Some Listeria monocytogenes Outbreak Strains Demonstrate ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2009, p. 5647–5658 0099-2240/09/$08.00⫹0 doi:10.1128/AEM.00367-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 17

Some Listeria monocytogenes Outbreak Strains Demonstrate Significantly Reduced Invasion, inlA Transcript Levels, and Swarming Motility In Vitro䌤 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 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 invasionattenuated 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 swarming, 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. serotype 4b (18, 29, 57). Various molecular-subtyping approaches, including ribotyping and pulsed field gel electrophoresis, 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 showing that humans are more frequently exposed to L. monocytogenes 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 associated with the 1986–1987 outbreak in Philadelphia (58), only an

Listeria monocytogenes is a gram-positive facultative intracellular pathogen of humans and animals and is the etiologic agent of the disease listeriosis. Nearly all cases of human listeriosis (99%) have been estimated to be attributable to consumption 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 approximately 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 compared 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

* 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) 4915326. E-mail: [email protected]. 䌤 Published ahead of print on 6 July 2009. 5647

5648

ROBERTS ET AL.

ECI strain associated with this outbreak was characterized in the study reported here. ECII includes serotype 4b strains that are genetically distinct from ECI and ECIa strains and that have been implicated in two multistate outbreaks in the United States; the 1998–1999 outbreak associated with hot dogs and the 2002 outbreak in the Northeastern United States associated with sliced deli meats (12). A serotype 1/2a strain that caused a sporadic listeriosis case associated with turkey franks in 1988 and persisted in the environment of a ready-to-eat meat-processing plant for more than 12 years to cause a multistate outbreak associated with deli meats in 2000 was designated ECIII (29, 46). Epidemic clone strains associated with listeriosis outbreaks are frequently included in L. monocytogenes growth, molecular genetics, and pathogenesis studies under the a priori assumption that they represent strains with enhanced in vitro and in vivo virulence phenotypes (15, 40, 49). Although epidemic clone strains, and serotype 4b strains in particular, have been shown to be highly clonal (29), some evidence suggests that the diversity in virulence-associated phenotypes among these strains may be greater than expected. For example, Werbrouck et al. observed considerable diversity in the abilities of serotype 4b strains to invade both Caco-2 human intestinal epithelial cells and HepG2 human hepatic cells (61). Furthermore, a food isolate from the 1985 outbreak in California associated with Jalisco cheese (strain F2365), an ECI strain, has been shown to be invasion attenuated in Caco-2 cells, as well as to contain several atypical point mutations leading to premature stop codons, including premature stop codon mutations in the transcriptional regulator sigL and in the virulence gene inlB (42). While extensive data on molecular subtyping and genetic characterization of outbreak-associated strains exists, little information is available about the in vitro and in vivo virulence phenotypes of these strains. The purposes of this study thus were (i) to more comprehensively examine and compare in vitro virulence-associated phenotypes for L. monocytogenes strains from human clinical cases involved in listeriosis outbreaks and (ii) to probe the mechanistic and genetic bases of observed phenotypic differences. MATERIALS AND METHODS Bacterial strains. A set of 15 human clinical L. monocytogenes strains associated with listeriosis outbreaks that have occurred worldwide was assembled to assess differences in in vitro virulence-associated phenotypes among outbreak strains (Table 1). L. monocytogenes strains isolated from the food vehicle associated with outbreaks in which a human clinical strain demonstrated consistent attenuated invasiveness were also selected (Table 1). The standard laboratory control strain 10403S (3) and a flaA null mutant in a 10403S background (45) were included as controls in assays probing virulence-associated phenotypes as appropriate. Cell culture invasion assays. Invasion assays were performed in both Caco-2 and HepG2 cell lines, as efficient invasion of both of these human cell lines requires internalin A (InlA) (31). Internalin B also plays a role in the invasion of HepG2 cells when L. monocytogenes is grown at 37°C with shaking (31). Invasion assays were performed essentially as previously described (31, 43), using inocula grown under two different conditions (i.e., 30°C without shaking and 37°C with shaking). Duplicate wells of semiconfluent cell monolayers were inoculated with approximately 2 ⫻ 107 L. monocytogenes cells/well, and the exact inoculum numbers were determined by plating appropriate serial dilutions on brain heart infusion (BHI) (Becton Dickson) agar in duplicate. Inoculated monolayers were incubated at 37°C for 30 min to allow attachment, followed by three washes with phosphate-buffered saline to remove nonadherent bacteria and the addition of fresh medium without antibiotics. Medium containing 150 ␮g/ml gentamicin was added 45 min postinoculation to kill extracellular bacteria. At 90 min postinoculation, the cell monolayers were washed three times with phosphate-buffered

