed food item.

Unpasteurized milk, yoghurt, water, apple cider (juice), sprouts, lettuce, salami, venison,

bathing water, etc. have likewise been reported as sources of outbreaks, and all potentiallyfaecally-contaminated foods must be considered likely to cause outbreaks. Accordingly, the

majority ofE. coliO157:H7 infections are classical food-borne zoonotic infections.

Cattle infected with E. coliO157:H7 will rarely exhibit clinical symptoms. In many herds,

individual animals excrete bacteria, but the excretors will be different animals in each case.

However, there is a need of studies to provide sufficient insight in the spreading of the

bacteria in the animal reservoir and to form the basis of a regular eradication plan. As yet,

methods for an adequately certain identification of infected animals are not available.

Results of mapping studies during the period 1996-1997 are shown in Table 5.

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Table 5.Occurrence of Escherichia coli O157 in fresh meat products from the retail stage,1996 and 1997.

Product type Number ofsamples

1997 / 1996

Results (% positive samples)

VT E. coliO157 non-VT E. coliO1571997 1996 1997 1996

Minced beef 1,100 /1,584 0.1 0.1 0.4 0.1

Minced pork 300 / 528 - 0.4 - 0.4

Mutton and lamb 300 / 0 0.7 - - -

Deer 200 / 0 1.0 - - -

Total number ofsamples

1,900 / 2,112 0.3 0.2 0.2 0.1

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7. LISTERIA MONOCYTOGENES

7.1 Importance

Listeriosis is a severe disease caused by L. monocytogenes. Listeriosis manifests itself as

meningitis and encephalitis, or as septicaemia. The disease mainly affects persons who are

decrepit or immunodeficient due to other disorders; but also healthy persons may be attacked.

The mortality rate may be above 50% for patients in high-risk groups, but only a few per cent

in otherwise healthy patients. Pregnant women constitute a particular high-risk group. The

woman rarely has any symptoms, or only slight flu-like symptoms, whereas the foetus will be

infected and is aborted or born prematurely with infection. L. monocytogenesmay also cause

transitory gastric infection, but this disease form is rarely diagnosed.

The incubation period of human listeriosis is reported to be between 1 and 70 days.

The infective dose for humans is more or less unknown; but the general presence of the

bacteria in numbers below 100/g in many raw foods is not assumed to constitute any risk of

disease, even for specially exposed persons. When the occurrence of L. monocytogenes in

foods is compared with the number of listeriosis cases, it seems obvious that many people are

capable of tolerating far higher bacterial levels without being ill. No actual dose/response

curves exist; but internationally there is a general agreement on a safety limit of 100/g.

However, some countries still require the absence of L. monocytogenes in ready-to-eat

products.

In the first half of the 1980es, approx. 10-20 cases of human listeriosis were registered in

Denmark every year. In 1985-1986, an epidemic was registered, after which the number of

diagnosed listeriosis cases increased to approx. 30-40 per year. This level remained

unchanged until 1992. Prior to the first epidemic in 1986, listeriosis was not registered

systematically, so it is not known whether the number of listeria infections actually did

increase during the 1980es.

Many of the cases in 1985-1986 and in 1989-1990 were caused by a particular type ofL.

monocytogenes: serotype 4b, the so-called EPI type. This type is also known to have caused

several major outbreaks in other countries. From 1992 through 1995, less than 30 cases were

registered per year, but in 1996 a total of 39 cases was diagnosed, the largest number since the

1986 epidemic. Typing of isolates from 1996 has shown many different types, but only a few

of the EPI type. 33 cases were diagnosed in 1997.

The majority of human infections are sporadic, but several major outbreaks have been

described. Dairy products, particularly including soft cheeses, coleslaw, meat products such

as pt, sausages, and gas-packed delicatessen goods, are examples of food items which have

caused outbreaks of human listeriosis in other countries. In Denmark, it has not been possible

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to demonstrate any relation between the intake of any particular food item and cases of human

listeriosis.

