Outbreak Strains Demonstrate Significantly Reduced Invasion ... · Texas Wesleyan University, Fort...
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2009, p. 5647–5658 Vol. 75, No. 17 0099-2240/09/$08.000 doi:10.1128/AEM.00367-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Some Listeria monocytogenes Outbreak Strains Demonstrate Significantly Reduced Invasion, inlA Transcript Levels, and Swarming Motility In Vitro A. J. Roberts, 1,2 S. K. Williams, 1 M. Wiedmann, 3 and K. K. Nightingale 1 * Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 80523 1 ; Department of Biology, Texas Wesleyan University, Fort Worth, Texas 76105 2 ; and Department of Food Science, Cornell University, Ithaca, New York 14853 3 Received 15 February 2009/Accepted 27 June 2009 Listeria monocytogenes can cause a severe invasive food-borne disease known as listeriosis, and large outbreaks of this disease occur occasionally. Based on molecular-subtype data, epidemic clone (EC) strains have been defined, including ECI and ECIa, which have caused listeriosis outbreaks on different continents. While a number of molecular-subtyping studies of outbreak strains have been reported, few comprehensive data sets of virulence-associated characteristics of these strains are available. We assembled a set of human clinical isolates from 15 outbreaks that occurred worldwide between 1975 and 2002. Initial characterization of these strains showed significant variation in the ability to invade human Caco-2 intestinal epithelial cells and HepG2 hepatic cells; four strains showed consistently reduced invasion in both cell lines. DNA sequencing of inlA, which encodes a protein required for efficient Caco-2 and HepG2 invasion, showed that none of the invasion-attenuated strains contained known virulence-attenuating mutations in inlA. Phylogenetic analyses of inlA sequences revealed a well-supported clade containing a fully invasive ECI strain and three invasion- attenuated ECI strains, along with a fully invasive ECIa strain and an invasion-attenuated ECIa strain. Of the four invasion-attenuated strains, one strain showed both reduced inlA transcript levels and impaired swarm- ing, one strain showed reduced inlA transcript levels, and two strains showed reduced swarming. Overall, our data show that (i) L. monocytogenes strains from outbreaks vary significantly in invasion efficiency and (ii) different mechanisms may contribute to reduced invasion efficiency. Association between EC strains and listeriosis outbreaks may involve characteristics other than virulence phenotypes, including survival and growth in food-associated environments. Listeria monocytogenes is a gram-positive facultative intra- cellular pathogen of humans and animals and is the etiologic agent of the disease listeriosis. Nearly all cases of human lis- teriosis (99%) have been estimated to be attributable to con- sumption of foods contaminated by L. monocytogenes, and ready-to-eat foods are typically implicated as the food vehicles associated with listeriosis outbreaks (33, 53). There are approx- imately 2,500 cases of listeriosis annually in the United States, about 90% of which result in hospitalization and 20 to 30% of which are fatal (37). Although listeriosis is a rare disease com- pared to other food-borne diseases, such as salmonellosis, the high mortality rate associated with listeriosis makes the disease one of the leading causes of death due to known food-borne pathogens each year in the United States (37). Listeriosis occurs as sporadic cases and small clusters of cases and in occasional large outbreaks, and evidence exists suggesting that L. monocytogenes strains differ in their abilities to cause human listeriosis. For example, more than 90% of human listeriosis cases are caused by L. monocytogenes strains belonging to only 3 of 13 serotypes, 1/2a, 1/2b, and 4b (23, 35), and most strains associated with listeriosis outbreaks belong to serotype 4b (18, 29, 57). Various molecular-subtyping ap- proaches, including ribotyping and pulsed field gel electro- phoresis, have been used to group L. monocytogenes isolates into at least three genetic lineages, termed lineages I, II, and III, which also correlate with serotype classifications (39, 50, 62). Of the three major L. monocytogenes genetic lineages, isolates belonging to lineage I are generally overrepresented among human clinical listeriosis cases despite evidence show- ing that humans are more frequently exposed to L. monocyto- genes strains belonging to lineage II through consumption of contaminated foods (24, 38, 60, 62). The majority of listeriosis outbreaks have been caused by strains belonging to a few genetically related groups, defined as epidemic clone I (ECI), ECIa, ECII, and ECIII (28); while some have designated ECIa ECIV (13), we use the designation ECIa here. Serotype 4b ECI and ECIa groups belong to EcoRI ribotypes DUP-1038B and DUP-1042B, respectively, and have been most frequently linked to listeriosis outbreaks worldwide. Specifically, ECI strains were responsible for the 1975–1976 outbreak in France, the 1981 outbreak in Nova Scotia, the 1983–1987 outbreak in Switzerland, the 1985 outbreak in Los Angeles, and the 1986–1987 outbreak in Philadelphia. ECIa strains were implicated in the 1979 outbreak in Boston, the 1983 outbreak in Massachusetts, the 1988–1989 outbreak in the United Kingdom, and the 2000–2001 outbreak in North Carolina (29). While both ECI and ECIa strains were associ- ated with the 1986–1987 outbreak in Philadelphia (58), only an * Corresponding author. Mailing address: Department of Animal Sciences, Colorado State University, 108B Animal Sciences Building, Fort Collins, CO 80523-1171. Phone: (970) 491-1556. Fax: (970) 491- 5326. E-mail: [email protected]. Published ahead of print on 6 July 2009. 5647 on June 1, 2020 by guest http://aem.asm.org/ Downloaded from
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2009, p. 5647–5658 Vol. 75, No. 170099-2240/09/$08.00�0 doi:10.1128/AEM.00367-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Some Listeria monocytogenes Outbreak Strains DemonstrateSignificantly Reduced Invasion, inlA Transcript Levels,
and Swarming Motility In Vitro�
A. J. Roberts,1,2 S. K. Williams,1 M. Wiedmann,3 and K. K. Nightingale1*Department of Animal Sciences, Colorado State University, Fort Collins, Colorado 805231; Department of Biology,
Texas Wesleyan University, Fort Worth, Texas 761052; and Department of Food Science, Cornell University,Ithaca, New York 148533
Received 15 February 2009/Accepted 27 June 2009
Listeria monocytogenes can cause a severe invasive food-borne disease known as listeriosis, and largeoutbreaks of this disease occur occasionally. Based on molecular-subtype data, epidemic clone (EC) strainshave been defined, including ECI and ECIa, which have caused listeriosis outbreaks on different continents.While a number of molecular-subtyping studies of outbreak strains have been reported, few comprehensivedata sets of virulence-associated characteristics of these strains are available. We assembled a set of humanclinical isolates from 15 outbreaks that occurred worldwide between 1975 and 2002. Initial characterization ofthese strains showed significant variation in the ability to invade human Caco-2 intestinal epithelial cells andHepG2 hepatic cells; four strains showed consistently reduced invasion in both cell lines. DNA sequencing ofinlA, which encodes a protein required for efficient Caco-2 and HepG2 invasion, showed that none of theinvasion-attenuated strains contained known virulence-attenuating mutations in inlA. Phylogenetic analyses ofinlA sequences revealed a well-supported clade containing a fully invasive ECI strain and three invasion-attenuated ECI strains, along with a fully invasive ECIa strain and an invasion-attenuated ECIa strain. Of thefour invasion-attenuated strains, one strain showed both reduced inlA transcript levels and impaired swarm-ing, one strain showed reduced inlA transcript levels, and two strains showed reduced swarming. Overall, ourdata show that (i) L. monocytogenes strains from outbreaks vary significantly in invasion efficiency and (ii)different mechanisms may contribute to reduced invasion efficiency. Association between EC strains andlisteriosis outbreaks may involve characteristics other than virulence phenotypes, including survival andgrowth in food-associated environments.