APPL. ENVIRON. MICROBIOL. saline and lysed by the addition of cold sterile deionized water and vigorous pipetting. Intracellular L. monocytogenes bacteria were enumerated by spread plating appropriate dilutions of the lysed-cell suspensions on BHI agar in duplicate. The standard control strain L. monocytogenes 10403S and uninoculated BHI broth were included as controls in each invasion assay. At least three independent assays for each strain were performed in each of the two cell lines for both bacterial growth conditions. The average invasion efficiency for duplicate wells in each independent invasion assay was reported as the percentage of initial inoculum recovered by enumeration of intracellular bacteria for each assay. Statistical analyses of invasion assay data. Plots of the Studentized residuals against the predicted values (for each set of data, stratified by cell line and bacterial growth condition) were evaluated to identify a transformation of the raw percent invasion efficiency data that satisfied the assumption of normality. The square-root transformation of percent L. monocytogenes invasion data for the Caco-2 and HepG2 cell lines (stratified for the bacterial growth conditions described above) satisfied the assumption of normality. Transformed invasion efficiencies observed for each strain were compared through one-way analysis of variance as implemented using the general linear model procedure in Statistical Analyses Software (SAS, Cary, NC). The invasion efficiency of each outbreak strain was compared to that of the reference outbreak strain CSUFSL N1-054 (selected to represent a fully virulent outbreak strain based on intragastric guinea pig infection experiments [A. Van Stelten and K. Nightingale, unpublished data]) through a series of pairwise comparisons using Dunnett’s correction for multiple comparisons to a reference strain. The data are graphically presented as percent invasion efficiency for ease of interpretation. Statistically significant comparisons were identified by adjusted P values of ⬍0.05. Growth curves. To assess whether invasion-attenuated outbreak strains were impaired in general growth characteristics, growth curves were performed as previously described (42). Briefly, overnight cultures of L. monocytogenes were grown in tubes containing 5 ml of BHI broth at 37°C with shaking for 12 h, and 1% of each culture was then used to inoculate a second set of prewarmed tubes containing 5 ml of BHI broth. The bacteria were then grown to a target optical density 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 flask containing 50 ml of prewarmed BHI. The inoculated side arm flask was incubated at 37°C with shaking for 12 h. Cell numbers were determined by standard plate 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 outbreak strains were PCR amplified using primers AVS-1 and AVS-2 and sequenced using 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 sequenced the partial inlA promoter region using primers inlA proF and inlA proR (Table 2) (47). Since the invasion-attenuated food (strain F2365) and human clinical isolate (CSUFSL N1-044) associated with the 1985 listeriosis outbreak linked to Jalisco cheese carry a 5⬘ nonsense mutation in inlB (42), we also sequenced the 5⬘ region of inlB containing this previously described mutation for two strains demonstrating reduced invasion (i.e., CSUFSL N1-009 and CSUFSL N1-081) using primers inlB PMSC F and inlB PMSC R (42). Partial 5⬘ inlB sequence data for the remaining invasion-attenuated strain (CSUFSL N1-008) had previously been reported (42). Partial rpoB sequencing was performed, using primers rpoB F and rpoB R (Table 2), to confirm quantitative reverse transcriptase (qRT) PCR primer and probe binding sites for each strain. All PCR amplifications were performed using 1⫻ PCR buffer, 0.25 ␮M each primer, 50 ␮M deoxynucleotide triphosphates, 1.5 mM MgCl2, and 4 units of GoTaq Colorless Taq polymerase (Promega, Madison, WI). All DNA sequencing was performed on an Applied Biosystems 3100 DNA analyzer using Big Dye Terminator chemistry and AmpliTaq-FS DNA Polymerase (Applied Biosystems, Foster City, CA) at the Colorado State University Proteomics and Metabolomics Facility (Fort Collins, CO). Nucleotide sequences were assembled and aligned with Seqman and Megalign software (DNAStar; Lasergene, Madison, WI), respectively. Phylogenetic inference. To identify where L. monocytogenes outbreak-associated strains clustered in relationship to other outbreak strains and to nonoutbreak-associated strains, an unrooted maximum-likelihood phylogenetic tree was inferred from 55 full inlA sequences, including (i) the 15 human clinical L. monocytogenes outbreak strains characterized in this study and (ii) 40 additional isolates described in our previous study (47), which were selected to represent genetic diversity and different sources of isolation. MODELTEST was used to compare the likelihood estimates of phylogenetic trees constructed under 56 different models of DNA substitution (51). MODELTEST showed that the HKY85 DNA substitution model, along with parameters to estimate a gamma distribution and the proportion of invariate sites, best explained these sequence

