Yersinia enterocolitica and Yersinia pseudotuberculosis Detection in Foods

Yersinia enterocolitica and Y. pseudotuberculosis which can cause yersiniosis in humans and animals are thought to be significant food-borne pathogens and be important as hygiene indicator in food safety. The pathogenic Y. enterocolitica serotypes/biotypes are O:3/4 and 3 variant VP negative, O:5, 27/2, O:8/1b, and O:9/2, have been reported worldwide. Y. pseudotuberculosis is distributed less widely than Y. enterocolitica. Isolation methods usually involve selective and recovery enrichment of the food sample followed by plating onto selective media, confirmation of typical colonies and testing for virulence properties of isolated strains. Recently, DNA-based methods, such as PCR assays, have been developed to detect pathogenic Y. enterocolitica and Y. pseudotuberculosis in foods more rapidly, and sensitivity than can be achieved by conventional culture methods. This paper reviews commercially available conventional and PCR-based procedures for the detection of pathogenic Yersinia in food. These methods are effective as the isolation and detection methods to target pathogenic Y. enterocolitica and Y. pseudotuberculosis in foods.

It is therefore important to isolate and identify and differentiate food-borne pathogenic Yersinia from nonpathogenic Yersinia strains. Isolation methods usually involve enrichment of the food sample followed by plating onto selective media, confirmation of typical colonies, and testing for virulence properties of isolated strains [17]. This method is an effective method which may be employed to Yersinia 2 Journal of Pathogens enterocolitica and Y. pseudotuberculosis in foods. The procedure has been used especially to detect the pathogenic Y. enterocolitica and Y. pseudotuberculosis in Japan.

Procedures Currently to Quantify and Confirm Yersinia sp. in Food
The presence of Y. enterocolitica and Y. pseudotuberculosis in food can be determined quantitatively by a direct culture on selective agar plates. However, confirmatory tests require a combination of cold enrichment, selective enrichment, and subculture on selective agar plates. A conventional protocol for detection and identification of Y. enterocolitica and Y. pseudotuberculosis from foods is shown in Figure 1. Suspect food samples must however be pretreated to enable successful analysis.

Enumeration of Yersinia sp. by Direct Culture Method.
For this procedure, an aliquot of homogenate is inoculated onto selective agar plates (see below) after treatment with an alkali [9,19]. Alkaline treatment can be achieved by mixing 0.5 mL of homogenate with 0.5 mL of 0.72% KOH in 0.54% NaCl for 30 sec. Yersinia is able to resist weak alkaline treatment, and this property is used to select the organism while suppressing background flora such as Pseudomonas, Proteus and Serratia [20]. It is reported that Y. enterocolitica serotypes O:3, O:5, 27, O:8, and O:9 and Y. pseudotuberculosis serotype 5a strains in the artificially contaminated pork samples showed comparatively high resistance to KOH, and all Yersinia strains were recovered from the pork samples contaminated with more than 10 2 cells per g after direct KOH treatment, without enrichment [9]. However, food samples with low contamination (less than 10 2 cells per g) require an enrichment procedure for successful recovery of Yersinia. An alternative to cold enrichment is selective enrichment. Selective enrichment uses media containing antimicrobial agents. Several selective enrichment media for isolation of Y. enterocolitica at higher temperatures have been developed [17]. Generally, cold enrichment yields higher recovery rates of pathogenic Y. enterocolitica than selective enrichment. Moreover, an effective selective enrichment system for Y. pseudotuberculosis has not been developed, so its current selective enrichment procedures are especially low.

Cold and
Nevertheless, in case of outbreaks, selective enrichment procedures for isolation of pathogenic Y. enterocolitica are useful for rapid detection and confirmation of the pathogen. In such cases, 9 mL of Irgasan-ticarcillin-potassium chlorate (ITC, Merck, Darmstadt, Germany) is inoculated with 1 mL of medium from cold enrichment and aerobically incubated at 25-30 • C for 48 hr [22].