Even though the EPI type is predominant in relation to outbreaks of listeriosis, all types ofL.

monocytogenes must be considered potentially pathogenic. Thus, in connection with sporadic

listeriosis cases, many different types are isolated. In experimental studies, the EPI type was

not found to be particularly virulent in comparison with other types.


A number of characteristics enable L. monocytogenes to grow in many different

environments. Its nutrient demands are very modest, it grows in the presence as well as the

absence of oxygen (facultatively anaerobic), has a wide pH range for growth (approx. 4.5-9),

grows at temperatures between 0 and 45C, and is able to multiply at high salt concentrations

(10% salt in the aqueous phase, corresponding to an awvalue of 0.92). It does not survive low

pasteurizing (72C for 15 seconds) and is furthermore usually sensitive to most disinfectants.

L. monocytogenesis widely distributed in the nature: in wildlife, plants, and soil, its natural

habitat being earth and decaying plant material. Survival and growth of L. monocytogenesin

the earth depends on soil type, moisture, and temperature. L. monocytogenes occurs with

varying frequencies in faeces from healthy animals and humans; consequently, it is also

usually found in sewage and surface water. The occurrence in faeces varies considerably

between different animal species. Danish studies have shown that among cattle, approx. one-

third of the animals are healthy excretors of L. monocytogenes. Among pigs, on the otherhand, only a few per cent are healthy excretors, and among humans, only a few per cent of the

healthy population excreteL. monocytogenesin faeces.

L. monocytogenesoccurs naturally in all raw foods.

In the mapping studies of the municipal food control units concerning vegetables, lettuce, and

sprouts in 1996-1997, L. monocytogenes was demonstrated in 7% of 351 samples by

qualitative analysis. In analyses of 737 samples by a semi-quantitative method, L.

monocytogeneswas demonstrated in 11 samples at levels above 10/g, including 9 samples at

levels above 100/g. Two samples containedL. monocytogenesat levels above 10,000/g.

The average occurrence ofL. monocytogenesin raw milk is 2.2%, determined on the basis of

5,100 samples collected worldwide.L. monocytogeneshas favourable conditions of growth in

milk and soft cheeses.

In raw meat, it is usually found in low numbers, due to the fact that pigs and cattle are natural

carriers ofL. monocytogenesin the throat and intestinal tract. SinceL. monocytogenesis cold-

tolerant and at the same time relatively resistant to unfavourable external conditions, it may

readily settle in the food production environment and is often isolated from production

premises and cold-storage rooms at slaughterhouses. It adheres easily to surfaces such asstainless steel, forming a bio-film that may be difficult to remove.

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L. monocytogenesis often present in minced beef and minced pork, where it has occasionally

(3-4%) been found in numbers above 100/g.

A Danish study has shown that 9% of heat-treated meat products from the retail stage contain

L. monocytogenes(see Table 6). This incidence is predominantly due to cross-contamination

of meat products after the heat treatment, especially through handling such as slicing and

packing. The occurrence of L. monocytogenes in heat-treated products constitutes a special

problem, because it has particularly favourable conditions of growth when the competing

flora has been eliminated. This is illustrated by a Danish study showing that 3% of samples of

heat-treated meat products contain more than 100 per g. For preserved, non heat-treated meat

products, the corresponding percentage was only 0.6, in spite of the fact that 23.5% of the

products in this category containedL. monocytogenes(see Table 6).

L. monocytogenesis not likely to occur on raw fish caught in non-contaminated waters; but

on the other hand, it quite readily settles in the cool fish processing environment, on crates,tools, machinery, etc.

L. monocytogenes is often found in cold-smoked salmon. Studies from many different

countries show that 10-15% of analysed samples are L. monocytogenes positive. A few

studies show occurrences in as much as one-third of samples.

In a Danish study in 1995-1996, L. monocytogeneswas isolated in 34% of 190 samples of

cold-smoked salmon from 10 different manufacturers immediately after production. The

levels were low, however; usually less than 10/g. At the expiry of the use-by date (after 3-8

weeks), 41% of the samples wereL. monocytogenespositive. In 8% of the samples, the levelswere higher than 100/g (see Table 6).