5649 L. MONOCYTOGENES OUTBREAK STRAIN VIRULENCE CHARACTERISTICS VOL. 75, 2009

Deaths/cases (% mortality)b Unknown Raw vegetables Dairy products

Food vehicle CSUFSL N1-009 CSUFSL N1-064 CSUFSL N1-006

Previous ID(s)c

Human clinical Human clinical Human clinical

Source

4b 4b 1/2a

Serotype

DUP-1038B (I) DUP-1042B (I) DUP-1030A (II)

Ribotype (lineage)d

Epidemic clonee

Reference(s)

4 25 55

4b

DUP-1042B (I) DUP-1053A (II)

DUP-1044A (I)

II

Ia III

II

9

8 7

6, 10, 37

56 56 21 2 34 58 5 36 36 14 Human clinical

4b 1/2a

DUP-1044A (I)

FSL R2-575, G3992/L4491b FSL J1-220, C7942 FSL R2-568, G3984/L745

DUP-1038B (I) DUP-1038B (I) DUP-1042B (I) DUP-1038B (I) DUP-1038B (I) DUP-1038B (I) DUP-1053A (II) DUP-1042B (I) DUP-1042B (I) DUP-1051B (I)

TABLE 1. Listeriosis outbreaks and L. monocytogenes strains from listeriosis outbreaks characterized in this study Listeriosis outbreaka NK/162 (NK) 3/20 (15) 5/11 (45)

Strain ID

Anjou, France 1975–1976 Boston, MA 1979 Carlisle, England 1981

clinical

4b 4b 4b 4b 4b 4b 1/2a 4b 4b 1/2b FSL N1-225, H7550

Human clinical Human clinical

4b

clinical

clinical clinical clinical clinical clinical clinical

CSUFSL N1-054

FSL R2-501, J0211 FSL R2-499, J0161

Human clinical

Human Food Human Human Human Human Human Human Food Human

CSUFSL N1-022 CSUFSL N1-023

FSL R2-763, J1735

FSL J1-108, TS27/L4738/DD6304 TS50/L4760/1042-TS50 FSL J1-225, Scott A FSL J1-123, TS55/L4486a/DD6332 FSL J1-119, TS43/F4565/DD6320 FSL J1-012, DD2360 FSL J1-101, TS14/F.6900/DD6292 FSL J1-116, TS38/L3306/DD6315 TS45/L.3350/1050-TS45 FSL R2-503, G6054

I Ia Not an EC strain I I Ia I I I III Ia Ia Not an EC strain

CSUFSL N1-079

N1-008 N1-014 N1-035 N1-072 N1-044 N1-074 N1-031 N1-081 N1-060 N1-003

CSUFSL CSUFSL CSUFSL CSUFSL CSUFSL CSUFSL CSUFSL CSUFSL CSUFSL CSUFSL

21/101 (21)

Coleslaw Coleslaw Pasteurized milk Cheese Jalisco soft cheese Ice cream, salami Turkey franks Pate Pate Chocolate milk

United States 1998–1999 5/12 (42) 7/29 (24)

Hot dogs, packed meat Mexican cheese Delicatessen sliced turkey Sliced deli meat

17/41 (42) 17/41 (42) 14/32 (44) 31/122 (25) 48/142 (34) 16/36 (44) Sporadic case 94/355 (26) 94/355 (26) 0/45 (0)

North Carolina 2000–2001 United States 2000 7/63 (11)

Nova Scotia, Canada 1981 Nova Scotia, Canada 1981 Massachusetts 1983 Switzerland 1983–1987 Los Angeles, CA 1985 Philadelphia, PA 1986–1987 Oklahoma 1988 United Kingdom 1988–1989 United Kingdom 1988–1989 Illinois 1994

Northeastern United States 2002

a CSUFSL N1-031 is an isolate from a single listeriosis case associated with turkey franks in 1988. This isolate was included because it belongs to the same molecular subtype that caused a multistate outbreak of listeriosis in the United States in 2000 linked to deli meats produced at the same processing facility. b For all outbreaks but the 1976 Anjou, France, outbreak, the number of cases, number of deaths, and references describing the original outbreaks were obtained from the 2003 Food-Borne Listeriosis Risk Assessment, Tables II-4 and II-5 (19). NK indicates “not known.” c Additional information about each strain is available under the previous FSL ID (e.g., FSL R2-275) for a given strain at http://www.pathogentracker.net. The genetic lineage was designated based on EcoRI ribotypes as detailed previously (62). As described previously (12, 29). d

e

5650

ROBERTS ET AL.

APPL. ENVIRON. MICROBIOL.

