)JOEBXJ1VCMJTIJOH$PSQPSBUJPO … · 2019. 5. 13. · Laborat ´orio de Zoonoses Bacterianas, Fundac ¸ao Instituto Oswaldo Cruz, Avenida Brasil , Manguinhos, RiodeJaneiro,RJ,Brazil

Research ArticlePhenotypic and Genotypic Characterization of AtypicalListeria monocytogenes and Listeria innocua Isolated fromSwine Slaughterhouses and Meat Markets

Luisa Zanolli Moreno12 Renata Paixatildeo2 Debora Dirani Sena de Gobbi2

Daniele Cristine Raimundo2 Thais Sebastiana Porfida Ferreira2

Andrea Micke Moreno2 Ernesto Hofer3 Cristhiane Moura Falavina dos Reis3

Glavur Rogeacuterio Matteacute1 and Maria Helena Matteacute1

1 Laboratorio Pratica de Saude Publica Faculdade de Saude Publica Universidade de Sao Paulo Avenida Doutor ArnaldoNo 715 01246 904 Sao Paulo SP Brazil

2 Laboratorio de Sanidade Suına e Virologia Faculdade de Medicina Veterinaria e Zootecnia Universidade de Sao PauloAvenida Professor Doutor Orlando Marques de Paiva No 87 Cidade Universitaria 05508 270 Sao Paulo SP Brazil

3 Laboratorio de Zoonoses Bacterianas Fundacao Instituto Oswaldo Cruz Avenida Brasil 4365 Manguinhos21040 360 Rio de Janeiro RJ Brazil

Correspondence should be addressed to Luisa Zanolli Moreno luzanolligmailcom

Received 23 February 2014 Accepted 3 May 2014 Published 28 May 2014

Academic Editor Stanley Brul

Copyright copy 2014 Luisa Zanolli Moreno et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

In the last decade atypical Listeria monocytogenes and L innocua strains have been detected in food and the environment Becauseof mutations in the major virulence genes these strains have different virulence intensities in eukaryotic cells In this study weperformed phenotypic and genotypic characterization of atypical L monocytogenes and L innocua isolates obtained from swineslaughterhouses and meat markets Forty strains were studied including isolates of L monocytogenes and L innocua with low-hemolytic activity The isolates were characterized using conventional phenotypic Listeria identification tests and by the detectionand analysis of L monocytogenes-specific genes Analysis of 16S rRNA was used for the molecular identification of the Listeriaspecies The L monocytogenes isolates were positive for all of the virulence genes studied The atypical L innocua strains werepositive for hly plcA and inlC Mutations in the InlC InlB InlA PI-PLC PC-PLC and PrfA proteins were detected in the atypicalisolates Further in vitro and transcriptomic studies are being developed to confirm the role of these mutations in Listeria virulence

1 Introduction

Listeria monocytogenes and L innocua are closely relatedspecies of the Gram-positive genus Listeria They are widelydistributed in the environment and frequently isolated fromfood L monocytogenes is the causative agent of listeriosisa foodborne disease with a high fatality rate (20ndash30)that mostly affects the elderly neonates and immunocom-promised individuals [1 2] L monocytogenes cannot bedistinguished from other Listeria species using conventionalisolation methods Standard biochemical methods and selec-tive and differential media are used for the identification of Lmonocytogenes [3 4] however some L ivanovii L innocua

and L seeligeri strains generate similar results to L mono-cytogenes in these tests [5ndash7] Therefore it is necessary toconfirm the virulence characteristics of L monocytogenes todistinguish the Listeria species

The best-characterized L monocytogenes virulence fac-tors are listeriolysin O (LLO) phosphatidylinositol phospho-lipase C (PI-PLC) and the internalins A and B (InlA andInlB) LLO and PI-PLC are encoded by the hly and plcAgenes respectively which belong to the virulence gene clusterListeria pathogenicity island 1 (LIPI-1) which contains themajor virulence genes of L monocytogenes [8] Few atypicalL innocua strains have been reported to contain L monocy-togenes-specific genes and exhibit phenotypic characteristics

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 742032 12 pageshttpdxdoiorg1011552014742032

2 BioMed Research International

similar to L monocytogenes such as weak hemolysis [6 7 9]Furthermore certain low-hemolytic Lmonocytogenes strainsretain their virulence despite the presence of mutations inmajor virulence genes [10ndash12]The existence of these atypicalstrains indicates that traditional phenotypic and genotypiccharacterization methods must be used with care and thatfurther studies are required to improve the identification ofListeria isolates

This study used phenotypic and genotypic methods tocharacterize atypical Lmonocytogenes and L innocua isolatesobtained from swine slaughterhouses and meat markets inSao Paulo State Brazil

2 Material and Methods

21 Bacterial Strains and Culture Conditions Forty Listeriasp isolates were studied Of these 25 were isolated fromporkslaughterhouses andmarkets (15 isolates of L monocytogenesand 10 of L innocua) 11 isolates of L monocytogenes wereobtained from human infections and four were controlstrains (L monocytogenes ATCC 19115 and ATCC 19111 andL innocua ATCC 33090 and CLIP 12612) (Table 1) Theenvironmental and pork isolates were isolated as describedby Moreno et al [13] the clinical strains and Listeria controlswere obtained from the Public Health Laboratory (School ofPublic Health University of Sao Paulo) and Laboratory ofSwine Health (School of Veterinary Medicine and AnimalScience University of Sao Paulo) collections The environ-mental and pork isolates were obtained from different swabsamples taken from the slaughterhouses environment andcarcasses from Sao Paulo State the clinical isolates wereobtained from the blood placenta and cerebrospinal fluidsamples of different patients from different Brazilian states(Tables 1 and 2)

The isolates were maintained in a stock medium con-taining glycerol at minus80∘C The isolates were reactivated inbrain-heart infusion (BHI) medium (Difco Sparks MDUSA) and plated on tryptone soy agar supplemented withyeast (TSAYE) (Oxoid Lenexa USA) to isolate pure coloniesbefore use

22 Conventional Listeria Identification Tests The isolateswere serotyped using polyclonal antisera produced againstListeria somatic and flagellar antigens in rabbits accord-ing to the method described by Seeliger and Hohne[14] The isolates were also characterized by catalasemotility and biochemical tests including acid productionfrom D-xylose D-mannitol L-rhamnose and 120572-methyl-D-mannoside Cultivation in selective agar Listeria accordingto Ottaviani and Agosti (ALOA) (Biolife Milan Italy) wasused to identify L monocytogenes isolates and 120573-hemolysiswas assessed by sting inoculation on 5 sheep bloodagar

23 Detection of L monocytogenes-Specific Genes GenomicDNA extraction was performed as described by Ausubel etal [15] All isolates were screened for the inlA inlB inlCinlJ hly prfA plcA and plcB genes The primers described

by Johnson et al [6] Liu et al [16] and Jung et al [17]were used for detection of prfA inlC and inlJ and inlArespectively Specific primers were designed for the completeamplification of the virulence genes (Table 3)The PCRs wereperformed using an Eppendorf Mastercycler gradient thermalcycler Each reaction (25120583L) contained 5 120583L of genomicDNA MilliQ water 10X PCR buffer 15mM MgCl

2200120583M

of dNTPs (Fermentas Burlington Canada) 200120583M of eachprimer and 125U of Taq DNA polymerase (Promega) ThePCR programs were as follows 30 cycles of denaturation at94∘C for 1min annealing at primer-specific temperature for1ndash15min elongation at 72∘C for 1min per 1 Kb and finalextension at 72∘C for 10min The amplified products wereseparated by electrophoresis on 15 agarose gels and stainedwith ethidium bromide (1120583gmL) The molecular weightsof the products were determined using the 1 Kb Plus DNALadder (Fermentas Burlington Canada)

24 DNA Sequencing The amplified fragments were purifiedusing the Illustra GFX PCR DNA and Gel Band Purificationkit (GE Healthcare) according to the manufacturerrsquos protocoland sequenced directly at Genomic (Genomic EngenhariaMolecular Sao Paulo Brazil) DNA sequencing was per-formed on an Applied Biosystems 3130xl DNA analyzer usingthe BigDye Terminator v31 cycle sequencing kit

