Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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appendicitis-like attack, diarrhea (lasting several

weeks), fever, rash, nausea, headache, and vomiting.

The minimum infectious dose is unknown. Illness

onset is usually between 24 and 48 h after intake,

although the incubation period can be as long as

11 days.

0002 Y. enterocolitica is widely distributed in the envir-

onment, in lakes and stream waters, which are

sources of the organisms to warm-blooded animals.

Animals from which Y. enterocolitica has been isol-

ated include cats, birds, dogs, guinea-pigs, rats,

horses, chickens, cattle, swine, lambs, and fish. Cer-

tain biotypes and serogroups are associated with a

specific host. A principal source or reservoir of viru-

lent Y. enterocolitica are pigs. Virulent strains of

Y. enterocolitica often reside in the oral cavity and

the gastrointestinal tract of pigs. Serogroups 0:3 and

0:9 are mainly associated with pork tongues

and tonsillar area. Despite this, it has been shown

that pork meat has accidentally been a vehicle for

yersiniosis.

0003 There are four serogroups of Y. enterocolitica asso-

ciated with pathogenicity: 0:3, 0:5, 0:8, and 0:9. The

genes encoding for invasion of mammalian cells

are located on the chromosome, while a 40–50-

MDa plasmid encodes most of the other virulence-

associated phenotypes. The 40–50-MDa plasmid is

present in almost all pathogenic Yersinia species,

and the plasmids appear to be homologous. Although

heat-stable enterotoxin has been isolated from most

clinical isolates, it is uncertain whether this toxin

plays a role in the pathogenicity of the organism.

0004 Y. enterocolitica have been detected in a wide var-

iety of processed foods, particularly pasteurized milk,

dairy products, vacuum-packaged meats, seafood,

vegetables, and many ready-to-use foods. Incidence

varies between countries. The most common Y. enter-

ocolitica serogroups in human infections are 0:3, 0.5,

27, 0:8, and 0:9. In Europe, Africa, and Japan,

serogroups 0:3 and 0:9 are the most common. These

serogroups are prevalent in Denmark, Belgium, and

Sweden, and are a significant cause of gastrointestinal

illnesses in those countries.

Growth Requirements

Temperature

0005 Y. enterocolitica is a psychrotroph and can grow at

temperatures as low as 0



C and as high as 44



C, the

optimum range being 22–29



C. For biochemical re-

actions, the optimum temperature is about 28



Cin

pure culture, but it is a poor competitor at this tem-

perature. Multiplication can occur between 2and



C, but growth is very slow. At 3



C, it takes 200 h

to increase 2 logarithmic cycles in culture medium.

Growth at 0–2



C in milk after 20 days has been

observed. Generation times at 4



C appear to be

strain-dependent, varying between 16 and 26 h in

pasteurized milk, and growth is preceded by an

extended lag time of up to 40 h. Increases in levels

of up to 5 log increments in 10–14 days have been

recorded in a variety of artificially contaminated

cooked foods stored at 4



C. Growth at 0–1



Con

pork and chicken has been observed and on raw

beef kept for 10 days at 0–1



C. Growth is slightly

slower in the presence of competitor organisms but at



C competes better against other psychrotrophic

bacteria. In milk at 4



C, Y. enterocolitica grow and

reach numbers of up to 10

CFU ml

1

in 7 days,

and compete well with the background flora.

0006The pH range for growth is 4.6–9.0, with an opti-

mum pH of 7.0–8.0. The minimum pH at which

growth can occur varies between strains and also

depends on storage temperature and the acidulant

used. The following values have been found for six

strains of Y. enterocolitica with the pH adjusted with

HCl and incubated for 21 days: 4.42–4.80 at 4



4.36–4.83 at 7



C; 4.26–4.50 at 10



C and 4.18–4.36

at 20



C. Growth inhibition at low pH is greater at



C than at 20



C, and the use of organic acids raises

the minimum pH for growth. When organic acids are

used to adjust pH, the order of their effectiveness is

acetic >lactic > citric. Acetic acid is the most inhibi-

tory organic acid commonly used in food, preventing

growth below 5.7 at 4



C and 5.1 at 20



C. It has also

been shown that tolerance to high pH values is de-

pendent on temperature and on the medium used.

Growth has been demonstrated at pH 9.5 at 10



but not at 6



C. Under the same pH and temperature

conditions, the inhibitory effects of acids are quite

different. At a high pH (5.8), the order of inhibition

of Y. enterocolitica 0:9 at 4



C is formic acid > acetic

acid > propionic acid > lactic acid, whereas at a

lower pH (4.2) the order is formic acid > lactic

acid > acetic acid > propionic acid. The mechanisms

of the interaction of these acids inside bacterial cells

differ under aerobic and anaerobic conditions. While

the inhibitory effect of lactic acid is enhanced under

anaerobic conditions, the acetic and formic acids

are less effective.

Heat Tolerance

0007According to available data, the heat resistance

of Y. enterocolitica seems to vary widely among

different strains. Strains of Y. enterocolitica are not

particularly heat-resistant. Sublethal injury to

6240

YERSINIA ENTEROCOLITICA

/Properties and Occurrence

Y. enterocolitica cells has been reported after heat

treatment at 47



C for more than 12 min. However,

the magnitude of the effect depends on growth

temperature. In the range of 4–20



C, the growth

temperature does not influence heat resistance at

54–66



C for Y. enterocolitica. However, when cells

are grown at 37



C, heat resistance increases fourfold.