TABLE 2. Primers and probes used for DNA sequencing and qRT-PCR characterization Primer or probe

Sequence (5⬘–3⬘)a

inlA proF inlA proR inlA F1 inlA S1R inlA F2 inlA S2R inlA seq inlB PMSC F inlB PMSC R rpoB F

TTT TAA AAG GTG GAA TGA CA GAA GCG TTG TAA CTT GGT CTA CAG GCA GCT ACA ATT ACA CA GGA CTG ATG TTA CTT ATT TGG T AAG ATA TAG GCA CAT TGG CGA GTT CGT ACT GAA ATY CCA KTT AGT TCC GTG GAC GGC AAA GAA ACA AC GCA CGT GCT AGT AAA TAG AAG TAG TG TGT TTT TYA CTG TRT TCG GAA CAA GGT TCC ATT GTT TTC GCA AC

rpoB R

CCG AGT ATT TCG GTT CTC CA

rpoB TqMnF rpoB TqMnR rpoB probe inlA TqMnF inlA TqMnR inlA probe

TGT AAA ATA TGG ACG GCA TCG T GCT GTT TGA ATC TCA ATT AAG TTT GG Fam-CTG ATT CGC GCA AAA CTT CTA CGC G-Bhq GGT CTC RCA AAC AGA TCT AGA CCA AGT TCA AGT ATT CCA MTC CAT CGA TAG ATT Fam-TAT CCC TAA TCT ATC CGC CTG AAG CGT-Bhq

Purpose

Reference

Sequencing of the inlA promoter region Sequencing of the inlA promoter region Sequencing of the inlA coding-domain region Sequencing of the inlA coding-domain region Sequencing of the inlA coding-domain region Sequencing of the inlA coding-domain region Sequencing of the inlA coding-domain region Sequencing of the 5⬘ portion of inlB Sequencing of the 5⬘ portion of inlB Sequencing rpoB region inclusive of the TaqMan primer and probe binding sequences Sequencing rpoB region inclusive of the TaqMan primer and probe binding sequences Primer for qRT-PCR of rpoB Primer for qRT-PCR of rpoB Probe for qRT-PCR of rpoB Primer for qRT-PCR of inlA Primer for qRT-PCR of inlA Probe for qRT-PCR of inlA

47 47 47 47 47 47 47 42 42 This study This study 59 59 59 This study This study 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, and C 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.

data. A maximum-likelihood phylogenetic tree was generated in PAUP* (54) using the parameters determined through MODELTEST, heuristic searches were performed using equal weights for all sites, and the tree bisection-reconnection branch-swapping algorithm was employed. Confidence measures for clustering of isolates within each clade were generated by performing 100 bootstrap replicates using the parameters defined by MODELTEST and the heuristic search algorithm described above. Selection of bacterial isolates for characterization by qRT-PCR and swarming assays 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 HepG2 cell lines. Furthermore, six L. monocytogenes strains that clustered to form a well-supported clade within the inlA phylogram, i.e., the four outbreak strains that demonstrated reduced invasion efficiencies, along with two outbreak strains that showed normal invasion efficiencies, were selected for further characterization by qRT-PCR to assess inlA transcript levels and swarming assays to assess motility. These strains comprised (i) one fully invasive and three invasion-attenuated ECI strains and (ii) one invasion-attenuated and one fully invasive ECIa strain. qRT-PCR. qRT-PCR was performed to determine transcript levels for inlA and for the housekeeping gene rpoB. For total-RNA collection, overnight cultures of L. monocytogenes were passaged by two 1% inoculum transfers as described for growth assays and grown in side arm flasks containing 50 ml of BHI broth 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 RNAeasy Mini kit (Qiagen) essentially as previously described (59), but using a Misonex XL-2000 sonicator (Misonex Inc., Farmingdale, NY) and the rigorous DNase treatment described in the manufacturer’s instructions accompanying the Turbo DNA-free Kit (Ambion). The RNA concentration and purity were determined by measuring absorbance on an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit without RNase inhibitor (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. qRT-PCR was performed using the TaqMan Universal Master Mix and the Applied Biosystems StepOne Plus sequence detection system with the TaqMan primers and probes for rpoB previously described by Sue et al. (59) (Table 2). To detect inlA, we designed the primers inlA TqMnF and inlA TqMnR, along with an inlA probe, to match binding sites for all strains tested (Table 2). We compared inlA and rpoB alignments to confirm that primer and probe binding sites matched for all strains characterized by qRT-PCR. Transcript levels for inlA were expressed as log cDNA copy numbers, which were normalized by subtracting the log cDNA copy numbers observed for the housekeeping gene rpoB. Two independent RNA isolations were performed, and each strain was analyzed in duplicate in two independent qRT-PCR experiments. The genome sequence for the laboratory