25 Detection of Mutations in L monocytogenes Viru-lence Genes Sequence analysis was performed using theBIOEDIT SequenceAlignment Editor 709 [18]Theobtainedsequences of the virulence genes were compared to pre-viously published L monocytogenes sequence accessionsfrom GenBank (NCBI Bethesda USA) The sequencingproducts were edited and compared with the sequencesavailable in the GenBank database by manual alignment andusing the ClustalW application The nucleotide sequencesobtainedwere translated into their corresponding amino acidsequences by the Nucleotide Translate application Subse-quently the amino acid sequences were analyzed to identifychanges in the compositions of their respective proteinswhich might modify or eliminate protein functions

26 Identification of Protein Domains and Prediction of Sec-ondary Structures The domains of InlC InlB InlA PI-PLCPC-PLC and Hly from reference strain L monocytogenesEGD-e were determined using the PROSITE database [19]of the ExPASy server (SIB Swiss Institute of Bioinformatics)The locations of these domains were compared to the muta-tions identified in the studied isolates

27 Species-Level Identification by 16S rRNAAmplification andPhylogenetic Analysis Species identity was confirmed using16S rRNA analysis The primers and amplification protocoldescribed by Thompson et al [20] were used to amplifycomplete 16S rRNA genes The fragments were sequencedand phylogenetic analysis was performed using theMega 510software [21] The dendrogram was constructed using themaximum-likelihood method with the Tamura-3-parametermodel

BioMed Research International 3

Table1Sourcesa

ndph

enotypicandgeno

typicc

haracteristicso

fthe

Liste

riamonocytogenesiso

latesu

sedin

thisstu

dy

Isolate

Species

Serotype

Orig

inSite

Year

ALO

AHem

olysis

inlA

inlB

inlC

inlJlowast

plcA

plcB

prfA

hly

Lm1

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm2

Lmonocytogenes

12b

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm3

Lmonocytogenes

4bMarket1

Floo

r2008

Halo

Positive

++

++

++

++

Lm21

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm22

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm23

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm25

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm26

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm27

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm28

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm29

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm30

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm31

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm34

Lmonocytogenes

12a

Hum

anBloo

d1989

Halo

Strong

positive

++

++

++

++

Lm35

Lmonocytogenes

4bHum

anBloo

d2004

Halo

Strong

positive

++

++

++

++

Lm36

Lmonocytogenes

4bHum

anBloo

d1977

Halo

Strong

positive

++

++

++

++

Lm37

Lmonocytogenes

4bHum

anCS

F1982

Halo

Strong

positive

++

++

++

++

Lm38

Lmonocytogenes

12b

Hum

anCS

F1983

Halo

Strong

positive

++

++

++

++

Lm39

Lmonocytogenes

12a

Hum

anPlacenta

1978

Halo

Strong

positive

++

++

++

++

Lm39a

Lmonocytogenes

12a

Hum

anPlacenta

1978

Halo

Positive

++

++

++

++

Lm40

Lmonocytogenes

12a

Hum

anBloo

d1985

Halo

Strong

positive

++

++

++

++

Lm41

Lmonocytogenes

4bHum

anCS

F1997

Halo

Strong

positive

++

++

++

++

Lm42

Lmonocytogenes

4bHum

anCS

F1997

Halo

Positive

++

++

++

++

Lm43

Lmonocytogenes

12a

Hum

anCS

F1983

Halo

Positive

++

++

++

++

Lm4

Lmonocytogenes

12a

Market2

Floo

r2008

Halo

Weakpo

sitive

++

++

++

++

Lm33

Lmonocytogenes

12a

Market2

Floo

r2008

Halo

Weakpo

sitive

++

++

++

++

Lm10

Lmonocytogenes

4bAT

CC19115

mdashmdash

Halo

Strong

positive

++

++

++

++

Lm15

Lmonocytogenes

12a

ATCC

19111

mdashmdash

Halo

Strong

positive

++

++

++

++

Slaught1slau

ghterhou

se1CS

Fcerebrospinalfl

uidlowast

Alliso

latesw

erep

ositive

forfragm

entsof

inlJbu

tpresented

varia

bler

esultsforh

oleg

enea

mplificatio

n(see

Table4

)


Table2Sourcesa

ndph

enotypicandgeno

typicc

haracteristicso

fthe

Liste

riainnocuaiso

latesu

sedin

thisstu

dy

Isolate

Species

Serotype

Orig

inSite

Year

ALO

AHem

olysis

inlA

xinlB

inlC

inlJ

plcA

plcB

prfA

hlyx

Lin5

Linnocua

6aMarket1

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin6

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin7

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin8

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin9

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin16

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin17

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin18

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin19

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin2

0Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin11

Linnocua

6aAT

CC33090

mdashmdash

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin4

6Linnocua

6aCL

IP12612

mdashmdash

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Slaught1slaug

hterho

use1Slaug

ht2sla

ughterho

use2

lowastSubtlehalolowastlowast

Very

weakpo

sitiveh

emolysis

x Atypicalisolatesw

erep

ositive

forfragm

entsof

inlABop

eron

andhlyb

utpresentedvaria

bler

esultsforinlA

andhlyc

ompletea

mplificatio

n(see

Table4

)


Table 3 Primers designed in this study for the amplification of the L monocytogenes virulence genes

Primer Sequence 51015840-31015840 Target Product (bp)inlA ext Fw CGGCTCCGTAGACAGATTAG

inlA

2884inlA ext Rv GTGATAGTCTCCGCTTGTACinlA In1-Fw GTGAGAAGAAAACGA 1200inlA Detec-Rv TGGTGTAAGATCGCTinlA Detec-Fw AAGTGATATAACTCC mdashinlB ext Fw GCTAGATGTGGTTTTCGGACT

inlB

2146inlB ext Rv TAAGCAGCGCAAAGGTGATTCCTACinlB In-Fw GTGAAAGAAAAGCAC 1227inlB Seq3-Rv ATTCCCGCGAATATAinlB Seq2-Fw TGATGGAACGGTAAT 900inlB End3-Rv TNATTTCTGTGCCCTplcB ext Fw CCATACGACGTTAATTCTTGCAATG plcB 1039plcB ext Rv TATCCACCCGCTAACGAGTGplcA ext Fw GAGGTTGCTCGGAGATATAC

plcA1100

plcA ext Rv CTGCTGTCCCTTTATCGTCGplcA Detec-Fw AACCATTATTATGCG 396plcA Detec-Rv TGCAGCATACTGACGhly ext Fw CGATAAAGGGACAGCAGGACT

hly

1796hly ext Rv AGCCTGTTTCTACATTCTTCACAAhly Detec-Fw TAACAACGCAGTAAA 566hly Detec-Rv CGTAAGTCTCCGAGGhly End-Fw CCTCCTGCATATATC 725hly End-Rv TTATTCGATTGGATTinlC In1-Fw ATGCTAGTNTTAATTGTA inlC 852inlC End2-Rv CTAATTCTTGATAGGTTGTGprfA Detec-Fw CTGCTAACAGCTGAGCTATG

prfA404

prfA Detec-Rv GCTACCGCATACGTTATCprfA End Rv ATGAACGCTCAAGCA mdashIn primers corresponding to the beginning of the gene End primers corresponding to the end of the gene Detec internal primers designed for gene detectionext external primers Seq internal primers designed for sequencing

28 Nucleotide Sequence Accession Numbers All DNAsequences from this study were deposited in GenBank underthe accession numbers KC618415-KC618420 KC666995-KC667019 KC808518-KC808549 and KC808567-KC808583

3 Results

31 Conventional Listeria Identification Tests Thephenotypiccharacterization of Listeria sp isolates is shown in Tables 1and 2 Five atypical L innocua isolates (Lin5ndash9) and six low-hemolytic L monocytogenes (Lm4 Lm33 and Lm28ndash31) iso-lates were observedThe atypical L innocua isolates exhibitedphenotypic characteristics similar to L monocytogenes withweak hemolysis and subtle halo in ALOA cultivation Theseisolates could be distinguished only by serotyping whichrevealed that the atypical isolates were L innocua serotype 6a