The heat resistance of Y. enterocolitica is also influ-

enced by the pH of the heating medium. The pH of

maximum heat resistance in citrate phosphate buffer

is pH 7 for cells grown at 37



C but pH 5 for those

grown at 4



C. In both, the magnitude of the pH effect

on heat resistance is constant at all heating tempera-

tures. Different studies have been carried out in milk as

pasteurization and thermalization are achieved

effectively when the inoculum does not exceed 10

CFU ml

1

Freezing

0008 Refrigeration reduces the multiplication capacity of

Y. enterocolitica but does not inhibit it completely.

Growth at low temperatures depends on other

factors, mainly on pH. Good growth has been ob-

served at 4



C, when the pH is neutral, but very little

growth has been detected at a slightly acidic pH (5.4).

The ability of Y. enterocolitica to withstand freezing

has been shown: cultures of Y. enterocolitica can

survive for several weeks at 20



C without any

reduction in the number of viable cells. Also, in stud-

ies with frozen foods, 3–4 logarithmic reductions

have been found to occur after 4 weeks of storage

at 18



C. Despite this, Y. enterocolitica has been

isolated from a wide range of frozen foods.

Irradiation

0009 The ability of Y. enterocolitica to survive g-radiation

depends on the food composition/treatment and

the presence of preservatives. As Y. enterocolitica is

more radiosensitive than other common foodborne

pathogens such as Salmonella and Listeria, it can be

completely eliminated at a dose range of 3–4 kGy.

With low numbers of Y. enterocolitica (D

values

between 0.25 and 0.30 kGy), a contamination of up

to 10

–10

CFU g

1

can be positively controlled by a

1–1.5-kGy dose. Thus, the average dose of 1 kGy

recommended by the FDA appears to be adequate

for eliminating Yersinia. While irradiation even at

6 kGy fails to prevent survival in raw pork, the bac-

terium is completely suppressed in cooked ham and

salami treated with lower doses of radiation. Faster

recovery of radiation-injured cells in raw meat than

in cured meat with spices or in cooked ham demon-

strates that Y. enterocolitica in irradiated products

can be inhibited by using combination processes

including irradiation.

Preservatives and Packaging

0010The behavior of Y. enterocolitica in response to vari-

ous preservatives depends on the temperature and

level of the chemical, pH, and amount of sodium

chloride present. At low pHs and temperatures, po-

tassium sorbate is very effective against Y. enteroco-

litica. In contrast, other preservatives such as sodium

phosphite and sodium hypophosphite are inhibitory

at relatively high pH values. Packaging containing

high levels of carbon dioxide is very effective at

delaying growth and can cause complete inhibition.

Y. enterocolitica can survive sodium chloride con-

centrations of 6–7% at specific temperature and

pH levels. Although 7% NaCl is inhibitory at 3



growth occurs with 5% NaCl. Other preservatives

such as potassium sorbate, sodium hypophosphite,

sodium phosphite, and curing salt delay or inhibit

the growth of this microorganism.

Predictive Modeling

0011Predictive models are used to describe the behavior of

microorganisms under different conditions such as

temperature, pH, water activity, oxidation–reduction

potential, and additives. They may be applied in vari-

ous ways to ensure the microbial safety and quality.

The main objective is to describe mathematically the

growth or inactivation of microorganisms in food

under prescribed food conditions.

0012Several models charting the growth and survival of

Yersinia enterocolitica have been developed by study-

ing the effect of different factors, such as: pH (4–8.5);

organic acids (acetic, lactic, sulfuric, and citric

acid); temperature (0–42



C); NaCl concentration

(0–10.5%); sodium nitrite (0–200 p.p.m.), strain

with and without plasmids related to virulence

agents, and mild heat treatment (50–60



C). These

models have been validated in different foods such

as pa

, packed cold smoked salmon, UHT milk,

yogurt, mayonnaise, meat and meat products, eggs,

fish, and tofu; in general, predictive models oversti-

mate growth rate values on the fail-safe side, i.e., the

prediction always describes faster growth rates than

the values observed in real foods.

Epidemiological and Clinical Features

0013From an epidemiological standpoint, Y. enterocolitica

is widespread in nature, occurring in the gastrointest-

inal tract of an array of animal hosts, including

mammals, avian species, and cold-blooded species,

and is occasionally isolated in humans. Of all sources,

swine appear to be the major source of strains

YERSINIA ENTEROCOLITICA

/Properties and Occurrence 6241

pathogenic for humans. Several outbreaks have

been reported where infection has been associated

with contact with pigs, and indirect contact with

contaminated pork products and other foods

(Table 1). Some outbreaks in families were attributed

to exposure to cats and dogs. On rare occasions, it

can be transmitted as a result of the bacterium passing

from stools or soiled fingers of one person to

the mouth of another person. This may happen

when basic hygiene and handwashing habits are in-

adequate.

0014 In general, sporadic or food-related outbreaks are

more frequent in regions where pigs are the carriers of

this microorganism. Seasonality seems to play a role

in the incidence of this microorganism in focal geo-

graphical areas, with a higher frequency noted during

cooler periods of the year.

0015 Children are infected more often than adults, and

confirmed cases or Y. enterocolitica in humans corres-

pond to 1 in 100 000 persons per year. In the USA, it is

estimated to cause 3000–20 000 cases each year.

Most recently, it has been shown that humans can

be asymptomatic, since clinically normal blood

donors have supplied Yersinia enterocolitica-infected

blood resulting in fatal septicemia in transfused

patients. Nevertheless, 90% of cases originate from

food.

0016Human clinical infections with Y. enterocolitica

occur most frequently after the intake of food or

water contaminated with this pathogen. The infec-

tious dose estimated to be sufficient to cause human

illness is approximately 10

cells, but consumer sus-

ceptibility will vary according to individual levels of

immunocompetence, age, and general health. After

intake, the bacteria pass through the stomach, colon-

ize the epithelial cell surface of the small intestinal

mucosa, and cause diarrhea, possibly by producing

the enterotoxin YST. This microorganism can also

invade epithelial cells, attach to M cells, and pene-

trate Peyer’s patches, where it multiplies with the

help of several surface components that enable it to

resist phagocytosis and survive complement-medi-

ated killing. The invasion process and virulence factor

production are complex processes that are thermore-

gulated by both plasmid and chromosomally encoded

information. Continued proliferation of bacteria

results in inflammation leading to the formation of

local microabscesses and ulceration of the overlying

epithelium. In a relatively short period, ingested

Y. enterocolitica reach the terminal ileum, spread to

the mesenteric lymph nodes, and enter the blood

stream.

