bacterial strains : in this study , we used 67 l. 
 monocytogenes strains [ 15 , 31 , 34 , 35 ] .
these strains were isolated from skin of beef cattle 
 from a japanese farm ( five strains ) , japanese patients with listeriosis ( seven strains ) and 
 meat produced in japan ( 37 strains ) or imported to japan from other countries ( 18 strains ) 
 ( table 1table 1.genetic classification of l. monocytogenes strains used in this 
 studystrainsourcecountryserotypemlstrflppfgeiapsigbactamlst typebpacc . # iap typebpacc .
# acta typexbaiclai ( baniii)pstiiap - rflp typeapaiascipulsotypeegd - erabbitengland1/2a407al5919750662al5919771561al59197411x1c1p111113e1skin of beef cattlejapan1/2a407ab2945750662lc158691 34561lc158758 187x1c2p22161323e2skin of beef cattlejapan1/2a407ab2945760662lc158692 34561lc158759187x1c2p2216132h3patientjapan1/2a395ab3656801662lc15869312561lc158760 6710x2c3p33107376p1porkjapan1/2c413ab3656662657lc15869436561lc15876116x1c4p14410478p1porkjapan1/2c413ab3656672662lc1586951561lc15876212x1c4p14n.d.n.d.n.d.173b3beefjapan1/2a413ab3656472662lc1586961561lc15876312x1c4p14n.d.n.d.n.d.23c1chickenjapan1/2c413ab3656693662lc1586971561lc158764689x3c5p45217576p2porkjapan1/2a389ab3656494662lc15869812561lc1587652115x4c6p5664678p5porkjapan1/2a389ab3656524662lc15869912561lc1587662115x4c6p5674789c5chickenjapan1/2a389ab3656504662lc15870012561lc1587672115x4c6p56n.d.n.d.n.d.yc35p1porkireland1/2a389ab3656954662lc15870112561lc1587682115x4c6p56n.d.n.d.n.d.80c1chickenjapan1/2a395ab3656555662lc1587021561lc158769116x5c7p6712168h1patientjapan1/2a395ab3656825662lc1587031561lc158770116x5c7p67n.d.n.d.n.d.hm1patientjapan1/2a395ab3656535662lc1587041561lc158771116x5c7p6711159hm2patientjapan1/2a395ab3656565662lc1587051561lc158772116x5c7p67n.d.n.d.n.d.265c1chickenjapan1/2a395ab3656576662lc15870612561lc1587732713x6c8p7819810268c1chickenjapan1/2a401ab3656587662lc1587071561lc158774114x7c9p8917311104p5retail porkjapan1/2a395ab5177458662lc1587081561lc15877513x7c9p81017212221c1chickenjapan1/2a395ab3656598662lc1587091561lc15877613x7c9p810n.d.n.d.n.d.yc39b1beefu.s.a.1/2a395ab3656998662lc1587101561lc15877713x7c9p81018513223c3chickenjapan1/2a407ab3656629662lc1587111561lc1587781811x8c10p911n.d.n.d.n.d.yc35p6porkireland1/2a407ab3657029662lc1587121561lc1587791811x8c10p911n.d.n.d.n.d.yc35p8porkireland1/2a407ab3657709662lc1587131561lc1587801811x8c10p911n.d.n.d.n.d.yc35p12porkireland1/2a407ab3657019662lc1587141561lc1587811811x8c10p9115141412hpatientjapan1/2a401ab36566310662lc1587151561lc15878215x10c12p1112131115186c1chickenjapan1/2a401ab36566410662lc1587161561lc15878315x10c12p1112n.d.n.d.n.d.188c3chickenjapan1/2a401ab36566510662lc1587171561lc15878415x10c12p1112141216yc4p12porkdenmark1/2a389ab36570311662lc1587181561lc158785188x11c13p1213n.d.n.d.n.d.yc51p12porkdenmark1/2a389ab36570411662lc1587191561lc158786188x11c13p12133617yc51p13porkdenmark1/2a389ab36578011662lc1587201561lc158787188x11c13p1213n.d.n.d.n.d.72c1chickenjapan1/2b389ab36567