32 Detection and Analysis of L monocytogenes VirulenceGenes The detection and complete amplification of the inlBinlC plcA plcB hly and prfA genes were performed using

previously published primers and primers designed in thisstudy The inlA and inlJ genes were only partially amplifiedusing the primers inlA In-FwinlA Detec-Rv designed in thisstudyand inlJ-FwinlJ-Rv which were described by Liu etal [16] All L monocytogenes isolates including the six low-hemolytic isolates (Lm4 Lm33 and Lm28ndash31) contained thestudied genes The five atypical L innocua isolates (Lin5ndash9) contained inlC and plcA and fragments of the hly gene(Table 4)

Nucleotide substitutions were detected in inlC inlBinlA plcA plcB and prfA only in the six low-hemolytic Lmonocytogenes isolates (Lm4 Lm33 and Lm28ndash31) Sevensubstitutions were detected in the inlC gene however onlythe transition of adenine to cytosine and the inversion ofthiamine to adenine at codon 10 led to the mutation Ile10Hisand the transition of thiamine to cytosine at codon 12 resultedin the mutation Met12Thr Ten substitutions were detectedin plcA leading to the mutations Ile17Val and Phe119Tyrin the PI-PLC In the plcB sequence only two transitionsof thiamine to cytosine were identified at codon 13 which


Table 4 Distribution of the results of the virulence gene amplification from Listeria species

Primer Species Positive Negative119873 () 119873 ()

inlC Liu1 L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

inlC InndashEnd L monocytogenes 28 (1000) 0L innocua 5 (417) 7 (583)

prfA JohnsonndashEnd L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

prfA Johnson2 L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

prfA Detec L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

plcA ext L monocytogenes 28 (1000) 0L innocua 5 (417) 7 (583)

plcA Detec L monocytogenes 28 (1000) 0L innocua 5 (417) 7 (583)

plcB ext L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

inlB InndashSeq3L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

inlB Seq2ndashEndL monocytogenes 28 (1000) 0L innocua 0 12 (1000)

inlA InndashDetec L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

inlAB Jung3 L monocytogenes 28 (1000) 0L innocua 5 (417) 7 (583)

hly ext L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

hly End L monocytogenes 28 (1000) 0L innocua 5 (417) 7 (583)

hly Border4 L monocytogenes 28 (1000) 0L innocua 5 (417) 7 (583)

hly Detec L monocytogenes 28 (1000) 0L innocua 5 (417) 7 (583)

inlJ Liu1 L monocytogenes 28 (1000) 0L innocua 0 12 (1000)

inlJ ext L monocytogenes 23 (821) 5 (179)L innocua 0 12 (1000)

1Primers described by Liu et al [16] 2Primers described by Johnson et al [6] 3Primers described by Jung et al [17] 4Primers described by Border et al [22]

resulted in the mutation Ile13Thr Seven substitutions weredetected in inlB however only the transitions of adenineto guanine at codons 117 and 132 resulted in the mutationsAla117Thr and Val132Ile (Figures 1 and 2)

A deletion of five nucleotides was also detected in theprfA sequence leading to the deletion of codons 236 and 237in the Lm4 Lm33 and Lm28ndash31 isolates Eight substitutionswere detected in the inlA fragments of the low-hemolytic Lmonocytogenes isolates resulting in the mutations Thr51Alaand Ile157Leu (Figure 3) The Lm4 Lm33 and Lm28ndash31isolates also contained 15 substitutions in the hly sequencewhereas the Lin5 and Lin6ndash9 isolates only contained 14 and13 of these substitutions respectively However all these

atypical isolates contained only the mutations Val438Ile andLys523Ser (Figure 3)

33 Identification of ProteinDomains Of the identifiedmuta-tions only Ala117Thr and Val132Ile in InlB and Ile157Leu inInlA were located in the leucine-rich repeat (LRR) domainsthat are characteristic of these proteins The Phe119Tyrmutation in PI-PLC was also located in the PI-PLC X-boxdomainThe thiol-activated cytolysin signature motifs in Hlyand the zinc-dependent phospholipase C domain in PC-PLCpresented distinct locations of the mutations identified in therespective proteins


186220231 501510 833843844852

ATTATGGACGTGGGCCTACTTAACTGCAGCCA

CGAAGACCATTGCCTGAATTATTC

CGAAGACCTAATTGCCCTGAATATTC

CTC

CTC

TC

CGTCT

CGTCT

CGTCT

CGTCT

CATCT

inlC EGDeinlC SLCC2378inlC SLCC2540inlC 08-5578inlC 08-5923inlC SLCC7179inlC Lm28inlC Lm29inlC Lm30inlC Lm31inlC Lm4inlC Lm33 CATCT

111114117 162189213216250274 954AGATAAAAGAACACTCTTTAA

TAGGGGCT

TAGGGGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT

plcA EGDeplcA 08-5578plcA 08-5923plcA CNL895807plcA CNL895795plcA A23plcA Lm28plcA Lm29plcA Lm30plcA Lm31plcA Lm4plcA Lm33 GGATTTC ACT

3839 870ATTG

CCA

CCA

CCC

CC

CC

CC

CC

CC

plcB EGDeplcB 08-5578plcB 08-5923 plcB SLCC5850plcB Lm28plcB Lm29plcB Lm30plcB Lm31plcB Lm4plcB Lm33 CC

421 762 1053 1665 1893CTGGGGTTTGCG

TCAAAACCC

TCAAAACCC

AAAACCCTA

AAAACCCTA

AAAACCCATA

AAAACCC

AAAACCC

AAAACCC

AAAACCC

AAAACCC

08-557808-5923

inlB EGDeinlB 08inlBinlB CNL895807inlB CNL895795inlB A23inlB Lm28inlB Lm29inlB Lm30inlB Lm31inlB Lm4inlB Lm33 AAAACCC

1 282935 45 84 94 141 276306318 334345 390397415 426 478 629 702711716 735750760

1 1118203739 49 56 81 356 690 784795

1 15 787

1201 145 349324 394 399 1532 1656

Figure 1 Nucleotide substitutions detected in the inlC plcA plcB and inlB genes The Lm28ndash31 Lm4 and Lm33 isolates were aligned withL monocytogenes EGDe and the previously described mutant strains Asterisks indicate the start and stop codons dots represent identicalnucleotides and numbers indicate the positions of the substitutions

34 Species Confirmation by 16S rRNA Phylogenetic AnalysisFrom the amplification and analysis of the 16S rRNA genes adendrogram was constructed which allowed the distinctionof L monocytogenes and L innocua species The dendrogramcontained three major groups the first group consistedof L grayi and L murrayi the second group contained

L rocourtiae and the third group consisted of clusters of Lmonocytogenes and L marthii L innocua L welshimeri Lseeligeri and L ivanovii (Figure 4) The isolates Lin5ndash9 andLin11 were grouped with the standard strains of L innocuawhereas the isolates Lm28ndash31 Lm4 and Lm33 were groupedtogether with the standard strains of L monocytogenes


1 10 12 32 74 102112 133 210 239 254 278282283IMVQKVLKSDTK

TIKVRNNIQ

TIKNMVRNNIQ

T

T

T

T

H

H

119NQLIYFT

IRVC

IRVC

VYA

VYA

VYA

VY

VY

VY

VY

VY

PI-PLC EGDePI-PLC 08-5578 PI-PLC 08-5923 PI-PLC CNL895807PI-PLC CNL895795PI-PLC A23PI-PLC Lm28PI-PLC Lm29PI-PLC Lm30PI-PLC Lm31PI-PLC Lm4PI-PLC Lm33 VY

263 KIE

TK

TK

NT

T

T

T

T

T

PC-PLC EGDePC-PLC 08-5578PC-PLC 08-5923PC-PLC SLCC5850PC-PLC Lm28PC-PLC Lm29PC-PLC Lm30PC-PLC Lm31PC-PLC Lm4PC-PLC Lm33 T