Listeria monocytogenes is a gram-positive facultative intra-cellular pathogen of humans and animals and is the etiologicagent of the disease listeriosis. Nearly all cases of human lis-teriosis (99%) have been estimated to be attributable to con-sumption of foods contaminated by L. monocytogenes, andready-to-eat foods are typically implicated as the food vehiclesassociated with listeriosis outbreaks (33, 53). There are approx-imately 2,500 cases of listeriosis annually in the United States,about 90% of which result in hospitalization and 20 to 30% ofwhich are fatal (37). Although listeriosis is a rare disease com-pared to other food-borne diseases, such as salmonellosis, thehigh mortality rate associated with listeriosis makes the diseaseone of the leading causes of death due to known food-bornepathogens each year in the United States (37).
Listeriosis occurs as sporadic cases and small clusters ofcases and in occasional large outbreaks, and evidence existssuggesting that L. monocytogenes strains differ in their abilitiesto cause human listeriosis. For example, more than 90% ofhuman listeriosis cases are caused by L. monocytogenes strainsbelonging to only 3 of 13 serotypes, 1/2a, 1/2b, and 4b (23, 35),and most strains associated with listeriosis outbreaks belong to
serotype 4b (18, 29, 57). Various molecular-subtyping ap-proaches, including ribotyping and pulsed field gel electro-phoresis, have been used to group L. monocytogenes isolatesinto at least three genetic lineages, termed lineages I, II, andIII, which also correlate with serotype classifications (39, 50,62). Of the three major L. monocytogenes genetic lineages,isolates belonging to lineage I are generally overrepresentedamong human clinical listeriosis cases despite evidence show-ing that humans are more frequently exposed to L. monocyto-genes strains belonging to lineage II through consumption ofcontaminated foods (24, 38, 60, 62).
The majority of listeriosis outbreaks have been caused bystrains belonging to a few genetically related groups, defined asepidemic clone I (ECI), ECIa, ECII, and ECIII (28); whilesome have designated ECIa ECIV (13), we use the designationECIa here. Serotype 4b ECI and ECIa groups belong to EcoRIribotypes DUP-1038B and DUP-1042B, respectively, and havebeen most frequently linked to listeriosis outbreaks worldwide.Specifically, ECI strains were responsible for the 1975–1976outbreak in France, the 1981 outbreak in Nova Scotia, the1983–1987 outbreak in Switzerland, the 1985 outbreak in LosAngeles, and the 1986–1987 outbreak in Philadelphia. ECIastrains were implicated in the 1979 outbreak in Boston, the1983 outbreak in Massachusetts, the 1988–1989 outbreak inthe United Kingdom, and the 2000–2001 outbreak in NorthCarolina (29). While both ECI and ECIa strains were associ-ated with the 1986–1987 outbreak in Philadelphia (58), only an
* Corresponding author. Mailing address: Department of AnimalSciences, Colorado State University, 108B Animal Sciences Building,Fort Collins, CO 80523-1171. Phone: (970) 491-1556. Fax: (970) 491-5326. E-mail: [email protected].
� Published ahead of print on 6 July 2009.
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ECI strain associated with this outbreak was characterized inthe study reported here. ECII includes serotype 4b strains thatare genetically distinct from ECI and ECIa strains and thathave been implicated in two multistate outbreaks in the UnitedStates; the 1998–1999 outbreak associated with hot dogs andthe 2002 outbreak in the Northeastern United States associ-ated with sliced deli meats (12). A serotype 1/2a strain thatcaused a sporadic listeriosis case associated with turkey franksin 1988 and persisted in the environment of a ready-to-eatmeat-processing plant for more than 12 years to cause a mul-tistate outbreak associated with deli meats in 2000 was desig-nated ECIII (29, 46). Epidemic clone strains associated withlisteriosis outbreaks are frequently included in L. monocyto-genes growth, molecular genetics, and pathogenesis studies un-der the a priori assumption that they represent strains withenhanced in vitro and in vivo virulence phenotypes (15, 40, 49).
Although epidemic clone strains, and serotype 4b strains inparticular, have been shown to be highly clonal (29), someevidence suggests that the diversity in virulence-associatedphenotypes among these strains may be greater than expected.For example, Werbrouck et al. observed considerable diversityin the abilities of serotype 4b strains to invade both Caco-2human intestinal epithelial cells and HepG2 human hepaticcells (61). Furthermore, a food isolate from the 1985 outbreakin California associated with Jalisco cheese (strain F2365), anECI strain, has been shown to be invasion attenuated in Caco-2cells, as well as to contain several atypical point mutationsleading to premature stop codons, including premature stopcodon mutations in the transcriptional regulator sigL and inthe virulence gene inlB (42). While extensive data on molecu-lar subtyping and genetic characterization of outbreak-associ-ated strains exists, little information is available about the invitro and in vivo virulence phenotypes of these strains. Thepurposes of this study thus were (i) to more comprehensivelyexamine and compare in vitro virulence-associated phenotypesfor L. monocytogenes strains from human clinical cases in-volved in listeriosis outbreaks and (ii) to probe the mechanisticand genetic bases of observed phenotypic differences.
MATERIALS AND METHODS
Bacterial strains. A set of 15 human clinical L. monocytogenes strains associ-ated with listeriosis outbreaks that have occurred worldwide was assembled toassess differences in in vitro virulence-associated phenotypes among outbreakstrains (Table 1). L. monocytogenes strains isolated from the food vehicle asso-ciated with outbreaks in which a human clinical strain demonstrated consistentattenuated invasiveness were also selected (Table 1). The standard laboratorycontrol strain 10403S (3) and a flaA null mutant in a 10403S background (45)were included as controls in assays probing virulence-associated phenotypes asappropriate.