012662lc1587225561lc1587891221x13c15p141525211874c1chickenjapan1/2b389ab36572112662lc1587235561lc1587901221x13c15p1415n.d.n.d.n.d.42c1chickenjapan4b389ab36571912662lc1587212561lc1587881624x12c14p1314n.d.n.d.n.d.82b1beefjapan4b389ab36572612662lc1587242561lc1587911624x12c14p1314n.d.n.d.n.d.338b2beefjapan4b389ab45759712662lc1587252561lc1587921624x12c14p1314n.d.n.d.n.d.468b1beefjapan4b389ab36572512662lc1587262561lc1587931624x12c14p1314332519yc20c9chickenchina1/2b395ab36578413662lc1587385561lc158805317x15c17p1619202427yc36c2chickencanada1/2b395ab36574213662lc1587395561lc158806317x15c17p1619n.d.n.d.n.d.yc50c3chickenchina3b395ab36578613662lc1587405561lc158807317x15c17p1619n.d.n.d.n.d.1e1skin of beef cattlejapan1/2b395ab29457013662lc15872737561lc158794318x15c17p1716212020100p3porkjapan1/2b395ab36576213662lc1587333561lc158800819x16c18p1718261824112p3porkjapan4b395ab36573713662lc1587343561lc158801819x16c18p1718n.d.n.d.n.d.114p3porkjapan1/2b395ab36574413662lc1587353561lc158802819x16c18p1718n.d.n.d.n.d.66c3chickenjapan1/2b395ab36575613662lc1587285561lc158795922x14c16p1517n.d.n.d.n.d.69c3chickenjapan1/2b395ab36576413662lc1587295561lc158796922x14c16p151730232179c1chickenjapan1/2b395ab36567513662lc1587305561lc158797922x14c16p151730222291c3chickenjapan1/2b395ab36574713662lc1587315561lc158798922x14c16p151729222393c1chickenjapan1/2b395ab36574813662lc1587325561lc158799922x14c16p1517n.d.n.d.n.d.116c1chickenjapan1/2b395ab36573513662lc1587365561lc158803922x14c16p1517302225yc20c12chickenchina1/2b395ab36575213662lc1587375561lc158804 922x14c16p151727192663p1porkjapan1/2b404ab36567614662lc1587415561lc1588081420x17c19p1820242628h2patientjapan4b389ab36569115662lc1587422561lc158809228x18c20p192122302911hpatientjapan4b389ab36570716662lc15874335561lc158810226x1820p1922n.d.n.d.n.d.229c1chickenjapan4b389ab36570816662lc1587442561lc158811227x18c20p1922232930393p1porkjapan4b389ab45760316662lc1587452561lc158812227x18c20p1922n.d.n.d.n.d.499c5retail chickenjapan4b389ab51776416662lc1587462561lc158813227x19c20p19232328311e3skin of beef cattlejapan1/2a401ab29457220662lc1587471561lc15881414x1c25p24248932yc13c10chickenu.s.a.1/2a401ab36571120662lc1587481561lc15881514x1c25p2424n.d.n.d.n.d.yc13c11chickenu.s.a.1/2a401ab36571020662lc1587491561lc15881614x1c25p24249933yc17p13porkireland1/2a401ab36571220662lc1587501561lc15881714x10c12p1125151134yc21p8porkcanada1/2a407ab36571421662lc1587511561lc1588182112x23c26p2526n.d.n.d.n.d.yc21p12porkcanada1/2a407ab36571321662lc1587521561lc1588192112x23c26p2526n.d.n.d.n.d.yc21p14porkcanada1/2a407ab36578821662lc1587531561lc1588202112x23c26p2526n.d.n.d.n.d.2e1skin of beef cattlejapan1/2b401ab29457422662lc1587547561lc1588211023x24c27p2627282735241c1retail chickenjapan4b407ab51777624662lc1587552561lc1588226925x25c28p2728313136505c7chickenjapan4b371ab45760725662lc1587566561lc1588231129x26c29p2829n.d.n.d.n.d.508c6retail chickenjapan4b371ab51776925662lc1587576561lc1588241129x26c29p2829323237a ) n.d . : not done . ) .