1PSAVG

SPTI

SPTI

TI

TI

TID

TI

TI

TI

TI

TI

EGDeInlBInlB

08-5578InlB 08-5923InlB CNL895807InlB CNL895795InlB A23InlB Lm28InlB Lm29InlB Lm30InlB Lm31InlB Lm4InlB Lm33 TI

1 236237238LN

-X

-X

-X

-X

-X

PrfA EGDePrfA 08-5578PrfA 08-5923PrfA Lm28PrfA Lm29PrfA Lm30PrfA Lm31PrfA Lm4PrfA Lm33 -X

InlC EGDeInlC SLCC2378InlC SLCC2540InlC 08-5578InlC 08-5923InlC SLCC7179InlCInlCInlCInlCInlCInlC

Lm28Lm29Lm30Lm31Lm4Lm33

1 4 7 13 1719 262 318

1 5 13 290

6305111321174941

Figure 2 Amino acids substitutions in the InlC PI-PLC PC-PLC InlB and PrfA proteins The Lm28ndash31 Lm4 and Lm33 isolates werealigned with L monocytogenes EGDe and the previously describedmutant strains Asterisks indicate the start and stop codons dots representidentical amino acids and numbers indicate the positions of the substitutions


TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

hly EGDehly 08-5578hly 08-5923 hly Lm28hly Lm29hly Lm30hly Lm31hly Lm4hly Lm33hly Lin5hly Lin6hly Lin7hly Lin8hly Lin9 __ _ATTCATGACCTGT

1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

inlA EGDeinlA 08-5578inlA 08-5923 inlA ATCC19115inlA F2365inlA Lm28inlA Lm29inlA Lm30inlA Lm31inlA Lm4inlA Lm33 TCCGGCTG_

63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -

ACTTAAAGCGAAGACGAGTTTCGTC -

TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS

KTVNTISNAAATSPVHNASADLM

LDNATT

Hly EGDeHlyHlyHly Lm28Hly Lm29Hly Lm30Hly Lm31Hly Lm4Hly Lm33Hly Lin5Hly Lin6Hly Lin7Hly Lin8Hly Lin9

08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_

InlA EGDeInlA 08-5578InlA 08-5923 InlA ATCC19115InlA F2365 InlA Lm28InlA Lm29InlA Lm30InlA Lm31InlA Lm4InlA Lm33 AL_

__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)

Figure 3 Nucleotide substitutions detected in inlA and hly (a) and mutations identified in InlA and Hly (b) The Lm28ndash31 Lm4 Lm33and Lin5ndash9 isolates were aligned with L monocytogenes EGDe and previously described mutant strains Asterisks indicate the start and stopcodons dots represent identical amino acids and numbers indicate the positions of the substitutions Gaps represent the regions that werenot amplified

4 Discussion

Studies on Listeria virulence mechanisms have becomeimportant in recent decades because this microorganism isused as a model of intracellular infection L monocytogenesvirulence factors have been described and their mecha-nisms of action and respective genes have been studiedusing distinct molecular techniques and in vivo and in vitro

experiments In addition to the use of Listeria as a modelorganism there is great interest in studying this organismbecause of the increasing incidence of listeriosis in theUnitedStates of America (USA) and Europe [23 24]

Our results using conventional Listeria identificationtests are consistent with the subjectivity and ambiguity ofphenotypic tests that have been discussed in the last decade[6 7] Although these conventional methods are still utilized


L marthii NR-9579L marthii NR-9580L marthii NR-957T

L monocytogenes H3597L monocytogenes H3598

L monocytogenes EU545987L monocytogenes H3599

Lm4Lm33Lm28 Lm29 Lm30 Lm31

L monocytogenes NCTC 10357TL monocytogenes NCTC 10357

L monocytogenes H3508L monocytogenes Scott A

L monocytogenes EU545986L monocytogenes ATCC19115

L innocua ATCC33090TL innocua NCT 11288Lin8

Lin5Lin6Lin7 Lin9Lin11

L welshimeri ATCC35897L welshimeri ATCC35897

L seeligeri ATCC35967L seeligeri NCTC 11856

L ivanovii CLIP 12229TL ivanovii CLIP 12510TL ivanovii CLIP 12150

L rocourtiae CLIP 109804TL murrayi NCTC 10812

L grayi ATCC19120TL grayi H350695

100

85

88

88

89

99

3698

27

70

66

15

16

26

0005

Figure 4 Dendrogram showing the evolutionary relationships among the Listeria isolates based on the 16S rRNA nucleotide sequencesThe dendrogram was constructed using the maximum-likelihood method (Tamura-3-parameter model) with the MEGA 510 software Thebootstrap values presented at corresponding branches were evaluated using 500 replicates

biochemical and phenotypic tests yield variable results duringthe identification of Listeria species and serotypes and theemergence of atypical isolates has further increased theuncertainty of the application of these tests From a publichealth perspective a drastic measure could be adopted toclassify all isolates with doubtful hemolytic status as Lmonocytogenes or as isolates with pathogenic risk withoutmajor efforts to identify the species and serovars However

for better epidemiological microbiological and evolutionaryunderstanding it is important to identify and characterize thephenotypes and molecular features of these atypical isolates

This study aimed to detect the hly plcA plcB prfA inlAinlB inlC and inlJ genes in L monocytogenes and L innocuaisolates These genes are characteristic of L monocytogenesand are essential for intracellular infection The presenceof these genes in isolates from meat and the environment


suggests the pathogenic potential of these isolates and a riskto human health We detected these virulence genes in allL monocytogenes isolates including the six low-hemolyticisolates (Lm4 Lm33 and Lm28ndash31) additionally the fiveatypical L innocua isolates (Lin5ndash9) contained the inlC hlyand plcA genes

Our results are consistent with the data of Johnson etal [6] and Volokhov et al [7] who identified some Lmonocytogenes virulence genes in L innocua strains withatypical hemolysis Therefore the use of traditional PCRmethods based mostly on the detection of hly and plcAfor the distinction of Listeria pathogenic species shouldbe reconsidered because these methods do not enable thedistinction of atypical isolates Accurate identification ofListeria species was possible only by the complete sequenc-ing and phylogenetic analysis of the 16S rRNA gene (Fig-ure 4) We propose that the detection of prfA plcB andinlB might be a better and reliable alternative to enable therapid distinction of L monocytogenes and L innocua Wealso suggest that analysis of the complete 16S rRNA genesequences is important for the accurate identification ofListeria species

The inlC and plcA genes from the atypical L innocua iso-lates did not contain nucleotide substitutions and mutationsin their respective proteins The only mutations identified inthese isolates were theVal438Ile and Lys523Ser inHlyThe hlygene could not be completely amplified but this might be dueto insertions or deletions between the detected fragmentsHowever the hemolytic phenotypes of these atypical isolatessuggest that despite the difficulty in amplifying this locusthere were no gross alterations in Hly function Furtherstudies will be carried out to confirm and quantify hlyexpression

Because the atypical L innocua isolates presented thelow-hemolytic phenotype and halo in ALOA cultivation weconcluded that these isolates produce at least Hly and PI-PLC Since the only detected mutations were not located inthe thiol-activated cytolysin signature motifs in Hly the lowexpression of the hly and plcA genes might be due to alteredpromoter activity As the prfA gene was also not detected inthese isolates we suggest that a secondary promoter mightactivate the expression of hly and plcA and originate theobserved phenotype However further in vitro and proteomicstudies are necessary to verify the activity and integrity ofthese virulence factors

The mutations detected in InlB and PI-PLC in thelow-hemolytic L monocytogenes isolates (Lm4 Lm33 andLm28ndash31) are consistent with results from previous studieson low-virulent L monocytogenes field strains [10ndash12] Themutations Ala117Thr and Val132Ile in InlB are located inthe LRR domains of this protein which are directly relatedto the interaction of this internalin with the Met cellularreceptor and might compromise the adhesin function ofInlB [11 12] The Ile17Val and Phe119Tyr mutations in PI-PLC are located in the signal sequence and the X-boxdomain respectively whereas theThr262Alamutation causesthe introduction of an amino acid with different physic-ochemical properties which might inhibit PI-PLC activity[12]