0017From a clinical standpoint, Y. enterocolitica is

a common human pathogen that causes a variety

tbl0001 Table 1 Outbreaks of foodborne Y. enterocolitica infection

Vehicle Number of cases(confirmed) Biogroup(s) Country Year

Pasteurized milk 1000 (172)

13a, 13b USA 1982

3(3)

6,30 UK 1984

19(19)

10K UK 1985

17(17)

6,30 UK 1985

Chocolate milk 218 (38)

8 USA 1976

250

USA 1978

Cream deserts 4

Belgium 1975

Powered milk/turkey chow mein 239(159)

8 USA 1981

Bean sprouts 16

8 USA 1982

Tofu 87(56)

8, tacoma(21) USA 1981

Chitterlings 14

USA 1988

Pork 1

USA 1994

Pork (pork cheese) 5(3)

3 Hungary 1983

From Tacket C, Narain J, Sattin R et al. (1984) A multistate outbreak of infections caused by Yersinia enterocolitica transmitted by pasteurized milk. Journal

of the American Medical Association 251: 383–486.

From Greenwood MH (1993) Yersinia enterocolitica. Properties and occurrence In: Macrae R, Robinson RK and Sadler MJ (eds) Encyclopaedia of Food

Science, Food Technology and Nutrition. London: Academic Press.

From Black R, Jackson R, Tsai T et al. (1978) Epidemic Yersinia enterocolitica infection due to contaminated chocolate milk. New England Journal of

Medicine 298: 76–79.

From Silliker J (1986) Yersinia enterocolitica. Food Technology 8: 22.

From Richard J, Moiner J, Cornu C, Goor M and Dereume G (1975) Le

ndemie

Yersinia enterocolitica dans le Tournaisis. Lille Medical 20(1): 36–43.

From Cover T and Aber R (1989) Yersinia enterocolitica. New England Journal of Medicine 231: 16–24.

From Aulisio C, Stanfield J, Weagent S and Hill W (1983) Yersiniosis associated with tofu consumption: serological, biochemical and pathogenicity

studies of Yersinia enterocolitica isolates. Journal of Food Protection 46: 226–230.

From Lee L, Gerber A and Lonsway D et al. (1990) Yersinia enterocolitica 0:3 infections in infants and children, associated with the household preparation

of chitterlings. New England Journal of Medicine 322: 984–987.

From Stoddard J, Wechsler D, Nataro J and Casella J (1994) Yersinia enterocolitica infection in a patient with sickle cell disease after the exposure to

chitterlings. American Journal of Pediatric Hematology and Oncology 16: 153–155.

6242

YERSINIA ENTEROCOLITICA

/Properties and Occurrence

of intestinal and extraintestinal syndromes, with

varying degrees of severity, ranging from mild

gastroenteritis to mesenteric lymphadenitis (often

confused with appendicitis), and septicemia. Infec-

tions caused by this microorganism may also reveal

postinfectious immunological sequelae, including

erythema nodosum, arthritis, miocarditis, and glo-

merulonephritis.

Occurrence in Foods

0018 Foods analyzed for Y. enterocolitica show that the

distribution of this microorganism is fairly exten-

sive; it has been isolated in beef, lamb, pork,

oysters, shrimp, crabs, and water. The vast majority

of these isolates are avirulent, and virulent strains

seldom occur in any of these foods. Studies have

examined the fecal droppings of rats near slaughter-

houses and have found that the rats carried virulent

Y. enterocolitica probably picked up from the

pork that was being processed at the slaughter-

houses.

0019 The 68% positive samples of porcine tongues taken

from freshly slaughtered animals have been isolated,

and these represent six serogroups, 0:8 being the

most common and 0:6, 30 the second most common,

followed by other serogroups including 0:3, 0:13, 7,

0:18, and 0:46. Several studies performed in Brazil

have reported positive isolations of Yersinia entero-

colitica from raw milk (16.8%), pasteurized milk

(1.7%), raw beef (80%) and chicken (60%), ground

beef, liver, and pork (20%). In one study performed

in the USA, after examining 100 milk samples, 12

raw samples and one pasteurized sample yielded

Yersinia enterocolitica. Another study of this product

in France reported that 81% of raw milk samples

were positive, the predominant serovar being

0:5 (Table 2).

0020 Outbreaks of yersiniosis have been reported in

which chocolate, coleslaw, tofu, bean sprout, pas-

teurized milk, and pork chitterlings have been

singled out as vehicles (Table 1). One of the first

and most important outbreaks occurred in the USA,

where 220 schoolchildren were taken ill; it was

confirmed that Y. enterocolitica had caused the

problem and that chocolate milk was the vehicle of

infection. This milk was prepared at a small dairy by

means of vat pasteurization; the chocolate was

added after pasteurization and mixed with a paddle;

hence, there was plenty of potential postprocessing

contamination. In another case, the vehicle of trans-

mission was tofu (soyabean curd) that had been

packaged in untreated spring water contaminated

with Y. enterocolitica.