The mapping study showed comparative results for cold-smoked Greenland halibut and for

pickled salmon and pickled Greenland halibut.

A previous study of heat-treated fish products showed anL. monocytogenesoccurrence of 5%

of 74 samples analysed during the first days after production.

In a mapping study of delicatessen goods, L. monocytogeneswas demonstrated in 3-12% of

the heat-treated products (see Table 7). The lowest occurrence, 3%, was found in saveloy.None of the samples had contents above 10L. monocytogenesper g. The highest occurrence,

12%, was found in pork roll and poultry sausage. 2% of pork rolls and 7% of poultry sausages

contained more than 100L. monocytogenesper g. These studies indicate that for this type of

foods, which are often sold with a relatively long shelf-life, there is a need of initiatives to

prevent growth of L. monocytogenes. Similar considerations may apply to preserved, non

heat-treated fish products.

Results of the mapping studies are presented in Tables 6 and 7.

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Table 6.Occurrence of Listeria monocytogenes in foods from the retail stage in Denmark,1995.

Producttype Number of

samples

Found in 25 g

(%)

>10/g

(%)

>100/g

(%)Raw meat 343 30.9 12.6 3.6

Meat products, heat-treated 431 9.0 5.2 2.4

Pt 341 1.8 0.3 0.3

Preserved meat products,non heat-treated 328 23.5 2.4 0.6

Raw fish 232 14.2 3.2 0.5

Preserved fish products,

non heat-treated 335 10.8 5.1 1.8

Table 7.Findings of Listeria monocytogenes in delicatessen goods, 1994/95.

Product

type

Number

of

samples

Packing date Expiry date

Qualitatively Quantitatively, per g Quantitatively, per g

Number

of

positive

samples

%

positive

samples

100 100

Hot dog 175 12 7 175 0 0 160 7 8

Pork roll 155 18 12 154 1 0 150 2 3

Smoked

fillet

140 20 14 140 0 0 140 0 0

Saveloy 165 5 3 165 0 0 165 0 0

Poultry

sausage

130 16 12 125 1 4 121 0 9

Smokedbreast ofturkey

35 11 31 34 1 0 34 1 0

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8. STAPHYLOCOCCUS AUREUS

8.1 Importance

Staphylococcus aureus is an occasionally pathogenic micro-organism which is present as a

natural part of the flora on skin and mucous membranes, and which therefore often occurs in

low numbers in foods. Some S. aureus are capable of producing a toxin which is called

staphylococcal enterotoxin because the most pronounced symptoms affect the intestinal tract;

but the majority of S. aureus strains are not enterotoxin-producing. Actually, the toxin is a

neurotoxin which acts on the vomit centre of the central nervous system. The toxin is formed

during the growth of the bacterium, and intoxications occur when a food item containing pre-

formed toxin is ingested. Accordingly, S. aureuscauses food-borne intoxication in contrast to

food-borne infection.

S. aureus is known to cause wound infections and abscesses. Therefore, S. aureus

intoxications may also occur when a person, infected by an enterotoxin-producing strain on

his/her hands, transfers the bacteria to foods during handling or preparation.

Staphylococcal intoxications are relatively frequent as well as unpleasant. Characteristically,

intoxication symptoms set in shortly after the ingestion of a toxin-containing food item

(normally after 1-6 hours), with acute malaise, headache, shivering, vomiting, and diarrhoea.

Normally, the intoxication is of short duration, usually with full recovery within 24 hours.

It is difficult to state any number of cases. Every year a modest number of outbreaks is

reported, but due to the brief course of the intoxication, with full recovery after a day or so,

the great majority of cases will never come to the notice of the health authorities.

Normally it is assumed that the number of S. aureusmust reach 106per g of food in order to

cause an intoxication.