The mutations identified in PC-PLC InlC InlA PrfAand Hly are novel The Ile13Thr mutation in PC-PLC is notlocated at the zinc-dependent phospholipaseCdomain of thisprotein and the Ile10His and Met12Thr mutations in InlCare not located in the LRR domains of this internalin TheThr51Ala and Ile157Leu mutations in InlA are also novel andalthough they do not cause the truncation of InlA [11 12] theyare located in the LRR domains therefore these mutationsmight compromise the internalization of L monocytogenesin epithelial cells Further expression studies are required toconfirm whether these mutations affect the expression andfunction of these virulence factors

The low-hemolytic L monocytogenes isolates containedthe same Hly mutations as the atypical L innocua theconse-quence of this observation is unclear The deletion in prfA inthe low-hemolytic L monocytogenes isolates might underliethe reduced hemolytic activity in these strains because PrfAis the activator of the LIPI-1 cluster However the impairmentof prfA would result in the reduced expression of all LIPI-1genes Therefore further transcriptomic studies are requiredto completely characterize these atypical isolates enhance ourknowledge of their evolution and impact on public healthand developmore efficient methods for the identification anddistinction of Listeria species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was sponsored by FAPESP-Sao Paulo ResearchFoundation (Process nos 0655501-0 1019005-4 1013511-5)and CAPES-Coordenacao de Aperfeicoamento de Pessoal deNıvelSuperior

References

[1] J M Farber and P I Peterkin ldquoListeria monocytogenes a food-borne pathogenrdquo Microbiological Reviews vol 55 no 3 pp476ndash511 1991

[2] P S Mead L Slutsker V Dietz et al ldquoFood-related illness anddeath in the United Statesrdquo Emerging Infectious Diseases vol 5no 5 pp 607ndash625 1999

[3] J Rocourt A Schrettenbrunner and H P R Seeliger ldquoBio-chemical differenciation of the Listeria monocytogenes (sensulato)rdquo Annales de lrsquoInstitut Pasteur Microbiologie vol 134 no1 pp 65ndash71 1983

[4] E G Lemes-Marques and T Yano ldquoInfluence of environmentalconditions on the expression of virulence factors by Listeriamonocytogenes and their use in species identificationrdquo FEMSMicrobiology Letters vol 239 no 1 pp 63ndash70 2004

[5] E Gouin J Mengaud and P Cossart ldquoThe virulence gene clus-ter of Listeria monocytogenes is also present in Listeria ivanoviian animal pathogen and Listeria seeligeri a nonpathogenicspeciesrdquo Infection and Immunity vol 62 no 8 pp 3550ndash35531994


[6] J Johnson K Jinneman G Stelma et al ldquoNatural atypical Lis-teria innocua strains with Listeria monocytogenes pathogenicityisland 1 genesrdquoApplied and EnvironmentalMicrobiology vol 70no 7 pp 4256ndash4266 2004

[7] D V Volokhov S Duperrier A A Neverov J George CBuchrieser and A D Hitchins ldquoThe presence of the internalingene in natural atypically hemolytic Listeria innocua strainssuggests descent from L monocytogenesrdquo Applied and Environ-mental Microbiology vol 73 no 6 pp 1928ndash1939 2007

[8] P Cossart and M Lecuit ldquoInteractions of Listeria monocy-togenes with mammalian cells during entry and actin-basedmovement bacterial factors cellular ligands and signalingrdquoEMBO Journal vol 17 no 14 pp 3797ndash3806 1998

[9] M Perrin M Bemer and C Delamare ldquoFatal case of Listeriainnocua bacteremiardquo Journal of Clinical Microbiology vol 41no 11 pp 5308ndash5309 2003

[10] S M Roche P Gracieux E Milohanic et al ldquoInvestiga-tion of specific substitutions in virulence genes characterizingphenotypic groups of low-virulence field strains of Listeriamonocytogenesrdquo Applied and Environmental Microbiology vol71 no 10 pp 6039ndash6048 2005

[11] S M Roche O Grepinet Y Corde et al ldquoA Listeria mono-cytogenes strain is still virulent despite nonfunctional majorvirulence genesrdquo Journal of Infectious Diseases vol 200 no 12pp 1944ndash1948 2009

[12] S Temoin S M Roche O Grepinet Y Fardini and P VelgeldquoMultiple point mutations in virulence genes explain the lowvirulence of Listeria monocytogenes field strainsrdquoMicrobiologyvol 154 no 3 pp 939ndash948 2008

[13] L Z Moreno R Paixao D D Gobbi et al ldquoCharacterizationof atypical Listeria innocua isolated from swine slaughterhousesand meat marketsrdquo Research in Microbiology vol 163 no 4 pp268ndash271 2012

[14] H P R Seeliger and K Hohne ldquoSerotyping of Listeria mono-cytogenes and related speciesrdquoMethods in Microbiology vol 13pp 31ndash49 1979

[15] F M Ausubel R Brent and R E Kingston Short Protocols inMolecular Biology John Wiley amp Sons New York NY USA1995

[16] D Liu M L Lawrence A J Ainsworth and F W AustinldquoToward an improved laboratory definition of Listeria monocy-togenes virulencerdquo International Journal of Food Microbiologyvol 118 no 2 pp 101ndash115 2007

[17] Y S Jung J F Frank R E Brackett and J Chen ldquoPoly-merase chain reaction detection of Listeria monocytogenes onfrankfurters using oligonucleotide primers targeting the genesencoding internalin ABrdquo Journal of Food Protection vol 66 no2 pp 237ndash241 2003

[18] T A Hall ldquoBioEdit a user-friendly biological sequence align-ment editor and analysis program for Windows 9598NTrdquoNucleic Acids Symposium Series vol 41 pp 95ndash98 1999

[19] C J A Sigrist L Cerutti E de Castro et al ldquoPROSITE aprotein domain database for functional characterization andannotationrdquo Nucleic Acids Research vol 38 no 1 pp D161ndashD166 2009

[20] F LThompson BHoste KVandemeulebroecke and J SwingsldquoGenomic diversity amongst Vibrio isolates from differentsources determined by fluorescent amplified fragment lengthpolymorphismrdquo Systematic and Applied Microbiology vol 24no 4 pp 520ndash538 2001

[21] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 Molecular evolutionary genetics analysis

using maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[22] P M Border J J Howard G S Plastow and K W SiggensldquoDetection of Listeria species and Listeria monocytogenes usingpolymerase chain reactionrdquo Letters in AppliedMicrobiology vol11 no 3 pp 158ndash162 1990

[23] F J Angulo ldquoListeriosis in theUnited Statesrdquo FSIS USDA 2009httpwwwfsisusdagovPDFLm Angulo 062309pdf

[24] F Allerberger and M Wagner ldquoListeriosis a resurgent food-borne infectionrdquo Clinical Microbiology and Infection vol 16 no1 pp 16ndash23 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


similar to L monocytogenes such as weak hemolysis [6 7 9]Furthermore certain low-hemolytic Lmonocytogenes strainsretain their virulence despite the presence of mutations inmajor virulence genes [10ndash12]The existence of these atypicalstrains indicates that traditional phenotypic and genotypiccharacterization methods must be used with care and thatfurther studies are required to improve the identification ofListeria isolates

This study used phenotypic and genotypic methods tocharacterize atypical Lmonocytogenes and L innocua isolatesobtained from swine slaughterhouses and meat markets inSao Paulo State Brazil

2 Material and Methods

21 Bacterial Strains and Culture Conditions Forty Listeriasp isolates were studied Of these 25 were isolated fromporkslaughterhouses andmarkets (15 isolates of L monocytogenesand 10 of L innocua) 11 isolates of L monocytogenes wereobtained from human infections and four were controlstrains (L monocytogenes ATCC 19115 and ATCC 19111 andL innocua ATCC 33090 and CLIP 12612) (Table 1) Theenvironmental and pork isolates were isolated as describedby Moreno et al [13] the clinical strains and Listeria controlswere obtained from the Public Health Laboratory (School ofPublic Health University of Sao Paulo) and Laboratory ofSwine Health (School of Veterinary Medicine and AnimalScience University of Sao Paulo) collections The environ-mental and pork isolates were obtained from different swabsamples taken from the slaughterhouses environment andcarcasses from Sao Paulo State the clinical isolates wereobtained from the blood placenta and cerebrospinal fluidsamples of different patients from different Brazilian states(Tables 1 and 2)