0021Both virulent and avirulent strains of Y. enteroco-

litica have been isolated from a variety of dairy prod-

ucts. Three milkborne outbreaks occurred in the

USA, two attributable to serogroup 0:8 and one to

serogroup 0:13a, 13b. In 1982, there was an outbreak

linked to pasteurized milk; 170 cases were confirmed,

although epidemiological studies revealed that

more than 1000 people were infected. The strain

was also isolated from the plastic carrying cases

used to carry the milk after bottling. In this case,

outdated milk had been brought to a pig farm

in carrying cases. The cases were placed in mud

and manure, the milk poured into troughs for the

pigs, and the carrying cases brought back to the

plant and superficially washed. Although it was con-

firmed that the pigs were the source of infection,

it was impossible to determine exactly how the

microorganism had reached the milk from the

carrying cases.

Prevention of Food Contamination

0022Food-related toxic infections caused by Yersinia

enterocolitica seem to be related to foods in

cold storage. In this situation, it is difficult to define

preventive measures (repeated washing of vegetables,

mainly in collective restaurants, strengthening of

the cold chain, etc.) that enable the spread of this

food-related infection to be restricted. These limita-

tions to prevention highlight the importance of

monitoring human infections (networks of expert

doctors, control laboratories, and reference labora-

tories for studying strains). The prevention of food-

borne disease depends on careful food production,

handling of raw products, and preparation of fin-

ished foods. Hazards can be introduced at any

point from farm to table. Monitoring and control

technologies systematically applied to food produc-

tion by the HACCP are highly effective for prevent-

ing foodborne illness.

0023Primary control of the microorganism would

require changes to current slaughtering practice. The

‘bagging technique’–the method of enclosing the

anus and rectum of pigs in a plastic bag during

slaughter – has been introduced in several slaughter-

houses, mainly in Norway, and it has been shown that

this effectively reduced carcass contamination by

Yersinia enterocolitica. The removal of the head,

tongue, and pharynx at an early stage of slaughter

and improvements in the way in which intestines are

removed would also help to prevent carcass contam-

ination. Additional practices, such as the exclusion of

tonsillar tissue from sale or the scalding of tongue and

head meat prior to the dispatch of products may also

be necessary.

YERSINIA ENTEROCOLITICA

/Properties and Occurrence 6243

tbl0002 Table 2 Occurrence of Yersinia enterocolitica on food

Food Percentage of samples Serogroup(s) Country

Raw milk 81

0:5 France

USA

18.2

Canada

16.8

Brazil

Morocco

Raw milk, goat 71

Australia

Pasteurized milk 1

USA

13.7

Brazil

Traditional fermeted milk 6.3

Morocco

Cheese 9.2

Canada

Morocco

Turkish Feta cheese 28.8

Turkey

Salmon and trout 23

Raw beef and chicken 80

Brazil

Ground meat and liver 60

Brazil

Ground meat 48

Germany

Porcine tongues 68

0:8; 1:6, 30; 0:3; 0:13, 7; 0:8; 0:46 USA

50.8

USA

Chitterling >50

0:3 USA

Pork meat 26.7

USA

Brazil

10.83

0:3, 3B phage type 2; 0:3, 4 phage type 8; biotype 1; 2 Japan

0:3 Norway

Drinking water 5

Germany

USA

Vegetables 1

Italy

France

From Vidon D and Delmas C (1981) Incidence of Yersinia enterocolitica in raw milk in Eastern France. Applied and Environmental Microbiology 41: 353–359.

From Moustafa M, Ahmed A and Marth E (1983) Occurrence of Yersinia enterocolitica in raw and pasteurized milk. Journal of Food Protection 46: 276–278.

From Schiemann D (1978) Association of Y. enterocolitica with the manufacture of cheese and occurrence in pasteurized milk. Applied and Environmental

Microbiology 36: 274–277.

From Tibana A, Warnken M, Nunes M et al. (1987) Occurrence of Yersinia enterocolitica in raw and pasteurized milk in Rio de Janeiro, Brazil. Journal of

Food Protection 50: 580–583.

From Hamama A, El Marrakchi A and El Othmani F (1992) Occurence ofYersinia enterocolitica in milk and dairy products in Morocco. International Journal

of Food Microbiology 16: 69–77.

From Hughes J and Jensen N (1981) Yersinia enterocolitica in raw goat’s milk. Applied and Environmental Microbiology 41: 309–310.

From Simsek O and Arici M (1994) Pathogenic microorganisms in cheese and their foodborne disease. In: Cheese With All Aspects, 2nd edn, Tekirdag

Trakya University.

From Davies A, Capell C, Jehanno D, Nychas G and Kirby R (2001) Incidence of foodborne pathogens on European fish. Food Control 12: 67–71.

From Warnken A, Nunes M and Noleto A (1987) Incidence of Yersinia enterocolitica in meat samples purchased in Rio de Janeiro, Brazil. Journal of Food

Protection 50: 578–579, 583.

From Kleinlein N, Unterman F and Beissner H (1989) Occurrence of Salmonella and Ye r s i n ia species as well as Listeria monocytogenes in minced meat.

Fleischwirtschaft 69: 1474–1476.

From Doyle MP, Hugdahl MB and Taylor SL (1981) Isolation of virulentYersinia enterocolitica from porcine tongues. Applied and Environmental Microbiology

42: 661–666.

Harmon MC (1984) A study of the incidence and pathogenic potential of Yersinia enterocolitica in Indiana pork. Dissertation Abstracts International, B 44:

3707.

From Lee LA, Gerber DR, Lonsway JD et al. Yersinia enterocolitica O:3 infections in infants and children, associated with the household preparation of

chitterlings. New England Journal of Medicine 322: 984–987.

From Fukushima H, Hoshima K, Nakamura R and Ito Y (1987) Occurrence of Ye r s i n ia spp. in raw beef, pork and chicken. Zentralblatt fur Bakteriologie

Mikrobiologie und Hygiene (B) 184: 50–59.