Cell culture invasion assays. Invasion assays were performed in both Caco-2and HepG2 cell lines, as efficient invasion of both of these human cell linesrequires internalin A (InlA) (31). Internalin B also plays a role in the invasion ofHepG2 cells when L. monocytogenes is grown at 37°C with shaking (31). Invasionassays were performed essentially as previously described (31, 43), using inoculagrown under two different conditions (i.e., 30°C without shaking and 37°C withshaking). Duplicate wells of semiconfluent cell monolayers were inoculated withapproximately 2 � 107 L. monocytogenes cells/well, and the exact inoculumnumbers were determined by plating appropriate serial dilutions on brain heartinfusion (BHI) (Becton Dickson) agar in duplicate. Inoculated monolayers wereincubated at 37°C for 30 min to allow attachment, followed by three washes withphosphate-buffered saline to remove nonadherent bacteria and the addition offresh medium without antibiotics. Medium containing 150 �g/ml gentamicin wasadded 45 min postinoculation to kill extracellular bacteria. At 90 min postinocu-lation, the cell monolayers were washed three times with phosphate-buffered
saline and lysed by the addition of cold sterile deionized water and vigorouspipetting. Intracellular L. monocytogenes bacteria were enumerated by spreadplating appropriate dilutions of the lysed-cell suspensions on BHI agar in dupli-cate. The standard control strain L. monocytogenes 10403S and uninoculatedBHI broth were included as controls in each invasion assay. At least threeindependent assays for each strain were performed in each of the two cell linesfor both bacterial growth conditions. The average invasion efficiency for dupli-cate wells in each independent invasion assay was reported as the percentage ofinitial inoculum recovered by enumeration of intracellular bacteria for eachassay.
Statistical analyses of invasion assay data. Plots of the Studentized residualsagainst the predicted values (for each set of data, stratified by cell line andbacterial growth condition) were evaluated to identify a transformation of theraw percent invasion efficiency data that satisfied the assumption of normality.The square-root transformation of percent L. monocytogenes invasion data forthe Caco-2 and HepG2 cell lines (stratified for the bacterial growth conditionsdescribed above) satisfied the assumption of normality. Transformed invasionefficiencies observed for each strain were compared through one-way analysis ofvariance as implemented using the general linear model procedure in StatisticalAnalyses Software (SAS, Cary, NC). The invasion efficiency of each outbreakstrain was compared to that of the reference outbreak strain CSUFSL N1-054(selected to represent a fully virulent outbreak strain based on intragastric guineapig infection experiments [A. Van Stelten and K. Nightingale, unpublished data])through a series of pairwise comparisons using Dunnett’s correction for multiplecomparisons to a reference strain. The data are graphically presented as percentinvasion efficiency for ease of interpretation. Statistically significant comparisonswere identified by adjusted P values of �0.05.
Growth curves. To assess whether invasion-attenuated outbreak strains wereimpaired in general growth characteristics, growth curves were performed aspreviously described (42). Briefly, overnight cultures of L. monocytogenes weregrown in tubes containing 5 ml of BHI broth at 37°C with shaking for 12 h, and1% of each culture was then used to inoculate a second set of prewarmed tubescontaining 5 ml of BHI broth. The bacteria were then grown to a target opticaldensity at 600 nm of 0.4 and used for a second 1% adjusted inoculum transfer(based on actual reading of the optical density at 600 nm) to a side arm flaskcontaining 50 ml of prewarmed BHI. The inoculated side arm flask was incu-bated at 37°C with shaking for 12 h. Cell numbers were determined by standardplate counts at 0, 6, 8, 10, and 12 h in at least two independent experiments.
DNA sequencing. The inlA coding-domain sequences from all 15 outbreakstrains were PCR amplified using primers AVS-1 and AVS-2 and sequencedusing the PCR primers and the additional sequencing primers inlA F1, inlA S1R,inlA F2, inlA seq, and AVS-2 (Table 2). To obtain complete coverage of the 5�region of the inlA coding-domain sequence, we also PCR amplified and se-quenced the partial inlA promoter region using primers inlA proF and inlA proR(Table 2) (47). Since the invasion-attenuated food (strain F2365) and humanclinical isolate (CSUFSL N1-044) associated with the 1985 listeriosis outbreaklinked to Jalisco cheese carry a 5� nonsense mutation in inlB (42), we alsosequenced the 5� region of inlB containing this previously described mutation fortwo strains demonstrating reduced invasion (i.e., CSUFSL N1-009 and CSUFSLN1-081) using primers inlB PMSC F and inlB PMSC R (42). Partial 5� inlBsequence data for the remaining invasion-attenuated strain (CSUFSL N1-008)had previously been reported (42). Partial rpoB sequencing was performed, usingprimers rpoB F and rpoB R (Table 2), to confirm quantitative reverse transcrip-tase (qRT) PCR primer and probe binding sites for each strain. All PCR am-plifications were performed using 1� PCR buffer, 0.25 �M each primer, 50 �Mdeoxynucleotide triphosphates, 1.5 mM MgCl2, and 4 units of GoTaq ColorlessTaq polymerase (Promega, Madison, WI). All DNA sequencing was performedon an Applied Biosystems 3100 DNA analyzer using Big Dye Terminator chem-istry and AmpliTaq-FS DNA Polymerase (Applied Biosystems, Foster City, CA)at the Colorado State University Proteomics and Metabolomics Facility (FortCollins, CO). Nucleotide sequences were assembled and aligned with Seqmanand Megalign software (DNAStar; Lasergene, Madison, WI), respectively.
Phylogenetic inference. To identify where L. monocytogenes outbreak-associ-ated strains clustered in relationship to other outbreak strains and to non-outbreak-associated strains, an unrooted maximum-likelihood phylogenetic treewas inferred from 55 full inlA sequences, including (i) the 15 human clinical L.monocytogenes outbreak strains characterized in this study and (ii) 40 additionalisolates described in our previous study (47), which were selected to representgenetic diversity and different sources of isolation. MODELTEST was used tocompare the likelihood estimates of phylogenetic trees constructed under 56different models of DNA substitution (51). MODELTEST showed that theHKY85 DNA substitution model, along with parameters to estimate a gammadistribution and the proportion of invariate sites, best explained these sequence
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TA
BL
E1.
