food - borne pathogenic yersinia ( y. enterocolitica and y. pseudotuberculosis ) is facultative anaerobic , gram - negative enterobacteriaceae and is isolated frequently from soil , water , animals , and foods [ 14 ] . y. enterocolitica causes human infections whose symptoms include diarrhea , terminal ileitis , mesenteric lymphadenitis , arthritis , and septicemia . y. pseudotuberculosis causes mesenteric lymphadenitis , diarrhea , and septicemia in humans . as a psychrophilic organism , yersinia is able to grow at 4c , and cold chain food products could offer a potential food safety hazard [ 3 , 5 , 6 ] . 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 [ 7 , 8 ] . in japan , o:3/3 variant vp negative is the most frequent cause of human yersiniosis . in the united states , despite declining incidences of serotype o:8/1b infections , o:3/4 and o:5 , 27/2 infections are on the increase . in europe , y. pseudotuberculosis is distributed less widely than y. enterocolitica and , although frequently isolated from animals , is rarely isolated from soil , water , and food [ 912 ] . a large outbreak of y. pseudotuberculosis infection has been reported in japan [ 13 , 14 ] . in the far east including japan , y. pseudotuberculosis various serotypes ( 1b , 2a , 2b , 2c , 3 , 4a , 4b , 5a , 5b , and all that ) are isolated from patients with exanthematous systemic infection such as fever , and almost strains isolated produce a superantigenic toxin - designed ypma encoded by ypma gene [ 1416 ] . in europe , serotypes ( 1a , 1b , and 3 ) have been isolated from patients with gastroenteric symptoms and have an extremely low frequency of isolation . 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 . this method is an effective method which may be employed to yersinia enterocolitica and y. pseudotuberculosis in foods . the procedure has been used especially to detect the pathogenic y. enterocolitica and y. pseudotuberculosis in japan . 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 . pretreatment starts with the homogenizing the food sample ( 25  g ) in a stomacher for 2  min with 225  ml of phosphate - buffered saline ( pbs ) or other cold enrichment medium ( see below ) . the resulting homogenate is used for the direct culture , enrichment culture experiments . for rapid separation and concentration , a 25  g food sample is mixed with 225  ml of 0.02% tween 20-buffered peptone water ( bpw ) in a plastic bag ( stomafilter p type ; gunze , tokyo , japan ) , containing a teflon cloth ( 40  mesh ) on the inside and homogenized in a stomacher for 2  min . 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 . 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 cells per g after direct koh treatment , without enrichment . however , food samples with low contamination ( less than 10 cells per g ) require an enrichment procedure for successful recovery of yersinia . for cold enrichment , an inoculated medium ( examples shown below ) after 1 , 2 , and 3 weeks , 0.5  ml of the medium is treated with koh and inoculated onto selective agar plates . however if the medium has low selectivity , environmental yersinia species and other bacteria may also multiply during enrichment . cold enrichment media used for detection of yersinia in food and water samples are   phosphate - buffered saline ( pbs ; 880  ml of 0.061  m na2hpo4 , 120  ml of 0.061  m kh2po4 , and 0.85% nacl ) , pbs with 1% mannitol and 0.15% bile salts ( pmb ) , pbs with 0.5% peptone and 1% sorbitol , 0.15% bile salts ( psb ) , pbs with 0.25% peptone and 0.25% mannitol ( pmp ) , buffered peptone water ( bpw , merck , germany ) . selective enrichment uses media containing antimicrobial agents . several selective enrichment media for isolation of y. enterocolitica at higher temperatures 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 . 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 2530c for 48  hr . a conventional protocol for rapid separation and concentration of food - borne 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 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 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  l of 0.15  m nacl , and then viable - cell counts ( cfu / g ) , which are obtained by culturing each dilution ( 10  l ) using selective agar plates , are determined for these bdc - lysate pellets ( 50  l ) . the other portion is treated with 50  l 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 isolation of yersinia and pathogenic yersinia ( pathogenic y. enterocolitica and y. pseudotuberculosis ) can be done using cefsulodin - irgasan - novobiocin agar ( cin agar , difco , oxoid )   and cin agar containing 0.1% esculin and 0.05% ferric citrate ( modified virulent yersinia enterocolitica agar ( mvye agar ) ) . 