S. aureushas its growth temperature optimum around 37C and growth range from approx.10

to 45C. It is relatively sensitive to heat treatment and will be eliminated by a heat treatment

equivalent to low pasteurizing. The toxin, on the other hand, is highly heat stable and will not

be inactivated by less than prolonged boiling.

S. aureusis characterized by its ability to grow at very low water activities (aw), down to 0.86.

Thus, it is the most salt tolerant of the known pathogenic bacteria.

S. aureus is highly resistant to desiccation, which is part of the reason for its frequent

presence on surfaces such as skin and mucous membranes, as well as in dust. It is socommonly occurring that it must be assumed to be present in low numbers in most raw foods.

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S. aureusis capable of growth within the pH interval from approx. 4 to 9.

Salted foods, favouring salt tolerant micro-organisms, constitute a risk of growth of S. aureus,

resulting in potential toxin formation. Bacon, pasta products, and salted mushrooms are

examples of salted or desiccated products that have given rise to intoxications.

Staphylococci grow best in foods where the competition from other bacteria has been

eliminated, e.g. heat-treated foods. Characteristically, it does not compete successfully with

other bacteria, which is why it is normally not assumed to present any problem in raw,

unsalted foods. Whenever a heat-treated food item is handled, there is a risk of transferring

staphylococci from their primary reservoir, skin and mucous membranes. If such food items

are inadequately refrigerated or frozen, there is a risk that any staphylococci present will have

an opportunity for propagation and toxin formation.

Intoxications from many different foods have been seen. Among numerous examples, thefollowing may be mentioned: hash kept hot at too low a temperature; hard-boiled eggs used at

an Easter egg hunt; hot-stored barnaise and hollandaise sauce not boiled; desserts left to

stand too long on the kitchen worktop; and boiled potatoes kept outside the fridge overnight

and subsequently used in potato salad.

Results from the 1997 mapping study [4] of Staphylococcus aureus in hot-stored, ready-to-eat

dishes from the retail stage are shown in Table 8.

Table 8.Occurrence of Staphylococcus aureus in samples of hot-stored dishes from the retail

stage, 1997.

Staphylococcus aureus

Number of analyses 1,309

Number of positive analyses 1

% positive analyses 0.08

Viable counts in positive analyses 100

Storage temperature (C) 60

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9. CLOSTRIDIUM PERFRINGENS

9.1 Importance

Clostridium perfringensis one of the most frequent causes of food-borne disease. The disease

in an intoxication caused by a toxin which the bacterium liberates in the small intestine. The

symptoms mainly occur in the posterior part of the intestinal tract, with intense stomach pains

and violent diarrhoea. Furthermore, headache and fever are often seen, but rarely vomiting.

The course of the disease, which is caused by a sub-type of C. perfringens, type A, is usually

brief and non-complicated. The incubation period is normally from about 8 to 12 hours.

It takes a large number of bacteria, approx. 106per g of food, to bring about intoxication.

A less frequent type of food-borne C. perfringens infection is caused by type C. This type

causes an infection which gives rise to a necrotizing enteritis (i.e. necrosis of intestinal

tissues). The disease is very serious, often lethal. Symptoms set in suddenly in the form of

violent stomach pains. The disease is very rare and has been observed only a few times in

Europe since the early 1960es.


C. perfringens has its growth temperature optimum around 37C and growth range from

approx. 10 to 50C. The bacterium is spore-forming. When present in spore form in foods, it

is very resistant to heat treatment; however, the heat resistance of the spores varies

considerably. Thus, some spores are readily killed by boiling, whereas others will be able to

survive boiling for a long time. The vegetative bacteria are easily inactivated by heat

treatment. Under favourable conditions, the bacterium grows very rapidly, for which reason

spores, having survived heat treatment, will have a good opportunity of growth during the

cooling phase. Its very wide growth range enhances its opportunities of multiplication.

C. perfringens is anaerobic (i.e. growing best in the absence of oxygen), but tolerates the

presence of oxygen better than most other clostridia. Thus, it will be able to growimmediately below the surface of foods, preferably together with other bacteria helping to

reduce the oxygen concentration.