From Johannessen G, Kapperud G and Kruse H (2000) Occurrence ofYersinia enterocolitica in Norwegian pork products determined by PCR method and a

traditional culturing method. International Journal of Food Microbiology 54: 75–80.

From Schindler P (1984) Isolation of Yersinia enterocolitica from drinking water in South Bavaria. Zentralblatt fur Bakteriologie Mikrobiologie und Hygiene

180: 76–84.

From Langeland G (1983) Yersinia enterocolitica and Yersinia enterocolitica-like bacteria in drinking water and sewage sludge. Acta Pathologica,

Microbiologica et Immunologica Scandinavica 91: 179–185.

From Gola S, Previdi M, Mutti P and Belloli S (1990) Microbiological investigation of frozen vegetables, incidence of Listeria and other psychrotrophic

pathogens. Industria Conserve 65: 36–38.

From Catteau M, Krembel C and Wauters G (1985) Yersinia enterocolitica in raw vegetables. Science Aliments 5: 103–106.

From Darbas H, Riviere M and Obertic J (1985) Ye r s i ni a in refrigerated vegetables. Science Aliments 5: 81–84.

6244

YERSINIA ENTEROCOLITICA

/Properties and Occurrence

0024 Although normal detergents and disinfectants used

in the food industry are effective for eliminating Yer-

sinia enterocolitica, their effect is reduced if fatty

residues are present. The regular use of steam

cleaning instead of cleaning with hot water at 85



has been used successfully at a milk pasteurization

plant.

0025 Handlers of meal are the last critical control point

before foods reach the table. Therefore, several prac-

tical food-handling precautions could reduce the risk

of contamination of food by this microorganism.

Cross-contamination of foods can be avoided by sep-

arating cooked and raw food and also by washing

food handlers’ hands, cutting boards, and contamin-

ated surfaces. Practical recommendations can be

followed by consumers, such as avoiding the con-

sumption of raw or undercooked pork, raw milk, or

related byproducts, and washing hands carefully

before eating, preparing food after contact with

animals, and after handling raw meat. After handling

raw chitterlings, it is recommended that hands and

fingernails be cleaned thoroughly with soap and

water before touching children or their toys, bottles,

or pacifiers.

See also: Epidemiology; Freezing: Principles; Heat

Treatment: Ultra-high Temperature (UHT) Treatments;

Chemical and Microbiological Changes; Electrical

Process Heating; Irradiation of Foods: Basic Principles;

pH – Principles and Measurement; Preservatives:

Classifications and Properties; Yersinia enterocolitica:

Detection and Treatment

Further Reading

Adams MR and Moss MO (1995) Food Microbiology.

Cambridge: The Royal Society of Chemistry.

Bottone EJ (1999) Yersinia enterocolitica: overview and

epidemiological correlates. Microbes and Infection

1: 323–333.

Doyle MP and Cliver DO (1990) Yersinia enterocolitica. In:

Cliver DO (ed.) Foodborne Diseases, pp. 223–

228.San Diego, CA: Academic Press.

Eley AR (1992) Microbial Food Poisoning. London: Chap-

man & Hall.

Forsythe SJ (2000) The Microbiology of Safe Food. Oxford:

Blackwell Science.

Greewood MH (1993) Yersinia enterocolitica. Properties

and occurrence. In: Macrae R, Robinson RK and Sadler

MJ (eds) Encyclopaedia of Food Science, Food Technol-

ogy and Nutrition. London: Academic Press.

Jay JM (2000) Modern Food Microbiology, 6th edn.

Gaithersburg, MD: Aspen.

Schiemann DA (1989) Yersinia enterocolitica and Yersinia

pseudotuberculosis In: Doyle MP (ed.) Foodborne

Bacterial Pathogens, pp. 601–672. New York: Marcel

Dekker.

Detection and Treatment

A Ramesh, B P Padmapriya, S Bharathi and

M C Varadaraj, Central Food Technological Research

Institute, Mysore, India

Background

0001Yersinia enterocolitica is an invasive enteropathogen

prevalent in the environment and often isolated from

soil, water, food, and clinical sources. The serotypes

of Y. enterocolitica associated with disease in humans

are O:3, O:9, O:8, and O:5, 27, and all harbor a

70–75-kb virulence plasmid. The bacterium has

been recognized as a significant foodborne pathogen

owing to its ability to survive in both vacuum-packed

and refrigerated food samples. Thus, in the light of

food hygiene and public health, it is imperative to

develop rapid methods of detecting Y. enterocolitica.

Various methods consisting of conventional cultural

methods, detection of virulence plasmid, immu-

nological methods, and rapid nucleic acid-based

techniques have been described for the detection and

identification of Y. enterocolitica. In an infection, the

principal clinical feature in humans is abdominal pain

and diarrhea accompanied by fever. Reactive arthritis

and erythema nodosum have also been reported as

common complications. Several cases of diarrhea

resulting from Y. enterocolitica infection resolve on

their own, thus avoiding the need for an antibiotic

treatment. However, in the case of a severe infection,

antibiotics are administered. The following section

describes the methods of detection and treatment of

Y. enterocolitica.

Detection of

Yersinia enterocolitica

0002The common food samples associated with Y. enter-

ocolitica infection are pork, milk and milk products,

and raw vegetables. The detection of pathogenic

strains of Y. enterocolitica from food samples and

clinical specimens is mainly based on the isolation of

the bacterium. A scheme of the different detection

methods followed for Y. enterocolitica is shown in

Figure 1.

Conventional Methods of Detection

0003Yersinia enterocolitica is a Gram-negative rod or

ovoid, facultatively anaerobic, oxidase-negative,

catalase-positive nonsporulating motile bacterium.