serotypes of these strains included 1/2a ( 34 isolates ) , 1/2b ( 16 isolates ) , 
 1/2c ( three isolates ) , 3b ( one isolate ) and 4b ( 13 isolates ) .
rflp analysis : genomic dna from l. monocytogenes was 
 extracted and purified as previously described [ 24 , 
 25 , 31 , 
 33 ] . for rflp analysis , genomic dna was digested 
 with restriction enzymes xbai , clai 
 ( baniii ) or psti according to the manufacturer s 
 instructions ( takara bio , otsu , japan ) . the reactants were separated on 0.8% agarose gels . 

dna fragments were stained with ethidium bromide ( nacalai tesque , kyoto , japan ) and 
 visualized using an ultraviolet transilluminator ( uvp , upland , ca , u.s.a . ) .
rflp patterns with less than five differences were 
 considered to be of the same genotype .
sequence analysis : each strain was incubated in brain heart infusion broth 
 ( bd , franklin lakes , nj , u.s.a . ) at 37c for 18 hr .
after incubation , bacterial cells were 
 harvested by centrifugation , washed with sterilized milliq water and suspended in 400 
 l te solution ( 10 mm tris - hcl ph 8.0 and 1 mm edta ph 8.0 ) .
the 
 bacterial suspensions were boiled for 15 min to lyse the cells , followed by centrifugation 
 at 15,000 g for 10 min at 4c to remove denatured proteins and bacterial 
 membranes .
the supernatant containing dna was obtained and stored at 80c until use . in 
 addition , dna for the iap sequencing was extracted and purified as 
 previously described [ 24 , 25 , 31 , 33 ] . to determine the nucleotide sequence , partial iap , 
 sigb and acta
were amplified using specific primer 
 pairs , si3a / si4b [ 24 , 25 , 31 , 34 , 36 ] , lmsigb15/lmsigb16   and massf / massr [ 12 , 41 ] , respectively ( table 2table 2.primers used in this studytarget geneprimer namesequence ( 5 to 3)iapsi3aactggtttcgttaacggtaaasi4btttagtgtaaccagagcaatcsigblmsigb15aatatattaatgaaaagcaggtglmsigb16ataaattatttgattcaactgccactamassf gctgatttaagagatagaggaacmassrtttatgtggtaatttgctgtc ) . the size of iap , sigb and 
 acta amplicons ( 810 , 841 and 827 bp , respectively ) were confirmed by 1.0% 
 agarose gel electrophoresis .
cycle sequencing using iap amplicons was 
 performed with hitachi dna sequencer 5500 ( hitachi , tokyo , japan ) as previously described 
 [ 24 , 25 , 
 31 , 33 ] . 

sequence analyses of sigb and acta were carried out at 
 eurofins genomics ( tokyo , japan ) .
the comparative sequences of iap , 
 sigb and acta in the reference strain , egd - e , were 
 located at 1,1161,522 ( 407 bp ) , 41702 ( 662 bp ) and 1,3571,917 ( 561 bp ) positions , 
 respectively .
the sequence data were edited and aligned using dnasis pro ( hitachi software , 
 ver .
phylogenetic analyses were conducted using mega , version 7.0   and the unweighted - pair group method with arithmetic 
 mean ( upgma ) .
all sequence data were registered at the dna data bank of japan ( mishima , 
 japan ) ; accession numbers are indicated in table 
 1 .
unfortunately , the strains belonging to iap group c described 
 in the previous report   were not tested for mlst , 
 because their partial acta was not amplified using a massf / massr primer 
 pair .