Rapid Separation and Concentration of Yersinia from Food
Samples for Cell Counting and PCR [18]. A conventional protocol for rapid separation and concentration of foodborne pathogens in food samples using filtration, centrifugation, and buoyant density centrifugation (BDC) prior to quantification by viable-cell counting and real-time PCR is shown in Figure 2. A 25 g food sample is mixed with 225 mL of 0.02% Tween 20-BPW in a small plastic bag (Stomafilter P type) and homogenized in a stomacher for 2 min. Approximately 220 mL portions of filtered solutions of the homogenates are placed in sterilized 350 mL glass tubes and centrifuged at 1,880 ×g for 5 min at room temperature, using a swing rotor. The upper portion is transferred to a sterilized 500 mL plastic tube and then centrifuged at 16,000 ×g for 5 min at room temperature. The pellet is then suspended in 1.5 mL of 0.15 M NaCl and centrifuged at 14,500 ×g with a bench-top centrifuge for 5 min at room temperature. The resultant pellet is harvested and used for the second step.
The second step is flotation and sedimentation BDC for purification of food-borne pathogens. In the flotation assay, 0.5 mL portions of sample suspensions are mixed with 1 mL of a 1.050 g/mL Percoll solution (Pharmacia Biotech, Sweden) and centrifuged at 4,500 ×g for 15 min at room temperature. The upper portion, including the food matrix, is carefully removed. For the sedimentation assay, the bottom portion (about 0.5 mL), including organisms, food        Journal of Pathogens 5 particles, and the mass with the highest buoyant density, is homogenized and then placed on top of two layers (0.6 mL of a 1.050 g/mL Percoll solution and 0.6 mL of a 1.123 g/mL Percoll solution) in a 1.5 mL microtube to which two density markers (orange for 1.033 g/mL and green for 1.098 g/mL) are added. The preparations are centrifuged at 14,500 ×g for 5 min at room temperature, and then using sterile 1-mL pipettes, about 1 mL is taken from the interface between the two density makers and divided into two samples. The sample is added to 1 mL of 0.15 M NaCl in a 1.5 mL microtube.
The preparations are then centrifuged at 14,500 ×g for 5 min. The bottom portions (0.5 mL) are resuspended with 1 mL of 0.15 M NaCl and then centrifuged at 14,500 ×g for 5 min. Each pellet is used for viable-cell counting and DNA extraction with InstaGene matrix (Bio-Rad). One portion of the sample is resolved with 50 µL of 0.15 M NaCl, and then viable-cell counts (CFU/g), which are obtained by culturing each dilution (10 µL) using selective agar plates, are determined for these BDC-lysate pellets (50 µL). The other portion is treated with 50 µL of InstaGene matrix for DNA extraction prior to real-time qPCR by using yadA primer for pathogenic Y. enterocolitica and Y. pseudotuberculosis. The total volume of 25 g food sample is reduced to 0.1 mL, and the target organisms in the sample are theoretically concentrated 250-fold within 2 hr.
CIN agar is useful to expedite the recovery of Y. enterocolitica and mVYE agar to differentiate virulent from avirulent isolates (Figures 3 and 4). The characteristic deep red center ("bull's eye") with a transparent margin and diameter 2-4 mm appearance of Yersinia colonies on CIN incubated at 30 • C for 24 hr is important for identification and is due to the presence of mannitol. Yersinia ferments the mannitol in the medium, producing an acid pH which gives the colonies their red color and the "bull's eye" appearance.
The greatest advantage of mVYE agar is that pathogenic Y. enterocolitica, which forms red colonies, is easily differentiated from most nonpathogenic Yersinia organisms and other gram-negative bacteria, which form dark-red colonies with a dark peripheral zone as a result of mannitol fermentation and esculin hydrolysis. Y. pseudotuberculosis, which forms dark pin colonies as a result of esculin hydrolysis, is easily differentiated from most nonpathogenic Yersinia organisms.
The "bull's eye" colonies on CIN agar and red colonies on mVYE agar are suspected to virulent Y. enterocolitica (and sometimes Y. kristensenii). The red pin colonies on CIN agar and dark-red pin colonies on mVYE agar are suspected to Y. pseudotuberculosis.