Listeriosis
outbreaksand
L.m
onocytogenesstrains
fromlisteriosis
outbreakscharacterized
inthis
study
Listeriosis
outbreaka
Deaths/cases
(%m
ortality) bF
oodvehicle
StrainID
PreviousID
(s) cSource
SerotypeR
ibotype(lineage) d
Epidem
icclone
eR
eference(s)
Anjou,F
rance1975–1976
NK
/162(N
K)
Unknow
nC
SUF
SLN
1-009F
SLR
2-575,G3992/L
4491bH
uman
clinical4b
DU
P-1038B(I)
I4
Boston,M
A1979
3/20(15)
Raw
vegetablesC
SUF
SLN
1-064F
SLJ1-220,C
7942H
uman
clinical4b
DU
P-1042B(I)
Ia25
Carlisle,E
ngland1981
5/11(45)
Dairy
productsC
SUF
SLN
1-006F
SLR
2-568,G3984/L
745H
uman
clinical1/2a
DU
P-1030A(II)
Not
anE
Cstrain
55
Nova
Scotia,Canada
198117/41
(42)C
oleslawC
SUF
SLN
1-008F
SLJ1-108,T
S27/L4738/D
D6304
Hum
anclinical
4bD
UP-1038B
(I)I
56N
ovaScotia,C
anada1981
17/41(42)
Coleslaw
CSU
FSL
N1-014
TS50/L
4760/1042-TS50
Food
4bD
UP-1038B
(I)I
56M
assachusetts1983
14/32(44)
Pasteurizedm
ilkC
SUF
SLN
1-035F
SLJ1-225,Scott
AH
uman
clinical4b
DU
P-1042B(I)
Ia21
Switzerland
1983–198731/122
(25)C
heeseC
SUF
SLN
1-072F
SLJ1-123,T
S55/L4486a/D
D6332
Hum
anclinical
4bD
UP-1038B
(I)I
2L
osA
ngeles,CA
198548/142
(34)Jalisco
softcheese
CSU
FSL
N1-044
FSL
J1-119,TS43/F
4565/DD
6320H
uman
clinical4b
DU
P-1038B(I)
I34
Philadelphia,PA1986–1987
16/36(44)
Icecream
,salami
CSU
FSL
N1-074
FSL
J1-012,DD
2360H
uman
clinical4b
DU
P-1038B(I)
I58
Oklahom
a1988
Sporadiccase
Turkey
franksC
SUF
SLN
1-031F
SLJ1-101,T
S14/F.6900/D
D6292
Hum
anclinical
1/2aD
UP-1053A
(II)III
5U
nitedK
ingdom1988–1989
94/355(26)
PateC
SUF
SLN
1-081F
SLJ1-116,T
S38/L3306/D
D6315
Hum
anclinical
4bD
UP-1042B
(I)Ia
36U
nitedK
ingdom1988–1989
94/355(26)
PateC
SUF
SLN
1-060T
S45/L.3350/1050-T
S45F
ood4b
DU
P-1042B(I)
Ia36
Illinois1994
0/45(0)
Chocolate
milk
CSU
FSL
N1-003
FSL
R2-503,G
6054H
uman
clinical1/2b
DU
P-1051B(I)
Not
anE
Cstrain
14
United
States1998–1999
21/101(21)
Hot
dogs,packedm
eatC
SUF
SLN
1-054F
SLN
1-225,H7550
Hum
anclinical
4bD
UP-1044A
(I)II
6,10,37
North
Carolina
2000–20015/12
(42)M
exicancheese
CSU
FSL
N1-022
FSL
R2-501,J0211
Hum
anclinical
4bD
UP-1042B
(I)Ia
8U
nitedStates
20007/29
(24)D
elicatessensliced
turkeyC
SUF
SLN
1-023F
SLR
2-499,J0161H
uman
clinical1/2a
DU
P-1053A(II)
III7
Northeastern
United
States2002
7/63(11)
Sliceddelim
eatC
SUF
SLN
1-079F
SLR
2-763,J1735H
uman
clinical4b
DU
P-1044A(I)
II9
aC
SUF
SLN
1-031is
anisolate
froma
singlelisteriosis
caseassociated
with
turkeyfranks
in1988.T
hisisolate
was
includedbecause
itbelongsto
thesam
em
olecularsubtype
thatcauseda
multistate
outbreakoflisteriosis
inthe
United
Statesin
2000linked
todelim
eatsproduced
atthe
same
processingfacility.
bF
oralloutbreaks
butthe1976
Anjou,F
rance,outbreak,thenum
berofcases,num
berofdeaths,and
referencesdescribing
theoriginaloutbreaks
were
obtainedfrom
the2003
Food-B
orneL
isteriosisR
iskA
ssessment,
Tables
II-4and
II-5(19).N
Kindicates
“notknow
n.”cA
dditionalinformation
abouteach
strainis
availableunder
theprevious
FSL
ID(e.g.,F
SLR
2-275)for
agiven
strainat
http://ww
w.pathogentracker.net.
dT
hegenetic
lineagew
asdesignated
basedon
EcoR
Iribotypes
asdetailed
previously(62).
eAs
describedpreviously
(12,29).
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data. A maximum-likelihood phylogenetic tree was generated in PAUP* (54)using the parameters determined through MODELTEST, heuristic searcheswere performed using equal weights for all sites, and the tree bisection-recon-nection branch-swapping algorithm was employed. Confidence measures forclustering of isolates within each clade were generated by performing 100 boot-strap replicates using the parameters defined by MODELTEST and the heuristicsearch algorithm described above.
Selection of bacterial isolates for characterization by qRT-PCR and swarmingassays based on phylogenetic clustering. Four outbreak-associated strains (i.e.,CSUFSL N1-008, CSUFSL N1-009, CSUFSL N1-044, and CSUFSL N1-081)showed significantly reduced invasion efficiencies in both the Caco-2 and HepG2cell lines. Furthermore, six L. monocytogenes strains that clustered to form awell-supported clade within the inlA phylogram, i.e., the four outbreak strainsthat demonstrated reduced invasion efficiencies, along with two outbreak strainsthat showed normal invasion efficiencies, were selected for further characteriza-tion by qRT-PCR to assess inlA transcript levels and swarming assays to assessmotility. These strains comprised (i) one fully invasive and three invasion-atten-uated ECI strains and (ii) one invasion-attenuated and one fully invasive ECIastrain.
qRT-PCR. qRT-PCR was performed to determine transcript levels for inlAand for the housekeeping gene rpoB. For total-RNA collection, overnight cul-tures of L. monocytogenes were passaged by two 1% inoculum transfers asdescribed for growth assays and grown in side arm flasks containing 50 ml of BHIbroth to stationary phase (defined as 10 h postinoculation of the side arm flask)at 37°C with shaking. RNA was isolated using RNA Protect and the RNAeasyMini kit (Qiagen) essentially as previously described (59), but using a MisonexXL-2000 sonicator (Misonex Inc., Farmingdale, NY) and the rigorous DNasetreatment described in the manufacturer’s instructions accompanying the TurboDNA-free Kit (Ambion). The RNA concentration and purity were determined bymeasuring absorbance on an ND-1000 spectrophotometer (NanoDrop Technol-ogies, Wilmington, DE). cDNA synthesis was performed using the High CapacitycDNA Reverse Transcription Kit without RNase inhibitor (Applied Biosystems,Foster City, CA) according to the manufacturer’s instructions. qRT-PCR wasperformed using the TaqMan Universal Master Mix and the Applied BiosystemsStepOne Plus sequence detection system with the TaqMan primers and probesfor rpoB previously described by Sue et al. (59) (Table 2). To detect inlA, wedesigned the primers inlA TqMnF and inlA TqMnR, along with an inlA probe, tomatch binding sites for all strains tested (Table 2). We compared inlA and rpoBalignments to confirm that primer and probe binding sites matched for all strainscharacterized by qRT-PCR. Transcript levels for inlA were expressed as logcDNA copy numbers, which were normalized by subtracting the log cDNA copynumbers observed for the housekeeping gene rpoB. Two independent RNAisolations were performed, and each strain was analyzed in duplicate in twoindependent qRT-PCR experiments. The genome sequence for the laboratory
control strain 10403S (i.e., inlA and rpoB sequences to confirm primer/probebinding sites for qRT-PCR) was obtained from the Broad Institute (http://www.broadinstitute.org/annotation/genome/listeria_group/MultiHome.html).
Statistical analysis of inlA transcript data. inlA transcript levels that werenormalized to rpoB as described above satisfied the assumption of normality. Tocompare differences in normalized inlA transcript levels among L. monocytogenesoutbreak strains, one-way analysis of variance, including a comparison of leastsquares means and Tukey’s Studentized residuals to correct for multiple com-parisons, was performed as implemented through the general linear model pro-cedure in SAS. Adjusted P values of �0.05 were considered statistically signifi-cant.