C. perfringensoccurs naturally in the intestinal tract and in faecally contaminated material.

Due to its spore-forming ability, it is capable of surviving under unfavourable conditions

through long periods of time; consequently, it is often found in dry environments, e.g. in earth

and dust. It is common in raw meat, vegetables, and dried foods such as spices.

An interesting feature of C. perfringens is the fact that its toxin formation takes place

especially in relation to sporulation, i.e. the transformation of the vegetative bacterial cell to aspore. This process, which is effected only with difficulty in foods or in the laboratory,

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preferably takes place in the alkaline environment in the anterior part of the intestinal tract.

The highly acidic environment in the ventricle will kill a large proportion of the vegetative

bacteria, which fact explains why it takes approx. 106bacteria per g of food to bring about an

intoxication.

It follows from the above that a thorough re-heating of foods containing C. perfringenswill

eliminate the vegetative cells and thereby prevent intoxication. Furthermore, the toxin is heat

sensitive, so any pre-formed toxin will be inactivated by thorough heat treatment.

Analyses of foods that have given rise to intoxications often reveal only modest numbers of

bacteria, since C. perfringensoften dies off, autosterilizes, during storage of the food.

Food, e.g. meat, which gives occasion for a C. perfringensintoxication, often contains a low

initial number of spores and has been exposed to a slow and inadequate cooling during

preparation, or has been kept hot at too low a temperature. Furthermore, the food has typicallyoften been insufficiently re-heated. Large portions of mixed stew, difficult to cool down and

difficult to re-heat, is a typical example of a food type which has given rise to intoxication.

Lasagne is another typical example.

Results of the 1997 mapping study [4] of C. perfringens in hot-stored ready-to-eat dishes

from the retail stage are shown in Table 9.

Table 9.Occurrence of Clostridium perfringens in samples of hot-stored dishes from theretail stage, 1997.

Clostridium perfringens




Viable counts in positive analyses; average (min.-

max.)

1,200 (10-2,700)

Storage temperature (C); average (min.-max.) 57 (48-65)

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10. BACILLUS CEREUS

10.1 Importance

Bacillus cereusgives rise to two different types of food intoxication. The emetic syndrome,

dominated by rapidly occurring vomiting, and the diarrhoeic syndrome, dominated by later

occurring diarrhoea.

In the diarrhoeic syndrome, the symptoms usually set in 8-16 hours after the ingestion of food

containingB. cereus. Stomach pains, watery diarrhoea, and rectal cramps occur. Nausea and

vomiting are rare. The symptoms, seldom lasting for more than 12 hours, are caused by an

enterotoxin, a large protein molecule which is sensitive to trypsine.

In the emetic syndrome, vomiting occurs very soon, from less than 1 hour to approx. 5 hours,

after the intake of contaminated food. Diarrhoea is also observed in many cases. The emetic

syndrome is caused by a small peptide having a molecular weight of less than 5,000. The

toxin is highly heat resistant as well as stable towards trypsine and pepsine. It is not antigenic

and cannot be demonstrated by immunological methods.

A relatively large number of bacteria must be ingested before symptoms of intoxication set in.

For intoxications of the emetic type, a requirement of 105-108B. cereusper g of food has been

described; and according to various studies, the diarrhoeic type requires the ingestion of a

similar number.


B. cereusis capable of growth in the temperature interval from approx. 10 to 48C, with its

optimum at 37C. Some strains, however, are able to grow at lower temperatures, from 5-7C

to around 37C. B. cereusis spore-forming. The spores are resistant to physical effects and

can survive boiling. Spores of the cold-tolerant types survive boiling for some minutes,

whereas spores of the other types survive for somewhat longer, up to 30 or 60 minutes. B.

cereusdoes not compete successfully in mixed cultures, but grows better in pure culture.

B. cereus is widely distributed and is commonly found in a large number of foods such as

dairy products, rice, spices, and other dried products. Due to its spore-forming ability, it is

highly resistant to physical effects such as desiccation, which is why it is widespread in earth

and dust. Thus, it also forms a normal part of the flora in cereals, spices, and vegetables.