in addition to 68 strains used in this study , 211 strains registered in the food 
 microbe tracker database ( www.pathogentracker.net ) maintained by cornell university were 
 analyzed in silico for the classification of nucleotide sequences of 
 sigb ( 179 strains ) and acta ( 194 strains ) ( supplementary 
 table 1 ) .
serotypes included 1/2a ( 57 strains ) , 1/2b ( 35 strains ) , 1/2c ( seven strains ) , 3a 
 ( four strains ) , 3b ( six strains ) , 3c ( one strain ) , 4a ( 19 strains ) , 4b ( 50 strains ) and 4c 
 ( 10 strains ) .
additionally , 20 and two strains , whose serotypes were designated as 
 unspecified and untypeable , respectively .
pfge analysis : molecular subtyping of l. monocytogenes 
 strains by pfge was performed based on standardized laboratory protocol , pulsenet 
 ( https://www.cdc.gov/listeria ) .
bacterial suspensions solidified with seakem gold agarose 
 ( lonza , rockland , ny , u.s.a . ) were lysed , washed and digested with the restriction enzymes , 
 apai and asci ( new england biolab japan , tokyo , japan ) . 

diversity index : simpson s index of diversity ( sid ) was recommended to 
 evaluate the discriminative ability of genotyping methods [ 17 , 30 ] .
the sid and the 95% confidence 
 intervals ( ci ) are presented in the following equations : where n is the total number of sample strains , s is the total 
 number of different types described , nj is the number of 
 strains belonging to the jth type , and j is the frequency nj / n .
classification by sequence of iap and rflp analysis of l. monocytogenes genome combined 
 subtyping method : to establish the dna sequence - based subtyping method , we first 
 performed iap - rflp assay using 67 l. monocytogenes 
 isolates and egd - e ( table 1 ) . the target 
 sequence was located at the nucleotide position of iap in l. 
 monocytogenes , from 1,116 to 1,522 bp in the reference strain , egd - e .
so far , 26 
 iap types have been designated to 0 through 25 based on a comparison of 
 nucleotide sequences ( supplementary table 2 ) [ 15 , 
 34 , 35 ] . 

rflp patterns of l. monocytogenes 
 genomes digested with xbai , clai or psti 
 were classified into 26 ( x1 to x26 ) , 29 ( c1 to c29 ) and 28 ( p1 to p28 ) patterns , 
 respectively ( supplementary fig .
all strains were classified into 22 , 25 and 23 types according to genomic 
 rflp analyses , digested with xbai , clai and 
 psti , respectively ( table 1 ) . 

rflp analysis was repeated more than three times for each isolate , and its pattern did not 
 change depending on the year of experiment or researcher .
five rflp patterns , x1 , x7 , x10 , 
 x18 and x20 , contained multiple iap types determined by the 
 iap sequences , and iap types 9 , 12 , 13 , 16 and 20 were 
 found to have more than two rflp patterns using xbai .
strains assigned to 
 pattern x1 were further classified into three ( p1 , p2 and p24 ) and four ( c1 , c2 , c4 and c25 ) 
 patterns by psti and clai , respectively ( table 1 and supplementary fig .
consequently , l. monocytogenes strains used 
 in this study were classified into 29 iap - rflp types as a result of a 
 combination of nucleotide sequencing for partial iap and rflp analyses 
 digested with xbai , clai and psti .
sid of 
 this iap - rflp method was 0.967 ( 95% ci : 0.955/0.979 ) .
mlst analysis usingiap , sigb and acta : in order to 
 develop a dna sequence - based subtyping method that can refer to the data of 
 iap - based rflp analyses , we focused on two genes , sigb 
 and acta , in addition to iap . to investigate the 
 characteristics of sigb and acta ,
the nucleotide sequences 
 of these genes in strains registered in the food microbe tracker database were compared to 
 the egd - e sequence in silico .