The isolates were maintained in a stock medium con-taining glycerol at minus80∘C The isolates were reactivated inbrain-heart infusion (BHI) medium (Difco Sparks MDUSA) and plated on tryptone soy agar supplemented withyeast (TSAYE) (Oxoid Lenexa USA) to isolate pure coloniesbefore use

22 Conventional Listeria Identification Tests The isolateswere serotyped using polyclonal antisera produced againstListeria somatic and flagellar antigens in rabbits accord-ing to the method described by Seeliger and Hohne[14] The isolates were also characterized by catalasemotility and biochemical tests including acid productionfrom D-xylose D-mannitol L-rhamnose and 120572-methyl-D-mannoside Cultivation in selective agar Listeria accordingto Ottaviani and Agosti (ALOA) (Biolife Milan Italy) wasused to identify L monocytogenes isolates and 120573-hemolysiswas assessed by sting inoculation on 5 sheep bloodagar

23 Detection of L monocytogenes-Specific Genes GenomicDNA extraction was performed as described by Ausubel etal [15] All isolates were screened for the inlA inlB inlCinlJ hly prfA plcA and plcB genes The primers described

by Johnson et al [6] Liu et al [16] and Jung et al [17]were used for detection of prfA inlC and inlJ and inlArespectively Specific primers were designed for the completeamplification of the virulence genes (Table 3)The PCRs wereperformed using an Eppendorf Mastercycler gradient thermalcycler Each reaction (25120583L) contained 5 120583L of genomicDNA MilliQ water 10X PCR buffer 15mM MgCl

2200120583M

of dNTPs (Fermentas Burlington Canada) 200120583M of eachprimer and 125U of Taq DNA polymerase (Promega) ThePCR programs were as follows 30 cycles of denaturation at94∘C for 1min annealing at primer-specific temperature for1ndash15min elongation at 72∘C for 1min per 1 Kb and finalextension at 72∘C for 10min The amplified products wereseparated by electrophoresis on 15 agarose gels and stainedwith ethidium bromide (1120583gmL) The molecular weightsof the products were determined using the 1 Kb Plus DNALadder (Fermentas Burlington Canada)

24 DNA Sequencing The amplified fragments were purifiedusing the Illustra GFX PCR DNA and Gel Band Purificationkit (GE Healthcare) according to the manufacturerrsquos protocoland sequenced directly at Genomic (Genomic EngenhariaMolecular Sao Paulo Brazil) DNA sequencing was per-formed on an Applied Biosystems 3130xl DNA analyzer usingthe BigDye Terminator v31 cycle sequencing kit

25 Detection of Mutations in L monocytogenes Viru-lence Genes Sequence analysis was performed using theBIOEDIT SequenceAlignment Editor 709 [18]Theobtainedsequences of the virulence genes were compared to pre-viously published L monocytogenes sequence accessionsfrom GenBank (NCBI Bethesda USA) The sequencingproducts were edited and compared with the sequencesavailable in the GenBank database by manual alignment andusing the ClustalW application The nucleotide sequencesobtainedwere translated into their corresponding amino acidsequences by the Nucleotide Translate application Subse-quently the amino acid sequences were analyzed to identifychanges in the compositions of their respective proteinswhich might modify or eliminate protein functions

26 Identification of Protein Domains and Prediction of Sec-ondary Structures The domains of InlC InlB InlA PI-PLCPC-PLC and Hly from reference strain L monocytogenesEGD-e were determined using the PROSITE database [19]of the ExPASy server (SIB Swiss Institute of Bioinformatics)The locations of these domains were compared to the muta-tions identified in the studied isolates

27 Species-Level Identification by 16S rRNAAmplification andPhylogenetic Analysis Species identity was confirmed using16S rRNA analysis The primers and amplification protocoldescribed by Thompson et al [20] were used to amplifycomplete 16S rRNA genes The fragments were sequencedand phylogenetic analysis was performed using theMega 510software [21] The dendrogram was constructed using themaximum-likelihood method with the Tamura-3-parametermodel


Table1Sourcesa

ndph

enotypicandgeno

typicc

haracteristicso

fthe

Liste

riamonocytogenesiso

latesu

sedin

thisstu

dy

Isolate

Species

Serotype

Orig

inSite

Year

ALO

AHem

olysis

inlA

inlB

inlC

inlJlowast

plcA

plcB

prfA

hly

Lm1

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm2

Lmonocytogenes

12b

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm3

Lmonocytogenes

4bMarket1

Floo

r2008

Halo

Positive

++

++

++

++

Lm21

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm22

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm23

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm25

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm26

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm27

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm28

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm29

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm30

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm31

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm34

Lmonocytogenes

12a

Hum

anBloo

d1989

Halo

Strong

positive

++

++

++

++

Lm35

Lmonocytogenes

4bHum

anBloo

d2004

Halo

Strong

positive

++

++

++

++

Lm36

Lmonocytogenes

4bHum

anBloo

d1977

Halo

Strong

positive

++

++

++

++

Lm37

Lmonocytogenes

4bHum

anCS

F1982

Halo

Strong

positive

++

++

++

++

Lm38

Lmonocytogenes

12b

Hum

anCS

F1983

Halo

Strong

positive

++

++

++

++

Lm39

Lmonocytogenes

12a

Hum

anPlacenta

1978

Halo

Strong

positive

++

++

++

++

Lm39a

Lmonocytogenes

12a

Hum

anPlacenta

1978

Halo

Positive

++

++

++

++

Lm40

Lmonocytogenes

12a

Hum

anBloo

d1985

Halo

Strong

positive

++

++

++

++

Lm41

Lmonocytogenes

4bHum

anCS

F1997

Halo

Strong

positive

++

++

++

++

Lm42

Lmonocytogenes

4bHum

anCS

F1997

Halo

Positive

++

++

++

++

Lm43

Lmonocytogenes

12a

Hum

anCS

F1983

Halo

Positive

++

++

++

++

Lm4

Lmonocytogenes

12a

Market2

Floo

r2008

Halo

Weakpo

sitive

++

++

++

++

Lm33

Lmonocytogenes

12a

Market2

Floo

r2008

Halo

Weakpo

sitive

++

++

++

++

Lm10

Lmonocytogenes

4bAT

CC19115

mdashmdash

Halo

Strong

positive

++

++

++

++

Lm15

Lmonocytogenes

12a

ATCC

19111

mdashmdash

Halo

Strong

positive

++

++

++

++

Slaught1slau

ghterhou

se1CS

Fcerebrospinalfl

uidlowast

Alliso

latesw

erep

ositive

forfragm

entsof

inlJbu

tpresented

varia

bler

esultsforh

oleg

enea

mplificatio

n(see

Table4

)