control strain 10403S (i.e., inlA and rpoB sequences to confirm primer/probe binding 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 were normalized to rpoB as described above satisfied the assumption of normality. To compare differences in normalized inlA transcript levels among L. monocytogenes outbreak strains, one-way analysis of variance, including a comparison of least squares means and Tukey’s Studentized residuals to correct for multiple comparisons, was performed as implemented through the general linear model procedure in SAS. Adjusted P values of ⬍0.05 were considered statistically significant. Swarming assays. The abilities of select L. monocytogenes outbreak strains to swarm were evaluated on semisoft agar essentially as previously described (52). The strains were grown overnight in BHI broth at 30°C without shaking, and an inoculating needle dipped in each culture was used to stab inoculate BHI semisoft agar (0.4%) in duplicate. The plates were incubated at 30°C for 72 h, and the diameter of each resultant intra-agar colony was measured to assess swarming ability. The diameters of duplicate stabs were averaged and expressed as a percentage 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 at least three independent experiments, and L. monocytogenes strains 10403S and 10403S ⌬flaA (45) were included in each experiment as controls. Statistical analysis of swarming assay data. Swarming assay data for outbreak strains that were normalized to the laboratory control strain satisfied the assumption of normality. To compare differences in swarming among L. monocytogenes outbreak strains, one-way analysis of variance, including a comparison of least squares means and Tukey’s Studentized residuals to correct for multiple comparisons, was performed as implemented through the general linear model procedure in SAS. Comparisons with adjusted P values of ⬍0.05 were deemed statistically significant. Nucleotide sequence accession numbers. The sequences for the inlA genes of the 15 outbreak strains were deposited in GenBank under accession numbers FJ617522 to FJ617536. The sequences of the 5⬘ regions of inlB for strains CSUFSL N1-009 and CSUFSL N1-081 were deposited in GenBank under accession numbers FJ751916 and FJ751917.

RESULTS AND DISCUSSION Based on epidemiological evidence suggesting that certain L. monocytogenes serotypes and molecular subtypes are more frequently associated with human listeriosis (18, 29, 57), studies of L. monocytogenes growth and survival, molecular genet-

VOL. 75, 2009

L. MONOCYTOGENES OUTBREAK STRAIN VIRULENCE CHARACTERISTICS

ics, and virulence characteristics often use a single strain or a few strains from listeriosis outbreaks under the a priori assumption that outbreak-associated strains have enhanced in vitro and in vivo virulence phenotypes. We characterized a set of human clinical L. monocytogenes strains from 15 listeriosis outbreaks that occurred worldwide using invasion assays in Caco-2 human intestinal epithelial cells and HepG2 human hepatic cells, as well as by sequencing of the virulence gene inlA, which encodes a key virulence factor required for efficient invasion of these two cell lines (30). Outbreak strains with reduced invasion efficiencies, as well as fully invasive outbreak strains, clustering within a well-supported phylogenetic clade were further characterized for inlA transcript levels and the ability to swarm, a phenotype previously linked to increased invasion (1). Overall, our data show that (i) L. monocytogenes strains from human clinical cases associated with listeriosis outbreaks show significant variation in invasion efficiencies, with some strains demonstrating consistent significantly reduced invasion efficiencies across two different cell lines, and (ii) different mechanisms may contribute to the reduced invasion efficiencies observed for these strains. Furthermore, the strain (CSUFSL N1-081) associated with the 1988–1989 pate outbreak in the United Kingdom, which showed reduced invasion efficiencies in both cell lines and reduced swarming, was recently shown to have reduced virulence in intragastric guinea pig infection experiments (41). Specifically, unvaccinated guinea pigs challenged with CSUFSL N1-081 (previous strain identifier [ID], FSL J1-116) (Table 1) showed reduced L. monocytogenes counts in their livers compared to unvaccinated animals challenged with the laboratory control strain 10403S (41), further supporting the idea that some outbreak-associated strains have attenuated virulence in vivo. Outbreak-associated L. monocytogenes strains show significant variation in invasion efficiencies. L. monocytogenes outbreak strains showed variation in invasion efficiencies in both Caco-2 human intestinal epithelial and HepG2 human hepatic cells. InlA is required for efficient invasion of both cell lines when the bacteria are grown under either condition tested here (i.e., 30°C without shaking and 37°C with shaking), while InlB contributes to invasion of HepG2 cells when the bacteria are grown at 37°C with shaking (30). Invasion efficiencies in Caco-2 cells for bacteria grown at 30°C without shaking varied significantly across the human clinical outbreak-associated strains tested in this study (P ⬍ 0.0001; overall F test), where the mean invasion efficiency ranged from 0.01% (for the human isolate from the 1988–1989 United Kingdom outbreak, CSUFSL N1081) to 3.17% (for the human isolate from the 1981 United Kingdom outbreak, CSUFSL N1-006) (Fig. 1A). Four outbreak-associated strains (CSUFSL N1-008, CSUFSL N1-009, CSUFSL N1-044, and CSUFSL N1-081) and the laboratory control strain (10403S) showed significantly (P ⬍ 0.05) reduced invasion efficiencies in Caco-2 cells when the bacteria were grown at 30°C compared to the fully virulent (A. Van Stelten and K. Nightingale, unpublished data) reference outbreak strain CSUFSL N1-054 (Fig. 1A). L. monocytogenes invasion of HepG2 human hepatic cells has been shown to be less efficient than invasion of Caco-2 cells (30), but we also observed significant differences (P ⬍ 0.0001; overall F test) among invasion efficiencies for outbreak strains when this cell line was used. For example, using bacteria grown at 30°C