Swarming assays. The abilities of select L. monocytogenes outbreak strains toswarm were evaluated on semisoft agar essentially as previously described (52).The strains were grown overnight in BHI broth at 30°C without shaking, and aninoculating needle dipped in each culture was used to stab inoculate BHI semi-soft agar (0.4%) in duplicate. The plates were incubated at 30°C for 72 h, and thediameter of each resultant intra-agar colony was measured to assess swarmingability. The diameters of duplicate stabs were averaged and expressed as apercentage of the diameter observed for the laboratory control strain 10403S,which was set at 100%. The ability of each strain to swarm was measured in atleast three independent experiments, and L. monocytogenes strains 10403S and10403S �flaA (45) were included in each experiment as controls.
Statistical analysis of swarming assay data. Swarming assay data for outbreakstrains that were normalized to the laboratory control strain satisfied the as-sumption of normality. To compare differences in swarming among L. monocy-togenes outbreak strains, one-way analysis of variance, including a comparison ofleast squares means and Tukey’s Studentized residuals to correct for multiplecomparisons, was performed as implemented through the general linear modelprocedure in SAS. Comparisons with adjusted P values of �0.05 were deemedstatistically significant.
Nucleotide sequence accession numbers. The sequences for the inlA genes ofthe 15 outbreak strains were deposited in GenBank under accession numbersFJ617522 to FJ617536. The sequences of the 5� regions of inlB for strainsCSUFSL N1-009 and CSUFSL N1-081 were deposited in GenBank under ac-cession numbers FJ751916 and FJ751917.
RESULTS AND DISCUSSION
Based on epidemiological evidence suggesting that certainL. monocytogenes serotypes and molecular subtypes are morefrequently associated with human listeriosis (18, 29, 57), stud-ies of L. monocytogenes growth and survival, molecular genet-
TABLE 2. Primers and probes used for DNA sequencing and qRT-PCR characterization
Primer or probe Sequence (5�–3�)a Purpose Reference
inlA proF TTT TAA AAG GTG GAA TGA CA Sequencing of the inlA promoter region 47inlA proR GAA GCG TTG TAA CTT GGT CTA Sequencing of the inlA promoter region 47inlA F1 CAG GCA GCT ACA ATT ACA CA Sequencing of the inlA coding-domain region 47inlA S1R GGA CTG ATG TTA CTT ATT TGG T Sequencing of the inlA coding-domain region 47inlA F2 AAG ATA TAG GCA CAT TGG CGA GTT Sequencing of the inlA coding-domain region 47inlA S2R CGT ACT GAA ATY CCA KTT AGT TCC Sequencing of the inlA coding-domain region 47inlA seq GTG GAC GGC AAA GAA ACA AC Sequencing of the inlA coding-domain region 47inlB PMSC F GCA CGT GCT AGT AAA TAG AAG TAG TG Sequencing of the 5� portion of inlB 42inlB PMSC R TGT TTT TYA CTG TRT TCG GAA CAA Sequencing of the 5� portion of inlB 42rpoB F GGT TCC ATT GTT TTC GCA AC Sequencing rpoB region inclusive of the TaqMan
primer and probe binding sequencesThis study
rpoB R CCG AGT ATT TCG GTT CTC CA Sequencing rpoB region inclusive of the TaqManprimer and probe binding sequences
This study
rpoB TqMnF TGT AAA ATA TGG ACG GCA TCG T Primer for qRT-PCR of rpoB 59rpoB TqMnR GCT GTT TGA ATC TCA ATT AAG TTT GG Primer for qRT-PCR of rpoB 59rpoB probe Fam-CTG ATT CGC GCA AAA CTT CTA CGC G-Bhq Probe for qRT-PCR of rpoB 59inlA TqMnF GGT CTC RCA AAC AGA TCT AGA CCA AGT Primer for qRT-PCR of inlA This studyinlA TqMnR TCA AGT ATT CCA MTC CAT CGA TAG ATT Primer for qRT-PCR of inlA This studyinlA probe Fam-TAT CCC TAA TCT ATC CGC CTG AAG CGT-Bhq Probe for qRT-PCR of inlA This study
a Probes are flanked by the reporter and quencher dyes, respectively (FAM, 6-carboxyfluorescein; Bhq, black hole quencher 1). Characters other than A, T, G, andC represent degenerative nucleotides, which were included to accommodate polymorphisms in primer binding sites. Specifically, “K” indicates G/T, “R” indicates A/G,and “Y” indicates C/T.
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ics, and virulence characteristics often use a single strain or afew strains from listeriosis outbreaks under the a priori as-sumption that outbreak-associated strains have enhanced invitro and in vivo virulence phenotypes. We characterized a setof human clinical L. monocytogenes strains from 15 listeriosisoutbreaks that occurred worldwide using invasion assays inCaco-2 human intestinal epithelial cells and HepG2 humanhepatic cells, as well as by sequencing of the virulence geneinlA, which encodes a key virulence factor required for efficientinvasion of these two cell lines (30). Outbreak strains withreduced invasion efficiencies, as well as fully invasive outbreakstrains, clustering within a well-supported phylogenetic cladewere further characterized for inlA transcript levels and theability to swarm, a phenotype previously linked to increasedinvasion (1). Overall, our data show that (i) L. monocytogenesstrains from human clinical cases associated with listeriosisoutbreaks show significant variation in invasion efficiencies,with some strains demonstrating consistent significantly re-duced invasion efficiencies across two different cell lines, and(ii) different mechanisms may contribute to the reduced inva-sion efficiencies observed for these strains. Furthermore, thestrain (CSUFSL N1-081) associated with the 1988–1989 pateoutbreak in the United Kingdom, which showed reduced in-vasion efficiencies in both cell lines and reduced swarming, wasrecently shown to have reduced virulence in intragastric guineapig infection experiments (41). Specifically, unvaccinatedguinea pigs challenged with CSUFSL N1-081 (previous strainidentifier [ID], FSL J1-116) (Table 1) showed reduced L.monocytogenes counts in their livers compared to unvaccinatedanimals challenged with the laboratory control strain 10403S(41), further supporting the idea that some outbreak-associ-ated strains have attenuated virulence in vivo.