next , we evaluated whether the method 
 developed in the present study showed a high discriminatory ability in the classification of 
 l. monocytogenes . the nucleotide sequences for partial 
 sigb , approximately 660 bp in length , were determined and used for 
 genetic classification of 247 strains , which consisted of 68 strains used in this study and 
 179 food microbe tracker strains ( table 1 and 
 supplementary table 1 ) .
the number and type of point mutations in partial 
 sigb are presented in table 
 3table 3.sequence typing based on partial sigbmutationtype12345678910111213141516171819202122232425262728293031323334353637ac0222222222002212222222222222222220202ag05555565531066555555556555101010105551505tc0444444444004455644455435498984330404tg0001000000000000000000000011110000000ca0000000000000010000000000011110000000ct07775676660166566867777797131314136661706ga0555455444005444445544454466664440505gt0000000001001111111111111122221220101at0111111112001121111111111133331110101ta0333333332002222222222222233332220303cg0101111110000000000000000033330000101gc0111111111001111111111111111111110101total029283026283027272511282727272828272828282827302754535553262626230029atcg01111121111121111910121211121311111112121210121122212221111010111011cgat01212129111210101101121111111113121312121213141222222322111212113012at0444444444003343333333333366663330404cg0212222221001111111111111144441110202size ( bp)662662662662662662662662662662662662662662662662662662662662662662662662662662662662662662662662662662662657662 .
thirty - seven sigb types were determined using 
 sigb sequences ( table 1 and 
 supplementary table 3 ) . in total , 112 point mutations were found in partial 
 sigb of 246 strains as compared with that of egd - e .
there were no 
 insertions , however , deletion of five nucleotides was found in 76p1 .
in addition to 76p1 , 
 nonsense mutation in sigb was detected in 1e1 . in comparison
, partial 
 acta , 562 bp in length , was analyzed using the nucleotide sequences of 
 262 isolates , including 68 strains used in this study and 194 food microbe tracker strains . 
 in silico assay
was used for classification into 69 acta 
 types ( table 4table 4.sequence typing based on partial actamutationtype123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869ac033333333333334230000000000000000000002223221212222222255655552322003ag018191919191919181919171818191819202220231121201112200161617171816161618161615171615161623222424232323161716172318tc078777698767888781112200100101002111107775666666776766667777768888117tg011111111111111110000000100000000010001112211211211121222222221122011ca033333333333333330000000001010000100003333333333334343344444443333003ct077108777777877777002000000101110000101999889888910978998101010101011111291111007ga010111010101010101010109101099111111010120100111100101112101311131210101210121012111110101010101011101011101110gt033333333333333330000000001010001000002223333343222233333433333233003at010100000010000000000000000000020000011122222221111222220221110110000ta022332311332222320000000000000000000001123323332212222323323332232003cg011121111111111110000000000000000000001111112111111101200000002122101gc012221121221222221111100100101001111001211111111112111111111111111112total057606361575759565959565758605658537661271564613656612545659586257595958555953575558575968677370697070605863616758atcg029313030302932303029283030322831313440251131301324310262627272825242626252725262625252636363938373736272928293529cgat023242624232323232323242223232222113111010423210221201242526242726272625252724252328262527272827272829282428271123at032432311342222320000000000000020000012245545553323444543544442342003cg023342232332333331111100100101001111002322223222223212311111113233213size ( bp)561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561561 ) . in total , 152 point mutations were identified , compared with egd - e sequence . 
 no insertions or deletions were observed .
the 67 strains isolated in japan and 194 food 
 microbe tracker strains were classified into 17 and 65 acta types , 
 respectively ( table 1 and supplementary table 
 1 ) .
consequently , all strains used in this study were divided into 29 mlst types using 
 iap , sigb and acta sequences ( table 1 and fig . 