5651

without shaking, mean HepG2 invasion efficiencies ranged from 0.0009% (for the human isolate from the 1988–1989 United Kingdom outbreak, CSUFSL N1-081) to 2.19% (for the human isolate from the 1998 U.S. outbreak, CSUFSL N1054) (Fig. 1B). Interestingly, the same four outbreak-associated strains that showed significantly reduced invasion of Caco-2 cells when the bacteria were grown at 30°C without shaking also showed the least ability to invade HepG2 cells (P ⬍ 0.05) when the bacteria were grown under the same conditions. Two additional outbreak-associated strains (i.e., CSUFSL N1-023 and CSUFSL N1-031) and the laboratory control strain (10403S) also showed significantly (P ⬍ 0.05) reduced invasion efficiencies in HepG2 cells when the bacteria were grown at 30°C without shaking. L. monocytogenes invasion of Caco-2 and HepG2 cells has been shown to be reduced when the bacteria are grown at 37°C with shaking compared to the invasion efficiencies for both cell lines when the bacteria are grown at 30°C without shaking (30). Invasion efficiencies in Caco-2 cells when the bacteria were grown 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 the 2000 North Carolina outbreak, CSUFSL N1-022) (Fig. 2A). The same four outbreak-associated strains that showed significantly reduced invasion of both Caco-2 and HepG2 cells when the bacteria were grown at 30°C without shaking (i.e., CSUFSL N1-008, CSUFSL N1-009, CSUFSL N1-044, and CSUFSL N1081) also showed significantly (P ⬍ 0.05) reduced invasion in Caco-2 cells when the bacteria were grown at 37°C with shaking (Fig. 2A). When the bacteria were grown at 37°C with shaking, invasion efficiencies in HepG2 cells also varied significantly (P ⬍ 0.0001; overall F test) among strains and ranged from 0.007% (for the human isolate from the 1976 outbreak in France, CSUFSL N1-009) to 0.51% (for the human isolate from 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 when grown 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) increased invasion of HepG2 cells under the same bacterial growth 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 in Anjou, France; CSUFSL N1-008, from the 1981 outbreak in Nova Scotia, Canada; and CSUFSL N1-044, from the 1985 outbreak in Los Angeles) and one ECIa strain (CSUFSL N1081, from the 1988–1989 outbreak in the United Kingdom) showed particularly low abilities to invade both human cell types and under both bacterial growth conditions. These four strains showed significantly reduced invasion efficiencies in Caco-2 cells for both bacterial growth conditions evaluated here and in HepG2 cells when the bacteria were grown at 30°C; the four strains were thus classified as invasion attenuated and were further probed for underlying genetic and mechanistic differences to explain this in vitro invasion-attenuated phenotype. Virulence-associated phenotypes, such as invasion efficiency for human cell types, have previously been correlated with virulence in animal models (22, 32). In addition, Nightingale

5652

ROBERTS ET AL.


FIG. 1. Percent Caco-2 (A) and HepG-2 (B) cell invasion efficiencies for human clinical L. monocytogenes strains representing 15 listeriosis 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 was assayed in duplicate in each independent experiment, and at least three independent invasion experiments were performed to characterize each 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 invasion efficiency 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 without 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 without shaking, using Dunnett’s multiple-comparison correction (with reference to outbreak strain CSUFSL N1-054), is denoted by an asterisk above each strain that significantly differed from the reference strain for the Caco-2 and HepG2 cell lines.