Outbreak-associated L. monocytogenes strains show signifi-cant variation in invasion efficiencies. L. monocytogenes out-break strains showed variation in invasion efficiencies in bothCaco-2 human intestinal epithelial and HepG2 human hepaticcells. InlA is required for efficient invasion of both cell lineswhen the bacteria are grown under either condition tested here(i.e., 30°C without shaking and 37°C with shaking), while InlBcontributes to invasion of HepG2 cells when the bacteria aregrown at 37°C with shaking (30). Invasion efficiencies in Caco-2cells for bacteria grown at 30°C without shaking varied signif-icantly across the human clinical outbreak-associated strainstested in this study (P � 0.0001; overall F test), where the meaninvasion efficiency ranged from 0.01% (for the human isolatefrom the 1988–1989 United Kingdom outbreak, CSUFSL N1-081) to 3.17% (for the human isolate from the 1981 UnitedKingdom outbreak, CSUFSL N1-006) (Fig. 1A). Four out-break-associated strains (CSUFSL N1-008, CSUFSL N1-009,CSUFSL N1-044, and CSUFSL N1-081) and the laboratorycontrol strain (10403S) showed significantly (P � 0.05) re-duced invasion efficiencies in Caco-2 cells when the bacteriawere grown at 30°C compared to the fully virulent (A. VanStelten and K. Nightingale, unpublished data) reference out-break strain CSUFSL N1-054 (Fig. 1A). L. monocytogenes in-vasion of HepG2 human hepatic cells has been shown to beless efficient than invasion of Caco-2 cells (30), but we alsoobserved significant differences (P � 0.0001; overall F test)among invasion efficiencies for outbreak strains when this cellline was used. For example, using bacteria grown at 30°C
without shaking, mean HepG2 invasion efficiencies rangedfrom 0.0009% (for the human isolate from the 1988–1989United Kingdom outbreak, CSUFSL N1-081) to 2.19% (forthe human isolate from the 1998 U.S. outbreak, CSUFSL N1-054) (Fig. 1B). Interestingly, the same four outbreak-associ-ated strains that showed significantly reduced invasion ofCaco-2 cells when the bacteria were grown at 30°C withoutshaking also showed the least ability to invade HepG2 cells(P � 0.05) when the bacteria were grown under the sameconditions. Two additional outbreak-associated strains (i.e.,CSUFSL N1-023 and CSUFSL N1-031) and the laboratorycontrol strain (10403S) also showed significantly (P � 0.05)reduced invasion efficiencies in HepG2 cells when the bacteriawere grown at 30°C without shaking.
L. monocytogenes invasion of Caco-2 and HepG2 cells hasbeen shown to be reduced when the bacteria are grown at 37°Cwith shaking compared to the invasion efficiencies for both celllines when the bacteria are grown at 30°C without shaking (30).Invasion efficiencies in Caco-2 cells when the bacteria weregrown at 37°C with shaking also varied significantly (P �0.0001; overall F test) among strains and ranged from 0.07%(for the human isolate from the 1981 Nova Scotia outbreak,CSUFSL N1-008) to 1.19% (for the human isolate from the2000 North Carolina outbreak, CSUFSL N1-022) (Fig. 2A).The same four outbreak-associated strains that showed signif-icantly reduced invasion of both Caco-2 and HepG2 cells whenthe bacteria were grown at 30°C without shaking (i.e., CSUFSLN1-008, CSUFSL N1-009, CSUFSL N1-044, and CSUFSL N1-081) also showed significantly (P � 0.05) reduced invasion inCaco-2 cells when the bacteria were grown at 37°C with shak-ing (Fig. 2A). When the bacteria were grown at 37°C withshaking, invasion efficiencies in HepG2 cells also varied signif-icantly (P � 0.0001; overall F test) among strains and rangedfrom 0.007% (for the human isolate from the 1976 outbreak inFrance, CSUFSL N1-009) to 0.51% (for the human isolatefrom the 1994 U.S. outbreak, CSUFSL N1-003) (Fig. 2B).While one outbreak-associated strain (CSUFSL N1-009)showed significantly reduced invasion of HepG2 cells whengrown at 37°C with shaking, four outbreak-associated strains(i.e., CSUFSL N1-003, CSUFSL N1-022, CSUFSL N1-031,and CSUFSL N1-035) showed significantly (P � 0.05) in-creased invasion of HepG2 cells under the same bacterialgrowth conditions compared to the reference outbreak strain,CSUFSL N1-054 (Fig. 2B). Overall, three ECI strains(CSUFSL N1-009, isolated from the 1975–1976 outbreak inAnjou, France; CSUFSL N1-008, from the 1981 outbreak inNova Scotia, Canada; and CSUFSL N1-044, from the 1985outbreak in Los Angeles) and one ECIa strain (CSUFSL N1-081, from the 1988–1989 outbreak in the United Kingdom)showed particularly low abilities to invade both human celltypes and under both bacterial growth conditions. These fourstrains showed significantly reduced invasion efficiencies inCaco-2 cells for both bacterial growth conditions evaluatedhere and in HepG2 cells when the bacteria were grown at 30°C;the four strains were thus classified as invasion attenuated andwere further probed for underlying genetic and mechanisticdifferences to explain this in vitro invasion-attenuated pheno-type. Virulence-associated phenotypes, such as invasion effi-ciency for human cell types, have previously been correlatedwith virulence in animal models (22, 32). In addition, Nightingale
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FIG. 1. Percent Caco-2 (A) and HepG-2 (B) cell invasion efficiencies for human clinical L. monocytogenes strains representing 15listeriosis outbreaks and the laboratory control strain 10403S grown at 30°C without shaking. The invasion efficiency (percentage of L.monocytogenes inoculum recovered from Caco-2 or HepG-2 cells) of each L. monocytogenes strain is indicated on the y axis. Each strain wasassayed in duplicate in each independent experiment, and at least three independent invasion experiments were performed to characterizeeach strain. The bars represent mean invasion efficiencies, and the error bars indicate the standard deviation observed for each strain.One-way analysis of variance showed that that L. monocytogenes outbreak-associated strains differed significantly with respect to invasionefficiency for Caco-2 (P � 0.0001; overall F test) and HepG-2 (P � 0.0001; overall F test) cells when the bacteria were grown at 30°C withoutshaking. Statistically significant (adjusted P values of �0.05) pairwise comparisons of square-root-transformed percent invasion efficiencydata for each cell line where the bacteria were grown at 30°C without shaking, using Dunnett’s multiple-comparison correction (withreference to outbreak strain CSUFSL N1-054), is denoted by an asterisk above each strain that significantly differed from the reference strainfor the Caco-2 and HepG2 cell lines.
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FIG. 2. Percent Caco-2 (A) and HepG-2 (B) cell invasion efficiencies for human clinical L. monocytogenes strains representing 15 listeriosisoutbreaks and the laboratory control strain 10403S grown at 37°C with shaking. The invasion efficiency (percentage of L. monocytogenes inoculumthat invaded Caco-2 or HepG-2 cells) of each L. monocytogenes strain is indicated on the y axis. Each strain was assayed in duplicate in eachindependent experiment, and at least three independent invasion experiments were performed to characterize each strain. The bars representmean invasion efficiencies, and the error bars indicate the standard deviation observed for each strain. One-way analysis of variance showed thatL. monocytogenes outbreak-associated strains differed significantly with respect to invasion efficiency for Caco-2 (P � 0.0001; overall F test) andHepG-2 (P � 0.0001; overall F test) cells when the bacteria were grown at 37°C with shaking. Statistically significant (adjusted P values of �0.05)pairwise comparisons of square-root-transformed percent invasion efficiency data for each cell line where the bacteria were grown at 30°C withoutshaking, using Dunnett’s multiple-comparison correction (with reference to outbreak strain CSUFSL N1-054), is denoted by an asterisk above eachstrain that significantly differed from the reference strain for the Caco-2 and HepG2 cell lines.