 1 ) .
strains assigned to certain 
 iap types ( 0 , 2 , 12 and 16 ) and iap type 13 were further 
 classified into two and four mlst types , respectively ( table 1 ) .
the phylogenic tree of mlst types indicated that strains were roughly 
 clustered in two groups ( mlst clusters a and b ) .
mlst clusters a and b were consistent with 
 lineages ii and i , respectively .
pfge analysis using apai and asci : to 
 compare the mlst classification with that of pfge , 38 strains were selected .
pfge patterns 
 obtained using pulsenet protocol with restriction enzymes , asci and 
 apai , could be distinguished into 32 types .
finally , the 38 strains were 
 separated into 37 pulsotypes ( table 1 and 
 supplementary fig .
the analyses of both pfge patterns represented two major clusters 
 ( pfge clusters a and b ) associated with their lineages as well as the results from mlst .
compared with the egd - e sequence , the iap target region sequence was used 
 for classification into 26 types and three groups based on total point mutations .
the 
 iap mutation leads to the reduction of virulence , but systemic infections 
 are caused .
it is thought that the 
 virulence - promoting function of iap protein ( p60 ) has been due to its cell wall hydrolysis 
 ability .
the iap contains a c - terminal 
 endopeptidase domain , two n - terminal lysin motif ( lysm ) domains and a single n - terminal src 
 homology 3 ( sh3)-like domain .
group a 
 contained less than nine places of mutations , including 14 iap types ( 011 , 
 20 and 21 ) .
eight iap types ( 1216 , 22 , 24 and 25 ) , which contained 2225 
 places of mutations , belonged to group b. group c ( four iap types ; 1719 
 and 23 ) contained more than 50 places of mutation ( supplementary table 2 ) .
as described 
 previously [ 15 , 34 ] , groups a and b were suggested to correspond to lineages ii and i , 
 respectively [ 20 , 21 , 37 ] .
unfortunately , no isolate was 
 classified into lineage iii , which is supposed to consist of serotype 4a according to 
 rasmussen et al . .
in contrast , 
 we preserved images of rflp patterns of genomic dna obtained from l. 
 monocytogenes isolated since 1998 . l. 
 monocytogenes strains , including egd - e , were classified into 26 , 29 and 
 28 types using whole genomic rflp analyses digested with xbai , 
 clai and psti , respectively .
the classification based on 
 partial iap sequences agreed with the rflp - based classification .
these 
 results support our previous suggestion that iap - rflp subtyping is useful 
 for detailed differentiation of isolates for epidemiological purposes [ 24 , 25 , 31 , 36 ] .
however , certain 
 drawbacks remain in rflp analysis of genomic dna with regard to distinction of rflp patterns 
 and inter - laboratory sharing of data .
pfge classification is a valuable investigation tool 
 to recognize common sources of food - borne outbreaks .
however , pfge is hard to determine the evolutionary relatedness of isolates 
 , because pfge patterns are influenced by 
 changes in the accessory genome , including transient bacteriophages .
several techniques for genetic classification of l. 
 monocytogenes using dna sequences have been developed . 

repetitive - sequence - based pcr ( rep - pcr ) targets noncoding short repetitive sequences .
multiple - locus variable - number tandem 
 repeat analysis ( mlva ) is a size analysis of amplified regions of dna containing variable 
 numbers of tandem repeats .
this method requires normalization of sizing discrepancies for accurate and standardized 
 mlva on capillary electrophoresis . in comparison 
 with dna size - based subtyping methods including pfge and rflp
, the dna sequence - based 
 subtyping approach , such as mlst , is an informative tool for epidemiology and studies 
 involving evolutionary relationships between strains .
the data obtained from our mlst can be used to compare with or refer to previous 
 information .
traditional mlst is based on several housekeeping genes , because these are non - susceptible 
 to horizontal gene transfer and selection . 