The diarrhoeic syndrome is caused by a large number of foods, such as meat and vegetable

dishes, custards, puddings, and other desserts that have undergone a form of heat treatment.

Since the bacterium is so commonly occurring and resistant to heat treatment, it will be

present in many heat-treated dishes in relatively low numbers, but without any competingconcomitant flora. Slow cooling or too warm storage possibly in combination with

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insufficient re-heating will provide opportunity for growth and may result in finished dishes

containing very high numbers of B. cereus. While the emetic toxin, which is a small

molecule, is highly heat resistant, the diarrhoeic toxin sensitive to heat. Therefore, thorough

heating will inactivate vegetative bacteria as well as any diarrhoeic toxin that might be

present.

The emetic syndrome is almost invariably related to rice dishes that have been kept non-

refrigerated for too long.B. cereusis to be regarded as a natural part of the flora in raw rice.

The spores of serotype H1, which is most frequently associated with outbreaks caused by rice

dishes, are considerably more heat resistant than those of other serotypes.

Results from the 1997 mapping study [4] of the occurrence ofBacillus cereusare presented in

Table 10.

Table 10.Occurrence of Bacillus cereus in samples of hot-stored dishes from the retail stage,1997.

Bacillus cereus




Viable counts (per g) in positive analyses;

average (min.-max.)

1,500 (100-11,000)

Storage temperature (C); average (min.-max.) 69 (48-126*)

* Sampled directly from the oven.

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11. ANTIBIOTIC RESISTANCE

11.1 Monitoring of antibiotic resistance

In Denmark, a national monitoring of antibiotic resistance in bacteria from production animals,

foods, and humans has been carried out since 1996, as well as a monitoring of the consumption of

antibiotics. The monitoring is a joint co-operation between the Danish Veterinary Laboratory, the

Danish Veterinary and Food Administration, the Danish National Serum Institute, the Danish

Plant Directorate, and the Danish Medicines Agency.

As part of the monitoring, the trends in the consumption of antimicrobial agents for veterinary

and human therapeutic use are followed, as well as the trends in the consumption of thoseantimicrobial agents that are used as growth promoters in animal feedstuffs. Furthermore, the

incidence of resistance in a number of pathogenic bacteria, including Enterococci, Streptococci,

Staphylococci,Listeria monocytogenes, Salmonella, Campylobacter,and Yersinia enterocolitica,

is monitored. Likewise, resistance conditions in the indicator bacteria Escherichia coli,

Enterococcus faecium, and Enterococcus faecalis are monitored. Table 11 lists those bacterial

species from animals, foods, and humans, which are routinely monitored for antibiotic resistance.

The monitoring provides data that can be used as a support in the selection of antibiotics for the

treatment of diseased animals and humans. Further, the monitoring results can be used to identifyany resistance problems that might be due to inappropriate uses of antibiotics.

The monitoring results are summarized in annual reports: Consumption of antimicrobial agents

and occurrence of antimicrobial resistance in bacteria from food animals, food and humans in

Denmark [5].

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Table 11. List of bacteria included in the antibiotic resistance monitoring from stable totable in Denmark.

Bacteria Animals Foods Humans

Healthy Diseased Healthy Diseased

E. coli + + + (+) +

Enterococci + + (+) +

Streptococci + +

Staphylococci + + +

L. monocytogenes + +

Salmonella + + + +

Campylobacter + + +

Y. enterocolitica + +

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12. SUMMARY AND CONCLUSION

Sub-report No. 5, dealing with microbial contaminants, describes background data and

analysis programmes for the most important pathogenic food-borne bacteria in Denmark,carried out during the period 1993-1997. At present, actual monitoring programmes have

been initiated only for Salmonella and Campylobacter, which are the two bacterial species

causing the largest number of gastro-intestinal infections. The results for Yersinia

enterocolitica, Escherichia coli O157, Staphylococcus aureus, Bacillus cereus, Clostridium

perfringens, and Listeria monocytogenes were based on mapping studies illustrating the

current situation with respect to the occurrence in foods.