The Second Confirmation Test from the First Confirmation
Test. Strains suspected as pathogenic Y. enterocolitica and Y. pseudotuberculosis by the first confirmation tests are selected for the second confirmation tests (pyrazinamidase test [25] and autoagglutination test [26]). For pyrazinamidase test, the strain is inoculated onto pyrazinamidase test agar slants (see below) and incubated at 30 • C for 48 hr. For autoagglutination test, the strain is incubated in Trypticase soy broth (TSB; BBL) (or MR-VP medium; Difco) at 25 • C and 37 • C for 24 hr.
The 5 mL portions of culture medium are autoclaved and cooled to make slants.
The pyrazinamidase test is to date the chromosomal phenotypical criterion to distinguish potentially pathogenic from nonpathogenic strains. Pathogenic Y. enterocolitica and Y. pseudotuberculosis show negative reactions, and nonpathogenic Yersinia strains show positive reactions which turn brownish pink in the presence of ferrous salts ( Figure 5). Autoagglutination test is positive on the plasmid for Yersinia virulence-(pYV-) positive strains of Y. enterocolitica and Y. pseudotuberculosis which are incubated at 37 • C but not at 25 • C ( Figure 6). The pYV lost strains which are subcultured, especially at 37 • C, and stored, show negative reactions.

Further Biochemical and Serological Confirmation.
Pure strains of suspected pathogenic Y. enterocolitica and Y. pseudotuberculosis are prepared on blood agar or other nutrient agar. The strains are investigated oxidase activity (negative), carried out Gram staining (negative). Then the strains are performed biotyping of Y. enterocolitica or genetic grouping of Y. pseudotuberculosis according to the criteria shown in Table 1 [27].
The following parameters can be used to distinguish between Y. enterocolitica and other Yersinia species: sucrose (positive), rhamnose (negative), melibiose (negative), ornithine decarboxylase (positive) and Voges-Proskauer (VP) positive. However, VP and/or sucrose-negative strains of Y. enterocolitica [18,28,29] and melibiose-negative strains of Y. pseudotuberculosis [16] may occur. Y. enterocolitica serotype O:3/biotype 4 is distributed all over the world and is the dominant human pathogenic strain in western countries. However, serotype O:3/biotype 3 variant VP − is the dominant human pathogenic strain in China, Taiwan, and Japan, and serotype O:3/biotype 3VP − , sucrose negative (S − ) is also reported in Japan. Serotype O:5,27, which is reported in the USA, China, and Japan, and Serotype O:9, from the Nordic countries, China, and Japan, belong to biotype 2. Serotype O:8 from the USA and Japan belongs to biotype 1B. Biotype 1A comprises numerous serotypes which have not been associated with human illness and are common in food and the environment.
The ail gene, located in the chromosome of pathogenic Y. enterocolitica strains, and inv gene, located in the chromosome of Y. pseudotuberculosis strains, are the most frequently used targets. Multiplex PCR method using a mixture of primers against inv (5 -TAAGGGTACTATCGCGGCGGA-3 and 5 -CGTGAAATTAACCGTCACACT-3 ), ail (5 -ACTCGATGATAACTGGGGAG-3 and 5 -CCCCCAGTA-ATCCATAAAGG-3 ), and virF (5 -TCATGGCAGAAC-AGCAGTCAG-3 and 5 -ACTCATCTTACCATTAAGAAG-3 ) [31] has been designed to detect Y. enterocolitica and Y. pseudotuberculosis in food and water [32]. Real-time PCR is a powerful advancement of the basic PCR technique. At present, the most popular real-time PCR assays are based on "Taqman" and "SYBR Green" approaches. The Taqman system is a 5 -nuclease assay that utilizes specific hybridization of a dual-labelled Taqman probe to the PCR product. The SYBR Green system is based on the binding of the fluorescent SYBR Green dye to the PCR product [33]. Chromosomally encoded ail [34] and yst [35] genes, the plasmid-borne yadA gene [36,37] and a Yersiniaspecific region of the 16S rRNA gene [37,38] have been used in real-time PCR.

Conclusion
Yersinia enterocolitica and Y. pseudotuberculosis continue to be important in food safety. While Yersinia can survive in many types of food, there is no much information about its the prevalence. This paper covers commercially available conventional and PCR-based procedures for the detection of pathogenic Yersinia in food. These methods are effective as the detection methods to target for pathogenic Y. enterocolitica and Y. pseudotuberculosis in foods. However, 8 Journal of Pathogens development of rapid test methods is needed to facilitate more timely and cost-effective testing.