l. monocytogenes mlst database 
 ( http://bigsdb.web.pasteur.fr/listeria ) maintained by the pasteur institute ( paris , france ) 
 is based on seven housekeeping genes : abcz , bgla , 
 cat , dape , dat , ldh 
 and lhka [ 26 , 40 ] .
however , the evolution of virulence genes , which represent 
 well - characterized pathogenicity of l. monocytogenes , is 
 considered important .
previous mlst studies were performed using i ) three housekeeping genes 
 ( reca , prs and sigb ) , two virulence 
 genes ( acta and inla ) and two intergenic regions 
 ( hly - mpl and plca - hly ) 
 , ii ) four housekeeping genes 
 ( betl , dat , reca and 
 sigb ) and three virulence genes ( acta , 
 inla and inlb )   and iii ) five housekeeping genes ( gap , prs , 
 purm , ribc and sigb ) and two virulence 
 genes ( acta and inla ) .
these suggest that the nucleotide sequences of sigb , 
 acta and inla are useful for genetic classification .
it 
 was reported that there are 19 different mutations leading to premature stop codons in 
 inla and these mutations occur commonly in l. 
 monocytogenes lineages i and ii .
therefore , we chose sigb and acta in 
 addition to iap for mlst analysis in this study . the sigb sequence resulted in classification into 37 types and three 
 groups by total point mutations as compared to that of egd - e as well as 
 iap
the number of point mutations in groups a ( sigb types 
 1 , 11 , 12 , 34 and 36 ) , b ( sigb types 210 , 1326 , 3133 , 35 and 37 ) and c 
 ( sigb types 2730 ) was less than two , 2530 and more than 50 , 
 respectively ( table 3 ) .
although a partial 
 sequence of sigb derived from 67 isolates did not show diversity in 
 comparison with iap ; the iap types 0 , 2 , 12 and 13 could 
 be classified into two or three groups via sigb type .
this suggests that 
 the nucleotide sequence of sigb might be relatively conserved in 
 l. monocytogenes regardless of the geographical 
 distribution .
nonetheless , the acta sequences were classified into 69 types 
 ( table 4 ) .
these types were further divided 
 into two groups by total point mutations as compared to that of egd - e .
group a 
 ( acta types 0 , 1838 , 67 and 68 ) contained less than seven places of 
 mutation , whereas group b ( acta types 217 , 3966 and 69 ) contained more 
 than 50 places of mutation .
acta can be used as an evolutional indicator as 
 it appears to have undergone positive selection . 

the target sequence of acta was located at the c - terminal region of acta 
 protein .
this region consists of the membrane anchor domain and the cell wall penetration 
 domain .
acta is a natively unfolded protein , and the n - terminal region and central domain of 
 acta are responsible for its virulence .
mutations 
 in this region are unrelated to the virulence function for intracellular motility of 
 l. monocytogenes .
therefore , they are likely to identify 
 the genetic character of gene sequence , because several acta mutations are 
 found in a single strain alone . in total ,
194 strains registered in food microbe tracker 
 were classified into 65 acta types , whereas 67 strains isolated in our 
 laboratory were divided into 17 acta types .
the number of 
 acta types was less than that of iap type in our 
 isolates .
additional studies may be necessary to verify the diversity in 
 acta among strains , including the strains belonging to 
 iap group c , derived from 
 different sources , such as patients , environment and food . taken together , we conclude that 
 the sigb and acta are useful for genetic classification to 
 detect certain characteristic mutations .
the discriminatory ability of mlst using iap , sigb and 
 acta is the same as that of iap - rflp method
. strains 
 belonging to iap - rflp types 4 and 22 were further classified into two mlst 
 types ( table 1 ) . in contrast , the 
 iap - rflp types 22 ( except for 11h ) and 23 , 24 and 25 were integrated into 
 the results from mlst . strains of 229c1 ( iap - rflp type 22 and mlst type 27 ) 
 and 499c5 ( iap - rflp type 23 and mlst type 27 ) shared the same pfge patterns 
 digested with apai .
it is difficult to ascertain the reason for this 
 discrepancy in the present data .
these results suggest that mlst and 
 iap - rflp method have potential applications in epidemiology of l. 
 monocytogenes to trace the source of human infection .
although pfge provides greater 
 discrimination power than that of mlst , clustering and lineage distinction were consistent 
 with the results from pfge ( fig .
mlst profiles of 68 l. 