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et al. recently reported that the human clinical strain associatedwith the 1988–1989 outbreak in the United Kingdom, a strainthat showed reduced invasion efficiency in the current study,showed reduced bacterial numbers in the livers of unvacci-nated guinea pigs that were orally challenged with this straincompared to counts recovered from unvaccinated guinea pigschallenged with the fully virulent laboratory control strain10403S (41).
Although it has been previously observed that L. monocyto-genes isolates belonging to different serotypes, including sero-type 4b isolates, exhibit heterogeneous abilities to invade hu-man cells (11, 61), to our knowledge, this is the first reportshowing that significant variability in invasion capabilities alsoexists within epidemic clone strains associated with listeriosisoutbreaks. While it could be argued that the consistently lowinvasion efficiency observed for the four human clinical isolatesfrom listeriosis outbreaks (i.e., CSUFSL N1-008, CSUFSL N1-009, CSUFSL N1-044, and CSUFSL N1-081) is an artifact oflaboratory passage, there is no apparent correlation betweenthe invasiveness of a strain and the time since the outbreak(Table 1). For example, both the invasion-attenuated strainCSUFSL N1-008 and the fully invasive strain CSUFSL N1-006were isolated from outbreaks beginning in 1981. Furthermore,when we compared the invasion efficiency of an invasion-at-tenuated human clinical isolate to that of a food isolate fromthe same outbreak, when a food isolate was available (i.e.,strains CSUFSL N1-014, a food isolate from the coleslaw as-sociated with the 1981 outbreak in Nova Scotia, and CSUFSLN1-060, a food isolate from the pate associated with the 1988–1989 outbreak in the United Kingdom), we observed that foodisolates showed similarly low invasion efficiencies compared tohuman clinical strains from the same outbreak. Specifically, thefood isolate CSUFSL N1-014 (1981 Nova Scotia outbreak) hadan average invasion efficiency of 0.02% compared to an aver-age invasion efficiency of 0.55% for the human clinical isolatefrom the same outbreak (CSUFSL N1-008) in Caco-2 cellsusing bacteria grown at 30°C without shaking, while the foodisolate CSUFSL N1-060 (1988–1989 United Kingdom out-break) had an average invasion efficiency of 0.06% comparedto an average invasion efficiency of 0.01% for a human clinicalisolate from the same outbreak (CSUFSL N1-081) in Caco-2cells using bacteria grown at 30°C. We also previously showedthat the food isolate (strain F2365) from the 1985 outbreak inLos Angeles associated with Jalisco cheese demonstrated at-tenuated invasiveness similar to the invasiveness of the humanclinical isolate from the same outbreak (CSUFSL N1-044)characterized here (42). These data suggest that low invasionefficiency is a natural phenotype of strains associated withcertain outbreaks and that strain-specific phenotypic charac-teristics other than enhanced virulence characteristics, such asenhanced survival and growth in foods or food-associated en-vironments, may contribute to the association of some epi-demic clone strains with listeriosis outbreaks. This hypothesisis supported by ongoing work by our group that shows that asubset of the fully invasive ECI and ECIa strains characterizedin this study vary significantly with respect to growth rates onturkey breast formulated without antimicrobials (J. Corronand K. Nightingale, unpublished data). Until future animalstudies of additional outbreak strains are performed, we can-not exclude the possibility that, for some strains, other viru-
lence mechanisms (e.g., intracellular growth, intracellularspread, and the ability to evade host immune responses) mayalso contribute to the association of strains with attenuatedinvasion with human outbreaks and may possibly compensatefor reduced invasion efficiency. Furthermore, it is important tonote that factors unrelated to the pathogen, such as immuno-compromised host status, may still enable strains showing re-duced invasion to cause disease.
Different mechanisms contribute to the attenuated invasionefficiencies of some human outbreak strains. Four outbreakstrains demonstrated attenuated invasion efficiencies in bothCaco-2 and HepG2 cell lines, suggesting that mutations in inlAor differences in inlA transcript levels may at least partiallyexplain the attenuated invasion phenotype observed for thesestrains, since InlA is required for efficient invasion of both celllines (30). Diminished ability to invade human cells has beencommonly attributed to mutations leading to premature stopcodons in inlA, which result in the production of a truncatedand secreted form of InlA (20, 27, 41, 43, 44). To probe thecause of the variation in invasion efficiencies among the set ofL. monocytogenes outbreak-associated strains characterizedhere, inlA genes from all 15 strains were sequenced. None ofthe four invasion-attenuated strains contained a prematurestop codon mutation in inlA. In addition, all four invasion-attenuated strains (CSUFSL N1-008, CSUFSL N1-009,CSUFSL N1-044, and CSUFSL N1-081) clustered together,along with two fully invasive strains, CSUFSL N1-035 andCSUFSL N1-072, to form a clade with bootstrap support of100% in the inlA phylogram (Fig. 3). Five isolates in this clade,including the four invasion-attenuated strains and the fullyinvasive strain CSUFSL N1-072, showed 100% InlA aminoacid identity, while the other fully invasive strain (CSUFSLN1-035) differed by a single amino acid at codon 800. The fourinvasion-attenuated strains also clustered with fully invasivestrains in phylogenies created using concatenated sequencesthat included the L. monocytogenes housekeeping genes gap,prs, purM, and ribC (H. den Bakker and M. Wiedmann, un-published data), suggesting that these invasion-attenuatedstrains are genetically related to fully invasive strains at thecore level.
To screen for general defects in the cellular growth rate thatmight explain an attenuated invasion phenotype, we performedgrowth curves in BHI broth using the four invasion-attenuatedstrains and the laboratory control strain 10403S. The growthrates observed for the invasion-attenuated strains were similarto that of 10403S, and all of the strains reached similar result-ant population densities at 12 h postinoculation (data notshown), indicating that invasion-attenuated strains are not im-paired in general growth characteristics. Along with InlA, InlBis a well-known virulence factor that plays a role in the invasionof multiple human cell types (17, 26, 48). While one would notexpect the attenuated invasion efficiencies of the four strainsidentified here to be solely attributable to mutations in inlB (asCaco-2 cell invasion, which was attenuated in these strains, isInlB independent), a rare mutation leading to a prematurestop codon in the 5� region of inlB was previously identified ina food isolate (strain F2365) and a human clinical isolate(CSUFSL N1-044) from the 1985 outbreak in California asso-ciated with Jalisco cheese (42), which is one of the humanclinical outbreak-associated strains that showed reduced
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Caco-2 and HepG2 invasion efficiencies in this study. We thusanalyzed the inlB sequences of this region in the other threeoutbreak strains demonstrating attenuated invasion in thisstudy. The three invasion-attenuated outbreak strains newlyidentified in this study (CSUFSL N1-008, CSUFSL N1-009,and CSUFSL N1-081) did not carry this previously describedpremature stop codon mutation in inlB.