VOL. 75, 2009


5653

FIG. 2. Percent Caco-2 (A) and HepG-2 (B) cell invasion efficiencies for human clinical L. monocytogenes strains representing 15 listeriosis outbreaks and the laboratory control strain 10403S grown at 37°C with shaking. The invasion efficiency (percentage of L. monocytogenes inoculum that 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 each independent experiment, and at least three independent invasion experiments were performed to characterize each 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 L. monocytogenes outbreak-associated strains differed significantly with respect to invasion efficiency 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 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 without shaking, using Dunnett’s multiple-comparison correction (with reference to outbreak strain CSUFSL N1-054), is denoted by an asterisk above each strain that significantly differed from the reference strain for the Caco-2 and HepG2 cell lines.

5654

ROBERTS ET AL.

et al. recently reported that the human clinical strain associated with the 1988–1989 outbreak in the United Kingdom, a strain that showed reduced invasion efficiency in the current study, showed reduced bacterial numbers in the livers of unvaccinated guinea pigs that were orally challenged with this strain compared to counts recovered from unvaccinated guinea pigs challenged with the fully virulent laboratory control strain 10403S (41). Although it has been previously observed that L. monocytogenes isolates belonging to different serotypes, including serotype 4b isolates, exhibit heterogeneous abilities to invade human cells (11, 61), to our knowledge, this is the first report showing that significant variability in invasion capabilities also exists within epidemic clone strains associated with listeriosis outbreaks. While it could be argued that the consistently low invasion efficiency observed for the four human clinical isolates from listeriosis outbreaks (i.e., CSUFSL N1-008, CSUFSL N1009, CSUFSL N1-044, and CSUFSL N1-081) is an artifact of laboratory passage, there is no apparent correlation between the invasiveness of a strain and the time since the outbreak (Table 1). For example, both the invasion-attenuated strain CSUFSL N1-008 and the fully invasive strain CSUFSL N1-006 were isolated from outbreaks beginning in 1981. Furthermore, when we compared the invasion efficiency of an invasion-attenuated human clinical isolate to that of a food isolate from the same outbreak, when a food isolate was available (i.e., strains CSUFSL N1-014, a food isolate from the coleslaw associated with the 1981 outbreak in Nova Scotia, and CSUFSL N1-060, a food isolate from the pate associated with the 1988– 1989 outbreak in the United Kingdom), we observed that food isolates showed similarly low invasion efficiencies compared to human clinical strains from the same outbreak. Specifically, the food isolate CSUFSL N1-014 (1981 Nova Scotia outbreak) had an average invasion efficiency of 0.02% compared to an average invasion efficiency of 0.55% for the human clinical isolate from the same outbreak (CSUFSL N1-008) in Caco-2 cells using bacteria grown at 30°C without shaking, while the food isolate CSUFSL N1-060 (1988–1989 United Kingdom outbreak) had an average invasion efficiency of 0.06% compared to an average invasion efficiency of 0.01% for a human clinical isolate from the same outbreak (CSUFSL N1-081) in Caco-2 cells using bacteria grown at 30°C. We also previously showed that the food isolate (strain F2365) from the 1985 outbreak in Los Angeles associated with Jalisco cheese demonstrated attenuated invasiveness similar to the invasiveness of the human clinical isolate from the same outbreak (CSUFSL N1-044) characterized here (42). These data suggest that low invasion efficiency is a natural phenotype of strains associated with certain outbreaks and that strain-specific phenotypic characteristics other than enhanced virulence characteristics, such as enhanced survival and growth in foods or food-associated environments, may contribute to the association of some epidemic clone strains with listeriosis outbreaks. This hypothesis is supported by ongoing work by our group that shows that a subset of the fully invasive ECI and ECIa strains characterized in this study vary significantly with respect to growth rates on turkey breast formulated without antimicrobials (J. Corron and K. Nightingale, unpublished data). Until future animal studies of additional outbreak strains are performed, we cannot exclude the possibility that, for some strains, other viru-