 monocytogenes strains were based on partial sequences of 
 iap , sigb and acta .
phylogenic 
 analysis was performed using unweighted - pair group method analysis with arithmetic 
 mean ( upgma ) .
the distances were calculated using the number of differences method 
 based on the number of nucleotide differences per target sequence .
the pfge 
 patterns of 104p5 and 268c1 with regard to apai as well as 12h and yc17p13 
 with regard to asci were similar ; however , a clear distinction was obtained 
 in mlst results for these strains .
the differences between 104p5 and 268c1 included two 
 substitutions , a to g and t to c , in iap , whereas only one substitution ( g 
 to a ) in iap differentiated 12h and yc17p13 .
this suggests that mlst 
 analysis is suitable to detect single nucleotide polymorphisms . in the future ,
mlst analyses 
 using whole genome sequence technology have global applications in subtyping of 
 l. monocytogenes [ 9 , 16 , 23 ] .
mlst profiles of 68 l. 
 monocytogenes strains were based on partial sequences of 
 iap , sigb and acta .
phylogenic 
 analysis was performed using unweighted - pair group method analysis with arithmetic 
 mean ( upgma ) .
the distances were calculated using the number of differences method 
 based on the number of nucleotide differences per target sequence .
the sequences of three genes in almost all the test strains were consistent with those of 
 food microbe tracker strains .
the types of sigb and acta , 
 which were not seen in the food microbe tracker strains , have a single base substitution or 
 nonsense mutation .
unfortunately , the iap target sequences of many food 
 microbe tracker strains are unspecified . therefore , the specific character of the japanese 
 isolates could not be determined in this study .
however , the strains that share the same 
 type of genes with the foreign isolates derived from listeriosis patients are frequently 
 isolated in japan .
in addition , almost 60% of food supply in japan depends on imports from other countries . 

consequently , there is an urgent need to develop effective countermeasures against 
 l. monocytogenes infection , even though occurrences of human listeriosis 
 in japan are limited to sporadic infections .
simultaneous surveillance for l. 
 monocytogenes contamination in food and environment along with listeriosis 
 epidemiology is vital for maintenance of food hygiene .
the results from this study include 
 the strains isolated from imported meat ; therefore , our mlst scheme can provide valuable 
 epidemiological information during outbreaks caused by strains that have entered japan from 
 other countries .
 pulse field gel electrophoresis ( pfge ) is widely used for listeriosis surveillance . 
 
 although this technique is effective for epidemiology , the data among laboratories are 
 inconsistent . 
 we previously reported a method for listeria monocytogenes 
 subtyping combined with sequence analysis of partial iap and whole genome 
 restriction fragment length polymorphism ( rflp ) using xbai , 
 clai ( baniii ) and psti . 
 however , 
 distinguishing subtypes was challenging , because the output comprised complicated fragment 
 patterns . in this study , we aimed to establish a simple genotyping method that does not 
 depend on visual observation , rather it focuses on multi - locus sequence typing ( mlst ) 
 using three genes , iap , sigb and acta . 
 
 sixty - eight strains of l. monocytogenes including egd - e as a reference 
 strain were investigated to ensure consistency with previous data on the genetic 
 characterization . 
 all strains were grouped into 29 types by both analyses . although there 
 are some differences in classification , major clades included the same strains . 
 simpson 
 s 
 indices of diversity ( sid ) by mlst and iap - rflp - based typing were 0.967 
 ( 95% confidence interval [ ci ] : 0.955/0.978 ) and 0.967 ( 95% ci : 0.955/0.979 ) , respectively . 
 
 the discriminatory power of both methods can be considered almost identical . 
 compared with 
 the results of 38 selected strains , the strains within the mlst clusters in this study 
 coincided with those obtained using pfge . 
 thus , the mlst strategy could help differentiate 
 among l. monocytogenes isolates during epidemiological studies . 
MATERIALS AND METHODS
RESULTS
DISCUSSION
Supplementary Material