The isolation of this bacterium from food samples is

difficult, because it is present in low numbers and is

slow growing compared to other coexisting members

YERSINIA ENTEROCOLITICA

/Detection and Treatment 6245

of the enterobacteriaceae present in the background

microflora. This underscores the need for a selective

enrichment technique to facilitate the isolation of the

bacterium. A basic isolation scheme involves preen-

richment in a highly nutritious, well-buffered medium

containing peptone, yeast extract, and mineral salts at

temperatures ranging from 4 to 37



C over 1–9 days.

The salient features of the various types of enrichment

procedures are indicated in Table 1.

0004 Selective enrichment media like bile oxalate sor-

bose (BOS) broth, and phosphate buffer saline

(PBS) supplemented with sorbitol and bile salts

with a postenrichment alkali treatment has been

widely used to enrich Y. enterocolitica from food

samples. This method is simple, sensitive, and rapid,

eliminating a large number of contaminating

non-Yersinia species. Y. enterocolitica is also a psy-

chrotroph, capable of growing under refrigerated

conditions. Hence, cold enrichment can increase the

isolation rate of the bacterium. Irgasan–ticarcillin–

chlorate (ITC) enrichment has been shown to be

more efficient in isolating pathogenic bioserotype

from pork. The enriched culture is plated on to

the selective media cefsulodin–irgasan–novabiocin

tbl0001 Table 1 Enrichment broth and growth conditions used for the isolation of Yersinia enterocolitica

Primary

enrichment

Incubation

temperature (



Incubation

period(days)

Secondary

enrichment

Incubation

period (days)

Incubation

temperature (



MRB 23 5

PB 4 14

PB 4 14 MRB 23 5

CMB 23 28 MRB 23 5

SC 37 1

RMC 22 3

BHI 8–10 2 SB 22–28 2

ITC RT 2

PBS 4 2

PBSSB 4 21

BHI 8–10 2 KOH RT

BHI 8–10 2 SC RT 2

HOBS 4 5

PBS 4 21 MRB 27 2

BOS 22 3–5

BHI, brain–heart infusion broth; BOS, bile–oxalate–sorbitol broth; CMB, cooked meat broth; HOBS, hemoglobin–oxalate–bile–sorbitol

broth;ITC, irgasan–ticarcillin–chlorate; MRB, modified Rappaport broth; PB, Butterfields phosphate buffer; PBS, phosphate-buffered saline;

PBSSB, phosphate-buffered saline–sorbitol–bile salts; RMC, restrictive MgCl

broth; SB, sorbitol bile broth; SC, selenite cystine.

Detection of Yersinia enterocolitica

Conventional

method

Immunological

methods

Virulence plasmid

mediated methods

Nucleic acid

methods

Enrichment/Plating

on selective media

Agglutination Immunoassays

Low calcium

response

CV and CR

binding

Gene probe PCR

5' nuclease

assay

PCR-ELISA

fig0001 Figure 1 Schematic representation of the various detection methods for Yersinia enterocolitica.

6246

YERSINIA ENTEROCOLITICA

/Detection and Treatment

(CIN) agar and incubated for 24–48 h at 32–37



Cto

obtain characteristic colonies with a deep red center

(like a bull’s eye) with an entire edge and a transparent

border measuring 0.5–1.0 mm in diameter. Suspected

Yersinia colonies from CIN plates are selected and

suspended individually in trypticase soya broth

(TSB) for further characterization through diagnostic

biochemical tests, as indicated in Table 2. Yersinia

enterocolitica has been essentially classified into six

biogroups, i.e., 1A, 1B, 2, 3, 4, and 5. Biotype 6 is

rare.

0005 Current methods based on selective enrichment

merely detect the presence of Y. enterocolitica, and

these methods fail to recover all pathogenic biosero-

types. Further improvements in the specific detection

of the bacteria can be achieved through immuno-

logical and nucleic acid-based methods.

Immunological Detection of

Y. enterocolitica

0006 Immunological methods have been reported since the

early detection of Y. enterocolitica as an advancement

over conventional microbiological tests. The methods

that have been widely accepted include agglutination

tests and immunoassay.

0007 Agglutination tests Latex slide agglutination and

tube agglutination (Widal) tests are commonly used

and form the basis for many commercially available

kits for the rapid detection of Y. enterocolitica O:3

and O:9 antibodies in the serum. Latex of 0.9 mm

particle size was coated with specific antibodies

raised against serotypes O:3, O:5, O:6, O:8, and

O:9 of Y. enterocolitica. The latex reagents specific-

ally detected the O-antigen of Y. enterocolitica, with

no cross-reactivity between the serotypes. Alcohol-

treated bacterial cells are used as antigens in some

cases, and an agglutination titer of 640 or a fourfold

or greater increase in the titer is an indication of

recent or actual infection. Commercial kits are

available to detect O:3 and O:9 antigens of

Y. enterocolitica individually or in combination,

using the latex agglutination method.

0008Immunoassays The lack of sensitivity and specifi-

city of the agglutination tests hinders their ap-

plication as a routine detection method. However,

immunoassays like surface adhesion immunofluores-

cence (SAIF) technique, enzyme immunoassay (EIA),

immunoblot, and enzyme-linked immunosorbent

assay (ELISA) can alleviate this problem. SAIF has

been used to detect Y. enterocolitica in broth and

enriched meat cultures, with a polycarbonate mem-

brane to trap the cells on the surface of the membrane

prior to detection by immunofluorescence.

0009EIA was developed for the detection of Yersinia

immunoglobulin (Ig) complexes of known Ig class.