Table2Sourcesa

ndph

enotypicandgeno

typicc

haracteristicso

fthe

Liste

riainnocuaiso

latesu

sedin

thisstu

dy

Isolate

Species

Serotype

Orig

inSite

Year

ALO

AHem

olysis

inlA

xinlB

inlC

inlJ

plcA

plcB

prfA

hlyx

Lin5

Linnocua

6aMarket1

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin6

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin7

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin8

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin9

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin16

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin17

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin18

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin19

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin2

0Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin11

Linnocua

6aAT

CC33090

mdashmdash

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin4

6Linnocua

6aCL

IP12612

mdashmdash

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Slaught1slaug

hterho

use1Slaug

ht2sla

ughterho

use2


Very

weakpo

sitiveh

emolysis

x Atypicalisolatesw

erep

ositive

forfragm

entsof

inlABop

eron

andhlyb

utpresentedvaria

bler

esultsforinlA

andhlyc

ompletea

mplificatio

n(see

Table4

)




inlA


inlB


plcA1100


hly


prfA404



3 Results
































186220231 501510 833843844852




CTC

CTC

TC

CGTCT

CGTCT

CGTCT

CGTCT

CATCT



TAGGGGCT

TAGGGGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT


3839 870ATTG

CCA

CCA

CCC

CC

CC

CC

CC

CC


421 762 1053 1665 1893CTGGGGTTTGCG

TCAAAACCC

TCAAAACCC

AAAACCCTA

AAAACCCTA

AAAACCCATA

AAAACCC

AAAACCC

AAAACCC

AAAACCC

AAAACCC

08-557808-5923


1 282935 45 84 94 141 276306318 334345 390397415 426 478 629 702711716 735750760

1 1118203739 49 56 81 356 690 784795

1 15 787

1201 145 349324 394 399 1532 1656





1 10 12 32 74 102112 133 210 239 254 278282283IMVQKVLKSDTK

TIKVRNNIQ

TIKNMVRNNIQ

T

T

T

T

H

H

119NQLIYFT

IRVC

IRVC

VYA

VYA

VYA

VY

VY

VY

VY

VY


263 KIE

TK

TK

NT

T

T

T

T

T


1PSAVG

SPTI

SPTI

TI

TI

TID

TI

TI

TI

TI

TI

EGDeInlBInlB


1 236237238LN

-X

-X

-X

-X

-X




1 4 7 13 1719 262 318

1 5 13 290

6305111321174941



TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT


1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_


63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -


TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS


LDNATT


08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_


__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)


4 Discussion



















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table1Sourcesa

ndph

enotypicandgeno

typicc

haracteristicso

fthe

Liste

riamonocytogenesiso

latesu

sedin

thisstu

dy

Isolate

Species

Serotype

Orig

inSite

Year

ALO

AHem

olysis

inlA

inlB

inlC

inlJlowast

plcA

plcB

prfA

hly

Lm1

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm2

Lmonocytogenes

12b

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm3

Lmonocytogenes

4bMarket1

Floo

r2008

Halo

Positive

++

++

++

++

Lm21

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm22

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm23

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm25

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm26

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm27

Lmonocytogenes

12a

Slaught1

Floo

r2008

Halo

Positive

++

++

++

++

Lm28

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm29

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm30

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm31

Lmonocytogenes

12a

Market2

Pork

2008

Halo

Weakpo

sitive

++

++

++

++

Lm34

Lmonocytogenes

12a

Hum

anBloo

d1989

Halo

Strong

positive

++

++

++

++

Lm35

Lmonocytogenes

4bHum

anBloo

d2004

Halo

Strong

positive

++

++

++

++

Lm36

Lmonocytogenes

4bHum

anBloo

d1977

Halo

Strong

positive

++

++

++

++

Lm37

Lmonocytogenes

4bHum

anCS

F1982

Halo

Strong

positive

++

++

++

++

Lm38

Lmonocytogenes

12b

Hum

anCS

F1983

Halo

Strong

positive

++

++

++

++

Lm39

Lmonocytogenes

12a

Hum

anPlacenta

1978

Halo

Strong

positive

++

++

++

++

Lm39a

Lmonocytogenes

12a

Hum

anPlacenta

1978

Halo

Positive

++

++

++

++

Lm40

Lmonocytogenes

12a

Hum

anBloo

d1985

Halo

Strong

positive

++

++

++

++

Lm41

Lmonocytogenes

4bHum

anCS

F1997

Halo

Strong

positive

++

++

++

++

Lm42

Lmonocytogenes

4bHum

anCS

F1997

Halo

Positive

++

++

++

++

Lm43

Lmonocytogenes

12a

Hum

anCS

F1983

Halo

Positive

++

++

++

++

Lm4

Lmonocytogenes

12a

Market2

Floo

r2008

Halo

Weakpo

sitive

++

++

++

++

Lm33

Lmonocytogenes

12a

Market2

Floo

r2008

Halo

Weakpo

sitive

++

++

++

++

Lm10

Lmonocytogenes

4bAT

CC19115

mdashmdash

Halo

Strong

positive

++

++

++

++

Lm15

Lmonocytogenes

12a

ATCC

19111

mdashmdash

Halo

Strong

positive

++

++

++

++

Slaught1slau

ghterhou

se1CS

Fcerebrospinalfl

uidlowast

Alliso

latesw

erep

ositive

forfragm

entsof

inlJbu

tpresented

varia

bler

esultsforh

oleg

enea

mplificatio

n(see

Table4

)


Table2Sourcesa

ndph

enotypicandgeno

typicc

haracteristicso

fthe

Liste

riainnocuaiso

latesu

sedin

thisstu

dy

Isolate

Species

Serotype

Orig

inSite

Year

ALO

AHem

olysis

inlA

xinlB

inlC

inlJ

plcA

plcB

prfA

hlyx

Lin5

Linnocua

6aMarket1

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin6

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin7

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin8

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin9

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin16

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin17

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin18

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin19

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin2

0Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin11

Linnocua

6aAT

CC33090

mdashmdash

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin4

6Linnocua

6aCL

IP12612

mdashmdash

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Slaught1slaug

hterho

use1Slaug

ht2sla

ughterho

use2


Very

weakpo

sitiveh

emolysis

x Atypicalisolatesw

erep

ositive

forfragm

entsof

inlABop

eron

andhlyb

utpresentedvaria

bler

esultsforinlA

andhlyc

ompletea

mplificatio

n(see

Table4

)




inlA


inlB


plcA1100


hly


prfA404



3 Results
































186220231 501510 833843844852




CTC

CTC

TC

CGTCT

CGTCT

CGTCT

CGTCT

CATCT



TAGGGGCT

TAGGGGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT


3839 870ATTG

CCA

CCA

CCC

CC

CC

CC

CC

CC


421 762 1053 1665 1893CTGGGGTTTGCG

TCAAAACCC

TCAAAACCC

AAAACCCTA

AAAACCCTA

AAAACCCATA

AAAACCC

AAAACCC

AAAACCC

AAAACCC

AAAACCC

08-557808-5923


1 282935 45 84 94 141 276306318 334345 390397415 426 478 629 702711716 735750760

1 1118203739 49 56 81 356 690 784795

1 15 787

1201 145 349324 394 399 1532 1656





1 10 12 32 74 102112 133 210 239 254 278282283IMVQKVLKSDTK

TIKVRNNIQ

TIKNMVRNNIQ

T

T

T

T

H

H

119NQLIYFT

IRVC

IRVC

VYA

VYA

VYA

VY

VY

VY

VY

VY


263 KIE

TK

TK

NT

T

T

T

T

T


1PSAVG

SPTI

SPTI

TI

TI

TID

TI

TI

TI

TI

TI

EGDeInlBInlB


1 236237238LN

-X

-X

-X

-X

-X




1 4 7 13 1719 262 318

1 5 13 290

6305111321174941



TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT


1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_


63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -


TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS


LDNATT


08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_


__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)


4 Discussion



















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Sourcesa

ndph

enotypicandgeno

typicc

haracteristicso

fthe

Liste

riainnocuaiso

latesu

sedin

thisstu

dy

Isolate

Species

Serotype

Orig

inSite

Year

ALO

AHem

olysis

inlA

xinlB

inlC

inlJ

plcA

plcB

prfA

hlyx

Lin5

Linnocua

6aMarket1

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin6

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin7

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin8

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin9

Linnocua

6aSlaught2

Floo

r2008

Halolowast

Weakpo

sitivelowastlowast

minusminus

+minus

+minus

minus+

Lin16

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin17

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin18

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin19

Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin2

0Linnocua

6aSlaught1

Floo

r2006

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin11

Linnocua

6aAT

CC33090

mdashmdash

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Lin4

6Linnocua

6aCL

IP12612

mdashmdash

Negative

Negative

minusminus

minusminus

minusminus

minusminus

Slaught1slaug

hterho

use1Slaug

ht2sla

ughterho

use2


Very

weakpo

sitiveh

emolysis

x Atypicalisolatesw

erep

ositive

forfragm

entsof

inlABop

eron

andhlyb

utpresentedvaria

bler

esultsforinlA

andhlyc

ompletea

mplificatio

n(see

Table4

)