The results for the occurrence of Salmonellain chicken products (non heat-treated) from the

retail stage show a decrease from 16.1% in 1994 to 5.7% in 1997. In pork products (non heat-

treated), the occurrence was reduced from 2.5% in 1994 to 1.4% in 1997. Since Salmonella

TyphimuriumDT104, multi-resistant, has not caused any human disease cases in Denmark

during the period 1993-1997, the occurrence of this bacterium in foods has not been analysed.

The occurrence of Campylobacterin poultry products (non heat-treated) from the retail stage

was around 25% on an average throughout the period. In beef and pork products, the

occurrence of Campylobacterwas approx. 1% during the period 1995-1997.

In the mapping studies, occurrences of Yersinia enterocoliticain 3.5% of minced pork and in

1-2% of other pork products were demonstrated.Escherichia coliO157 was demonstrated in0.3% of samples of beef, pork, deer, mutton, and lamb.

In a mapping study, Listeria monocytogeneswas demonstrated in 23.5% of preserved, non

heat-treated meat products. In 0.6% of samples, L. monocytogeneswas present in numbers

above 100/g. In the same study,L. monocytogeneswas demonstrated in 10.8% of samples of

preserved, non heat-treated fish products, and 1.8% of samples had contents above 100/g.

In another mapping study, L. monocytogenes was demonstrated in 3-12% of heat-treated

delicatessen goods. The lowest occurrence, 3%, was found in saveloy. None of the samples

contained levels above 10/g. The highest occurrence, 12%, was found in pork roll and poultrysausage. 2% of pork rolls and 7% of poultry sausages contained levels above 100/g. These

studies seem to indicate that for this type of foods, often sold with a long shelf-life, initiatives

preventing the growth of L. monocytogenesare needed. Similar considerations may apply to

preserved, non heat-treated fish products.

In a mapping study concerning ready-to-eat, ready-prepared dishes from the retail stage,

Bacillus cereus, Staphylococcus aureus, and Clostridium perfringenswere demonstrated in

0.8%, 0.2%, and 0.08%, respectively, of the analysed samples.

Mapping studies and monitoring programmes for the most important pathogenic food-bornebacteria will be continued in the next period. Information on the occurrence of pathogenic

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bacteria in foods is essential with regard to the risk assessments that are to be carried out in

the years to come concerning relevant bacteria and foods. Thus, the risk assessment includes

information on the occurrence of the bacteria as well as data on dietary habits in an

assessment of the exposure of the population.

Likewise, updated information on the occurrence of pathogenic bacteria is of great

importance in relation to the implementation and evaluation of prevention and control

initiatives.

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13. REFERENCES

1. The National Food Agency of Denmark. Food monitoring in Denmark. Nutrients and

Contaminants, 1983-1987. Publication No. 195, September 1990.2. The National Food Agency of Denmark. Food monitoring, 1988-1992. Publication No.

232, December 1995.

3. National Food Agency of Denmark. Danish Food Monitoring Programme, 1996 Review,based on the report Food Monitoring 1988-1992. Publication No. 239, June 1997.

4. Veterinr- og Fdevaredirektoratet (Danish Veterinary and Food Administration).Aerobt kimtal samt forekomst af Bacillus cereus, Clostridium perfringens ogStaphylococcus aureus i varmholdte retter i detailleddet i 1997 (Aerobic viable countsand occurrence of Bacillus cereus, Clostridium perfringens, and Staphylococcus aureusin hot-stored dishes at the retail stage). Mad & Mikroorganismer, No. 4, ISSN 1397-5404.

5. Consumption of antimicrobial agents and occurrence of antimicrobial resistance inbacteria from food animals, food and humans in Denmark. Danish IntegratedAntimicrobial Resistance Monitoring and Research Programme (DANMAP), No. 1,February 1997, ISSN 1397-078X.

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Microbial Food Cantamination