Immune complexes are attached to polystyrene

microtiter plates by rabbit antihuman immunoglobu-

lins. Detection of Y. enterocolitica O:3 antigens was

demonstrated using Fab fragments of alkaline phos-

phatase-conjugate antibody against the same sero-

type. Methods to differentiate serological responses

to Y. enterocolitica O:9 from responses to Brucella

have been very successful. However, immunoblots

tbl0002 Table 2 Differentation of Yersinia enterocolitica biogroups

Test Biogroup

1A 1B2345 6

Indole production þ þ  

Esculin hydrolysis þ/  

Voges–Prausker test þ þ þþþþ 

L-Ornithine decarboxylase þ þ þþþþ/þ

Pyrazinamidase þ   þ

Lipase þ þ  

D-Glucosidase þ   

Acid from

Inositol þ þ þþþþ þ/

Salicin þ   

L-Sorbose þ þ þþþ þ

Tr e h a l o s e þ þ þþþ þ

D-Xylo se þþþþV þ

Sucrose þ þ þþþþ 

This biotype is relatively rare.

V, variable reacton.

Modified from Bottone EJ (1999) Yersinia enterocolitica: overview and epidemiological correlates. Microbes and Infection 1: 323–333; Wauters G (1981)

Antigens of Yersinia enterocolitica. In: Bottone EJ (ed.), pp. 41–53. Boca Raton, FL: CRC Press.

YERSINIA ENTEROCOLITICA

/Detection and Treatment 6247

and ELISA targeting the Yersinia outer membrane

proteins (YOPs) are more sensitive and specific than

other serological tests for yersiniosis.

0010 Monoclonal antibodies specific for the lipopolysac-

charide (LPS) O antigens of Y. enterocolitica serotype

O:3 can be used for the detection of Y. enterocolitica

in colony immunoblots. This method has been shown

to be sensitive in detecting a single colony of Y. enter-

ocolitica in the presence of 3.110

heterologous

organisms. Besides, the assay was specific with no

significant cross-reactivity against any related sero-

types. An immunoblot kit called recom Blot Yersinia

IgG and recom Blot IgM/IgA has been developed and

provides recombinant Yersinia antigens in a test strip

format to detect IgG, IgM, or IgA antibodies directed

against Y. enterocolitica and Y. pseudotuberculosis.

0011 ELISAs using either purified lipopolysaccharide

or formalinized whole cells expressing virulence

plasmid-coded surface antigen (pYV

cells) can be

performed to detect Y. enterocolitica. It has been

shown that ELISA can specifically detect pYV anti-

bodies in sera, cross-adsorbed to formalinized pYV-

cured Y. enterocolitica O:3 cells prior to the assay.

The serological response against YOPs using AB-

ELISA was used to detect the O:9 serotype of

Y. enterocolitica. In order to avoid interference by

Y. enterocolitica O:9 LPS with YOPs in serological

analysis, an antigenic preparation of YOPs was

obtained from Y. enterocolitica O:7. There is a high

degree of homology between YOPs of different Yersi-

nia species and an absence of cross-reactivity between

Y. enterocolitica O:7 LPS and Y. enterocolitica O:9

LPS. The commercially available Y. enterocolitica

ELISA kit provides a high diagnostic sensitivity

capable of differentiating IgG from IgA and IgM.

0012 Although a host of immunological methods are

available to detect Y. enterocolitica for clinical appli-

cations, the use of these assays in a food system is

comparatively limited. An immunomagnetic separ-

ation technique has been shown as an efficient device

to sequester Y. enterocolitica in food samples and

facilitate its sensitive detection. The major factors

limiting the use of immunological techniques for the

detection of Y. enterocolitica in food systems are

the cross-reactivity with the background microflora

present in food samples and the inhibitors present in

the food matrix itself.

Detection of Virulent Plasmid-bearing (pYV

)

Strains

0013 The pathogenic strains of Y. enterocolitica are char-

acterized by the presence of a 70–75-kb plasmid

which encodes for a number of virulence factors

manifested as conspicuous colony morphology, auto-

agglutination, dye binding capacity, and low calcium

response. Plasmid-mediated phenotypic expression

has been used to detect virulent strains and to

discern them from avirulent plasmid-less strains.

In a calcium-deficient medium like the brain–heart

infusion agarose (BHO) calcium response of viru-

lent strain was tested. At 37



C, plasmid-bearing

virulent cells (pYV

) appear as pin-point colonies

0.36 mm in diameter, whereas avirulent cells

(pYV



) form distinctly larger colonies. The CIN

agar medium incorporated with crystal violet can

distinguish between pathogenic and nonpathogenic

strains. At 32



C, the colony morphology appears

similar for both the strains. However it has been

demonstrated that at 37



C, plasmid-bearing patho-

genic strains appear as densely stained microcolo-

nies compared to the nonpathogenic strains. Direct

detection of a plasmid-bearing virulent strain of Y.

enterocolitica has been carried out in artificially

contaminated food samples like pork chops, ground

pork, cheese, and zucchini. Enriched samples plated

on BHO incorporated with Congo red and incu-

bated at 37



C form red pin-point colonies as a

manifestation of plasmid-mediated dye binding

and a low calcium response. Thus, the Congo red

binding technique is suitable for direct detection

and isolation of pathogenic Y. enterocolitica using

a single medium.

Detection by Nucleic Acid Probes

0014The physiological traits encoded by the virulent plas-

mid are only expressed at 37



C, which also favors

plasmid loss accompanied by elimination of virulent

traits. Thus, the instability of the plasmid limits its use

as a tool for detecting virulent Y. enterocolitica in

clinical and food samples. The emergence of nucleic

acid probes has provided an efficient tool to detect

genes in Y. enterocolitica that encode specific

pathogenic traits. Such gene probes can be used in

colony- or dot-blot hybridization to directly detect

Y. enterocolitica in the presence of high background

microflora.Additionally,these probes canbeevaluated

as a substitute to tissue culture-based assay to ascertain

the invasive phenotype of pathogenic strains.