inlA


inlB


plcA1100


hly


prfA404



3 Results
































186220231 501510 833843844852




CTC

CTC

TC

CGTCT

CGTCT

CGTCT

CGTCT

CATCT



TAGGGGCT

TAGGGGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT


3839 870ATTG

CCA

CCA

CCC

CC

CC

CC

CC

CC


421 762 1053 1665 1893CTGGGGTTTGCG

TCAAAACCC

TCAAAACCC

AAAACCCTA

AAAACCCTA

AAAACCCATA

AAAACCC

AAAACCC

AAAACCC

AAAACCC

AAAACCC

08-557808-5923


1 282935 45 84 94 141 276306318 334345 390397415 426 478 629 702711716 735750760

1 1118203739 49 56 81 356 690 784795

1 15 787

1201 145 349324 394 399 1532 1656





1 10 12 32 74 102112 133 210 239 254 278282283IMVQKVLKSDTK

TIKVRNNIQ

TIKNMVRNNIQ

T

T

T

T

H

H

119NQLIYFT

IRVC

IRVC

VYA

VYA

VYA

VY

VY

VY

VY

VY


263 KIE

TK

TK

NT

T

T

T

T

T


1PSAVG

SPTI

SPTI

TI

TI

TID

TI

TI

TI

TI

TI

EGDeInlBInlB


1 236237238LN

-X

-X

-X

-X

-X




1 4 7 13 1719 262 318

1 5 13 290

6305111321174941



TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT


1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_


63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -


TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS


LDNATT


08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_


__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)


4 Discussion



















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




inlA


inlB


plcA1100


hly


prfA404



3 Results
































186220231 501510 833843844852




CTC

CTC

TC

CGTCT

CGTCT

CGTCT

CGTCT

CATCT



TAGGGGCT

TAGGGGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT


3839 870ATTG

CCA

CCA

CCC

CC

CC

CC

CC

CC


421 762 1053 1665 1893CTGGGGTTTGCG

TCAAAACCC

TCAAAACCC

AAAACCCTA

AAAACCCTA

AAAACCCATA

AAAACCC

AAAACCC

AAAACCC

AAAACCC

AAAACCC

08-557808-5923


1 282935 45 84 94 141 276306318 334345 390397415 426 478 629 702711716 735750760

1 1118203739 49 56 81 356 690 784795

1 15 787

1201 145 349324 394 399 1532 1656





1 10 12 32 74 102112 133 210 239 254 278282283IMVQKVLKSDTK

TIKVRNNIQ

TIKNMVRNNIQ

T

T

T

T

H

H

119NQLIYFT

IRVC

IRVC

VYA

VYA

VYA

VY

VY

VY

VY

VY


263 KIE

TK

TK

NT

T

T

T

T

T


1PSAVG

SPTI

SPTI

TI

TI

TID

TI

TI

TI

TI

TI

EGDeInlBInlB


1 236237238LN

-X

-X

-X

-X

-X




1 4 7 13 1719 262 318

1 5 13 290

6305111321174941



TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT


1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_


63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -


TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS


LDNATT


08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_


__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)


4 Discussion



















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























186220231 501510 833843844852




CTC

CTC

TC

CGTCT

CGTCT

CGTCT

CGTCT

CATCT



TAGGGGCT

TAGGGGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT


3839 870ATTG

CCA

CCA

CCC

CC

CC

CC

CC

CC


421 762 1053 1665 1893CTGGGGTTTGCG

TCAAAACCC

TCAAAACCC

AAAACCCTA

AAAACCCTA

AAAACCCATA

AAAACCC

AAAACCC

AAAACCC

AAAACCC

AAAACCC

08-557808-5923


1 282935 45 84 94 141 276306318 334345 390397415 426 478 629 702711716 735750760

1 1118203739 49 56 81 356 690 784795

1 15 787

1201 145 349324 394 399 1532 1656





1 10 12 32 74 102112 133 210 239 254 278282283IMVQKVLKSDTK

TIKVRNNIQ

TIKNMVRNNIQ

T

T

T

T

H

H

119NQLIYFT

IRVC

IRVC

VYA

VYA

VYA

VY

VY

VY

VY

VY


263 KIE

TK

TK

NT

T

T

T

T

T


1PSAVG

SPTI

SPTI

TI

TI

TID

TI

TI

TI

TI

TI

EGDeInlBInlB


1 236237238LN

-X

-X

-X

-X

-X




1 4 7 13 1719 262 318

1 5 13 290

6305111321174941



TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT


1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_


63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -


TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS


LDNATT


08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_


__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)


4 Discussion



















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


186220231 501510 833843844852




CTC

CTC

TC

CGTCT

CGTCT

CGTCT

CGTCT

CATCT



TAGGGGCT

TAGGGGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCTCAGT

GGATTTCACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT

GGATTTC ACT


3839 870ATTG

CCA

CCA

CCC

CC

CC

CC

CC

CC


421 762 1053 1665 1893CTGGGGTTTGCG

TCAAAACCC

TCAAAACCC

AAAACCCTA

AAAACCCTA

AAAACCCATA

AAAACCC

AAAACCC

AAAACCC

AAAACCC

AAAACCC

08-557808-5923


1 282935 45 84 94 141 276306318 334345 390397415 426 478 629 702711716 735750760

1 1118203739 49 56 81 356 690 784795

1 15 787

1201 145 349324 394 399 1532 1656





1 10 12 32 74 102112 133 210 239 254 278282283IMVQKVLKSDTK

TIKVRNNIQ

TIKNMVRNNIQ

T

T

T

T

H

H

119NQLIYFT

IRVC

IRVC

VYA

VYA

VYA

VY

VY

VY

VY

VY


263 KIE

TK

TK

NT

T

T

T

T

T


1PSAVG

SPTI

SPTI

TI

TI

TID

TI

TI

TI

TI

TI

EGDeInlBInlB


1 236237238LN

-X

-X

-X

-X

-X




1 4 7 13 1719 262 318

1 5 13 290

6305111321174941



TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT


1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_


63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -


TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS


LDNATT


08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_


__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)


4 Discussion



















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 10 12 32 74 102112 133 210 239 254 278282283IMVQKVLKSDTK

TIKVRNNIQ

TIKNMVRNNIQ

T

T

T

T

H

H

119NQLIYFT

IRVC

IRVC

VYA

VYA

VYA

VY

VY

VY

VY

VY


263 KIE

TK

TK

NT

T

T

T

T

T


1PSAVG

SPTI

SPTI

TI

TI

TID

TI

TI

TI

TI

TI

EGDeInlBInlB


1 236237238LN

-X

-X

-X

-X

-X




1 4 7 13 1719 262 318

1 5 13 290

6305111321174941



TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT


1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_


63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -


TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS


LDNATT


08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_


__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)


4 Discussion



















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


TATTACCCATGCAGTTCAA

CA

CA

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

CATTCCATGACCTGT

__ _ATTCCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT

__ _ATTCATGACCTGT


1 150151 1143 2403

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_

TCCGGCTG_


63 848 213237 280 336342352 424 465469 516 560 621 660 906946972985987 1035

GTGGTTGTTGCATAG -

GTGGTTGTTGCATAG -


TTGATAAG -

TTGATAAG -

1 78 254 588 110112601263127812991312138113831422144315011542156815691590297 877

(a)

DNIVK

IS

IS

IS

IS

IS

IS

_IS

_IS

_IS

_IS

_IS


LDNATT


08-557808-5923

LDNATT

RLDSEPVANSAYDPTTNIV

RLDSEPVANSAYDPTTNIV

AL_

AL_

AL_

AL_

AL_


__

__

__

__

__

1 523 530

1 3 51 94 118 142 416420426454474476500530558 594648664 801738781790381187157

26 85 293 438

(b)


4 Discussion



















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















100

85

88

88

89

99

3698

27

70

66

15

16

26

0005















Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Acknowledgments


References


































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

)JOEBXJ1VCMJTIJOH$PSQPSBUJPO … · 2019. 5. 13. · Laborat ´orio de Zoonoses Bacterianas, Fundac ¸ao Instituto Oswaldo Cruz, Avenida Brasil , Manguinhos, RiodeJaneiro,RJ,Brazil