Since we did not identify any premature stop codon muta-tions in inlA, we hypothesized that reduced inlA transcriptionmight contribute to the attenuated invasion phenotype ob-served for some outbreak strains. qRT-PCR analysis of theinlA transcript levels of the four invasion-attenuated strainsand the two fully invasive outbreak-associated strains, whichformed a highly clonal clade in the inlA phylogram, showedmean normalized inlA transcript levels ranging from �2.49 forstrain CSUFSL N1-009 to �0.33 for strain CSUFSL N1-072(Fig. 4). Two invasion-attenuated ECI strains (CSUFSL N1-008 and CSUFSL N1-009) demonstrated significantly lower
inlA transcript levels than the fully invasive ECI strainCSUFSL N1-072 (P � 0.007 and P � 0.001, respectively) (Fig.4). The invasion-attenuated ECIa strain demonstrated inlAtranscript levels similar to those observed for the fully invasiveECIa strain (Fig. 4). These results confirm and extend thefindings of others who have also observed variation in inlAtranscript levels among and within L. monocytogenes serotypes(11, 61).
Swarming motility in L. monocytogenes is a phenotype thathas been shown to contribute to the ability to adhere to andinvade Caco-2 intestinal epithelial cells, as well as the ability tocause disease in a mouse infection model (1, 16, 45). Wetherefore compared the swarming abilities of the four invasion-attenuated strains and the closely related fully invasive strainsCSUFSL N1-035 and CSUFSL N1-072. We observed swarm-ing efficiencies ranging from 45.4% for strain CSUFSL N1-081(a human clinical strain from the 1988–1989 United Kingdomoutbreak) to 110.6% for strain CSUFSL N1-008 (a human
FIG. 3. Unrooted maximum-likelihood phylogram inferred from inlA coding-domain sequences for 15 L. monocytogenes outbreak strains, alongwith 40 additional L. monocytogenes isolates selected to represent different sources of isolation, including sporadic human cases, animal cases,foods, and the natural environment (47). The taxon labels at the branch tips include the isolate identification, followed by the serotype and EcoRIribotype in parentheses. Branching support was determined by 100 bootstrap replicates, and bootstrap values (if 70%) are shown at the nodelabels. L. monocytogenes outbreak strains are labeled in red, and strains that demonstrated reduced invasion efficiency in Caco-2 and HepG-2 cellsare flanked by asterisks. The dashed rounded rectangle indicates a phylogenetic clade composed of L. monocytogenes ECI and ECIa outbreakstrains demonstrating reduced or normal invasion efficiencies for Caco-2 and HepG-2 cells.
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clinical strain from the 1981 Nova Scotia outbreak) normalizedto the swarming efficiency for control strain 10403S, which wasset at 100% (Fig. 5). Three of the four invasion-attenuatedstrains, CSUFSL N1-009, CSUFSL N1-044, and CSUFSL N1-081, showed reduced swarming at 66.0%, 72.4%, and 45.4%,respectively, of that of 10403S (Fig. 5); one of these strains
(CSUFSL N1-009) also demonstrated reduced inlA transcriptlevels. Strain CSUFSL N1-081 was particularly attenuated inswarming ability compared to the fully invasive strainsCSUFSL N1-035 (P � 0.002) and CSUFSL N1-072 (P �0.002), suggesting that the attenuated invasion efficiencies inthese strains might be at least partially attributable to defects
FIG. 4. Normalized inlA transcript levels for six L. monocytogenes strains clustering in a single inlA phylogenetic clade that includes strainsdemonstrating either a reduced or normal invasion phenotype in Caco-2 and HepG-2 cells and for the standard laboratory control strain 10403S.The transcript levels were determined by qRT-PCR and are expressed as log inlA cDNA copy number normalized to the log cDNA copy numberof the housekeeping gene rpoB (log10 copy number inlA � log10 copy number rpoB). Each strain was assayed in duplicate in each independentexperiment, and at least two independent RNA isolations, cDNA syntheses, and qRT-PCR experiments were conducted to characterize eachstrain. The bars represent mean transcript levels for each strain, and the error bars represent the standard deviations around the mean transcriptlevels. Different letters in the bars indicate statistical significance (P � 0.05) based on one-way analysis of variance and comparison of strain meannormalized transcript levels with Tukey’s correction for multiple comparisons.
FIG. 5. Swarming abilities of six L. monocytogenes strains clustering within the same inlA phylogenetic clade, which includes strains demonstratingeither a reduced or normal invasion phenotype in Caco-2 and HepG-2 cells, normalized to that of the standard laboratory control strain 10403S.Swarming ability was determined by inoculating semisoft agar with each strain, followed by measuring the resultant intra-agar swarming diameters. Eachstrain was assayed in at least three independent swarming assays. The bars represent the mean normalized swarming ability for each strain, and the errorbars represent the standard deviations around the mean transcript levels. Different letters in the bars indicate statistical significance (P � 0.05) based onone-way analysis of variance and comparison of strain mean normalized transcript levels with Tukey’s correction for multiple comparisons.
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in flagellar motility. Future studies involving sequence andexpression analyses of flagellin structural and regulatory genesin invasion-attenuated and genetically similar fully invasiveoutbreak strains are needed to provide additional insight intothe mechanisms of reduced flagellar motility and to determineto what extent reduced flagellar motility contributes to theinvasion-attenuated phenotype observed in this study.
Conclusions. It has been well documented that considerablediversity in virulence-associated phenotypes exists among L.monocytogenes strains representing different serotypes and mo-lecular subtypes (18, 24, 29, 35, 57, 62). Our data demonstratethat this diversity in virulence-associated phenotypes (i.e., in-vasion, inlA transcript levels, and swarming motility) also ex-tends to strains belonging to highly clonal groups frequentlylinked to listeriosis outbreaks, such as ECI and ECIa. Specif-ically, L. monocytogenes epidemic clone strains examined in thecurrent study that demonstrated reduced in vitro invasion inboth Caco-2 and HepG2 cell lines also showed reduced inlAtranscript levels, reduced swarming, or both reduced inlA tran-script levels and reduced swarming. These findings suggest thatinvasion efficiency may depend, at least in part, on inlA tran-script levels and swarming ability; however, future experimentsare required to more precisely elucidate the association be-tween inlA transcript levels, swarming motility, and invasionefficiency. Although strains that demonstrated attenuated invitro virulence phenotypes were not characterized by notablyreduced morbidity rates for their respective listeriosis out-breaks, these findings suggest that the association of epidemicclone strains with a majority of the human listeriosis outbreaksthat have occurred worldwide may include factors other thanenhanced virulence, such as survival and growth characteristicsin food-associated environments and host immune status. Im-portantly, the outcomes of this study also support the idea thatoutbreak-associated L. monocytogenes epidemic clone strainscannot, a priori, be considered to demonstrate highly virulentin vitro phenotypes for molecular genetics and pathogenesisstudies.
ACKNOWLEDGMENTS
This project was supported by USDA Special Research Grants 2005-34459-15625 and 2006-34459-16952 (both to M.W.), USDA-CSREESHatch Funds (NYC-143451; to M.W.), and the Colorado State Uni-versity Agriculture Experiment Station.
We thank Jessica Corron for assistance with growth curve experi-ments and Julie Simpson and Anna Van Stelten for assistance withDNA sequencing.
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