0015The target regions in a probe-based diagnostic

assay can be unique sequences within chromosomal

DNA, ribosomal RNA, or plasmid DNA. The riboso-

mal RNA is widely preferred as a target gene owing to

its ubiquity and relatively high copy number, which

enhances the assay sensitivity. The GENE-TRAK col-

orimetric hybridization assay for Y. enterocolitica has

been developed, employing probes directed against

specific signature sequences of 16 s rRNA. This assay

has been shown to be a convenient alternative to

conventional microbiological procedures, yielding

presumptive results in a significantly shorter time.

6248

YERSINIA ENTEROCOLITICA

/Detection and Treatment

However, this method lacks the discriminatory power

to differentiate virulent from avirulent strains.

0016 Probes targeting virulence plasmid genes can be

employed to specifically detect pathogenic Y. entero-

colitica serogroups and distinguish them from en-

vironmental Yersinia species, which are frequently

encountered in food samples. A synthetic radio-

actively labeled 19-mer oligonucleotide probe based

on the plasmid-encoded Yersinia adhesin A (Yad A)

gene has been used in colony hybridization to

detect pathogenic Y. enterocolitica in pork samples.

The probe demonstrates a superior estimation of

virulent strains in the samples than conventional

methods. Polymerase chain reaction (PCR)-generated

nonradioactive probes are safe, inexpensive, and

shelf-stable, and can be used in the detection of

the organism. In colony hybridization, the DIG

(digoxigenin)-labeled PCR-generated Vir F and Yad

A amplicons are able to detect pathogenic Y. entero-

colitica in artifically contaminated food samples and

indicate equivalence with the PCR assay and pheno-

typic test for pathogenicity.

0017 Spontaneous loss of plasmids in Y. enterocolitica

during in-vitro replication is frequently encountered

either in the environment or in the laboratory. This

fosters the need to develop probes targeting the

chromosomally encoded virulence factors like the

attachment invasion locus (Ail) and the heat-stable

enterotoxin (Yst). The gene coding for the attachment

invasion locus (Ail) has been found uniquely in patho-

genic strains of Y. enterocolitica, whereas the invasion

(Inv) gene in all Yersinia spp. DIG-labeled Inv and

Ail probes has been used to differentiate pathogenic

from nonpathogenic strains. A PCR-amplified DIG-

labeled Yst probe has been used in colony hybridiza-

tion to detect and differentiate between pathogenic

and nonpathogenic strains in naturally and artificially

contaminated pork and milk samples following

enrichment in ITC enrichment broth. The hybridiza-

tion results are concordant with those from biochem-

ical and serological tests, and a detection limit of

10 colony-forming units (CFU) ml

1

in the original

sample has been achieved. The use of a multi-

plex riboprobe can be adopted as a comprehensive

strategy to detect multiple targets, thus enhancing

assay sensitivity and ensuring detection of virulent

Y. enterocolitica irrespective of plasmid loss.

Detection by Polymerase Chain Reaction

0018 The use of gene probe-based hybridization assays

to detect Y. enterocolitica is limited by the lack of

sensitivity. PCR allows rapid and selective identifica-

tion of Y. enterocolitica and fulfils the criteria for

specificity and sensitivity. The Yad A gene located

on the virulence plasmid (pYV) encoding for an outer

membrane polymeric protein has been used as a target

to detect the presence of pathogenic Y. enterocolitica in

retailed pig tongue and minced meat. However, loss of

pYV is frequently encountered under certain condi-

tions of handling, especially in food samples. This

leads to the possibility of false-negative results. The

chromosomally encoded attachment invasion locus

(Ail) is characteristically associated with virulence

and can be an appropriate target to detect pathogenic

Y. enterocolitica. Ail-specific primers have been used

in PCR to identify virulent strains in blood, and the

levels of sensitivity achieved indicate the potential of

PCR in the diagnosis of transmission-related bactere-

mia caused by Y. enterocolitica. A two-step PCR

targeting the Ail gene has been employed to detect

pathogenic strains in environmental water, and the

results have demonstrated a superior specificity and

sensitivity over cultural methods.

0019A PCR method based on the targeting of the rfb

C gene involved in the biosynthesis of the O side chain

of Y. enterocolitica O:3 has enabled the detection

and differentiation between pathogenic strain of

Y. enterocolitica of serotype O:3 and Y. pseudotuber-

culosis and other pathogenic Y. enterocolitica. PCR

enables reliable and sensitive detection when applied

to pure cultures, but its efficiency is markedly curtailed

when applied to food and clinical samples. This is

mainly due to the potential inhibitors or high levels of

background microflora present in such samples.

Therefore, adequate sample preparation is required

to either eliminate such inhibitors or concentrate target

cells to yield satisfactory detection levels. Selective

enrichment followed by buoyant density gradient cen-

trifugation provides an efficient sample preparation

method prior to PCR. This treatment method has

facilitated the detection of 10 CFU ml

1

of the bacter-

ium in pork using a multiplex PCR simultaneously

targeting the plasmid-borne Yad A gene and the Yersi-

nia-specific 16S rRNA region. Immunomagnetic sep-

aration (IMS) of the target cells from meat samples has

enabled a nested PCR assay to detect 10–30 CFU g

1

meat in the presence of 10

-fold excess background

microflora. Thus, IMS provides an efficient way of

excluding the PCR inhibitors from the sample and

has the potential to be exploited for automation.

0020Methods for detecting virulent Y. enterocolitica

based on PCR involve gel electrophoresis for product

analysis and the use of ethidium bromide to stain

the gels, which is hazardous and impractical for rou-

tine analysis in food monitoring laboratories. The

fluorogenic 5

nuclease assay is an attractive option

for rapid detection of virulent Y. enterocolitica. The

assay exploits the 5

–3

nuclease activity of Taq DNA

polymerase and releases a probe with a fluorescent

reporter dye and a quencher, which hybridizes to

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