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 24  mm appearance of yersinia colonies on cin incubated at 30c 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 . colonies showing typical morphology on cin and mvye agars ( at least four colonies from each of the agar plates ) are selected . the first confirmation test is carried out by inoculating urea broth , tsi medium , lim medium , and bile - esculin agar and incubating at 30c for 24  hr . depending on the target organism , colonies are selected for further examination . in the case of y. enterocolitica , colonies that are urea positive ; lysine negative in the lim medium ; no gas formation in the tsi medium ; glucose positive , sucrose positive ; lactose negative ( yellow slant and yellow agar column in the tsi medium ) should be selected . if the targets are y. pseudotuberculosis , y. enterocolitica biotype 3  vp , and sucrose negative / serotype o:3 , colonies that are glucose positive , sucrose negative , and lactose negative ( yellow slant and red agar column in the tsi medium ) should be selected for further examination . it should be noted that pathogenic y. enterocolitica strains ( serotypes o:3 , o:5 , 27 , o:8 , and o:9 ) are esculin negative while y. pseudotuberculosis strains and nonpathogenic y. enterocolitica strains ( biotype 1a / other numerous serotypes ) are esculin positive . strains suspected as pathogenic y. enterocolitica and y. pseudotuberculosis by the first confirmation tests are selected for the second confirmation tests ( pyrazinamidase test   and autoagglutination test ) . for pyrazinamidase test , the strain is inoculated onto pyrazinamidase test agar slants ( see below ) and incubated at 30c for 48  hr . for autoagglutination test , the strain is incubated in trypticase soy broth ( tsb ; bbl ) ( or mr - vp medium ; difco ) at 25c and 37c for 24  hr .   pyrazinamidase test agar . trypticase soy agar ( difco ) ( 30.0  g ) . yeast extract ( difco ) ( 3.0  g ) . tris - maleate ( 0.2  m , ph6 ) to ( 1,000  ml ) . 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 37c but not at 25c ( figure 6 ) . the pyv lost strains which are subcultured , especially at 37c , and stored , show negative reactions . 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 . the serotyping is carried out by slide agglutination using commercial antisera o:3 , o:5 , o:8 , and o:9 for y. enterocolitica ( denka seiken , tokyo , japan ) , and antisera o:1 , o:2 , o:3 , o:4 , o:5 , and o:6 ( denka seiken ) for y. pseudotuberculosis . the serotype and subserotype of y. pseudotuberculosis are carried out possibly by o - genotyping using o - antigen gene cluster - specific pcrs . 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   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 . biotype 1a comprises numerous serotypes which have not been associated with human illness and are common in food and the environment . y. pseudotuberculosis strains belong to genetic groups 1 to 6 and serotypes 1a , 1b , 1c , 2a , 2b , 2c , 3 , 4a , 4b , 5a , 5b , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , and 15 . the genetic group 3 ( far eastern systemic - pathogenicity type)/serotypes 1b , 1c , 2a , 2b , 2c , 3 , 4a , 4b , 5a , 5b , 6 , 7 , 8 , 10 , and 15 are the human pathogens in japan , china , and korea . the genetic group 2 ( european gastroenteric - pathogenicity type)/serotypes 1a and 1b and genetic group 5/serotype o:3 are the human pathogens in western countries . although most strains of y. pseudotuberculosis are melibiose positive , genetic group 4/serotypes o:1 , o:5 , o:6 , o:7 , o:9 , o:10 , o:11 , and o:12 ( nonpathogenic strains ) , which are distributed in the environment of japan , and genetic group 5/serotype o:3 are melibiose negative . using pcr , pathogenic y. enterocolitica can be detected in samples rapidly and with high specificity and sensitivity . several pcr assays have been developed to detect pyv - positive y. enterocolitica and y. pseudotuberculosis in clinical , food , and environmental samples . many of these samples use primers targeting the yada or virf gene located on pyv . because of possible plasmid loss on subculture and storage , 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-actcgatgataactgggg ag-3 and 5-cccccagtaatccataaagg-3 ) , and virf ( 5-tcatggcag aacagcagtcag-3 and 5-actcat cttaccattaagaag-3 )   has been designed to detect y. enterocolitica and y. pseudotuberculosis in food and water . 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 . chromosomally encoded ail   and yst   genes , the plasmid - borne yada gene [ 36 , 37 ] and a yersinia - specific region of the 16s rrna gene [ 37 , 38 ] have been used in real - time pcr . pathogenic y. enterocolitica and y. pseudotuberculosis strains yield positive pcr products from the yada gene . using sybrgreen real - time pcr assay , the tm values of this yada primer pair ( yada - f1757 : 5-acgagttgacaaaggtttagcc-3 and yada - r1885 : 5-gaaccaaccgctaatgcctga-3 ) are also different between the pathogenic y. enterocolitica ( 82.2c ) and y. pseudotuberculosis ( 81.5c ) strains ( figure 7 ) . therefore , this primer pair was confirmed to be useful for detection and differentiation of the two pathogenic yersinia species . yersinia enterocolitica and y. pseudotuberculosis continue to be important in food safety . while yersinia can survive in many types of food 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 , development of rapid test methods is needed to facilitate more timely and cost - effective testing .
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 .