lence mechanisms (e.g., intracellular growth, intracellular spread, and the ability to evade host immune responses) may also contribute to the association of strains with attenuated invasion with human outbreaks and may possibly compensate for reduced invasion efficiency. Furthermore, it is important to note that factors unrelated to the pathogen, such as immunocompromised host status, may still enable strains showing reduced invasion to cause disease. Different mechanisms contribute to the attenuated invasion efficiencies of some human outbreak strains. Four outbreak strains demonstrated attenuated invasion efficiencies in both Caco-2 and HepG2 cell lines, suggesting that mutations in inlA or differences in inlA transcript levels may at least partially explain the attenuated invasion phenotype observed for these strains, since InlA is required for efficient invasion of both cell lines (30). Diminished ability to invade human cells has been commonly attributed to mutations leading to premature stop codons in inlA, which result in the production of a truncated and secreted form of InlA (20, 27, 41, 43, 44). To probe the cause of the variation in invasion efficiencies among the set of L. monocytogenes outbreak-associated strains characterized here, inlA genes from all 15 strains were sequenced. None of the four invasion-attenuated strains contained a premature stop codon mutation in inlA. In addition, all four invasionattenuated 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 and CSUFSL N1-072, to form a clade with bootstrap support of 100% in the inlA phylogram (Fig. 3). Five isolates in this clade, including the four invasion-attenuated strains and the fully invasive strain CSUFSL N1-072, showed 100% InlA amino acid identity, while the other fully invasive strain (CSUFSL N1-035) differed by a single amino acid at codon 800. The four invasion-attenuated strains also clustered with fully invasive strains in phylogenies created using concatenated sequences that included the L. monocytogenes housekeeping genes gap, prs, purM, and ribC (H. den Bakker and M. Wiedmann, unpublished data), suggesting that these invasion-attenuated strains are genetically related to fully invasive strains at the core level. To screen for general defects in the cellular growth rate that might explain an attenuated invasion phenotype, we performed growth curves in BHI broth using the four invasion-attenuated strains and the laboratory control strain 10403S. The growth rates observed for the invasion-attenuated strains were similar to that of 10403S, and all of the strains reached similar resultant population densities at 12 h postinoculation (data not shown), indicating that invasion-attenuated strains are not impaired in general growth characteristics. Along with InlA, InlB is a well-known virulence factor that plays a role in the invasion of multiple human cell types (17, 26, 48). While one would not expect the attenuated invasion efficiencies of the four strains identified here to be solely attributable to mutations in inlB (as Caco-2 cell invasion, which was attenuated in these strains, is InlB independent), a rare mutation leading to a premature stop codon in the 5⬘ region of inlB was previously identified in a food isolate (strain F2365) and a human clinical isolate (CSUFSL N1-044) from the 1985 outbreak in California associated with Jalisco cheese (42), which is one of the human clinical outbreak-associated strains that showed reduced

VOL. 75, 2009


5655

FIG. 3. Unrooted maximum-likelihood phylogram inferred from inlA coding-domain sequences for 15 L. monocytogenes outbreak strains, along with 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 EcoRI ribotype in parentheses. Branching support was determined by 100 bootstrap replicates, and bootstrap values (if ⬎70%) are shown at the node labels. L. monocytogenes outbreak strains are labeled in red, and strains that demonstrated reduced invasion efficiency in Caco-2 and HepG-2 cells are flanked by asterisks. The dashed rounded rectangle indicates a phylogenetic clade composed of L. monocytogenes ECI and ECIa outbreak strains demonstrating reduced or normal invasion efficiencies for Caco-2 and HepG-2 cells.

Caco-2 and HepG2 invasion efficiencies in this study. We thus analyzed the inlB sequences of this region in the other three outbreak strains demonstrating attenuated invasion in this study. The three invasion-attenuated outbreak strains newly identified in this study (CSUFSL N1-008, CSUFSL N1-009, and CSUFSL N1-081) did not carry this previously described premature stop codon mutation in inlB. Since we did not identify any premature stop codon mutations in inlA, we hypothesized that reduced inlA transcription might contribute to the attenuated invasion phenotype observed for some outbreak strains. qRT-PCR analysis of the inlA transcript levels of the four invasion-attenuated strains and the two fully invasive outbreak-associated strains, which formed a highly clonal clade in the inlA phylogram, showed mean normalized inlA transcript levels ranging from ⫺2.49 for strain CSUFSL N1-009 to ⫺0.33 for strain CSUFSL N1-072 (Fig. 4). Two invasion-attenuated ECI strains (CSUFSL N1008 and CSUFSL N1-009) demonstrated significantly lower

inlA transcript levels than the fully invasive ECI strain CSUFSL N1-072 (P ⫽ 0.007 and P ⫽ 0.001, respectively) (Fig. 4). The invasion-attenuated ECIa strain demonstrated inlA transcript levels similar to those observed for the fully invasive ECIa strain (Fig. 4). These results confirm and extend the findings of others who have also observed variation in inlA transcript levels among and within L. monocytogenes serotypes (11, 61). Swarming motility in L. monocytogenes is a phenotype that has been shown to contribute to the ability to adhere to and invade Caco-2 intestinal epithelial cells, as well as the ability to cause disease in a mouse infection model (1, 16, 45). We therefore compared the swarming abilities of the four invasionattenuated strains and the closely related fully invasive strains CSUFSL N1-035 and CSUFSL N1-072. We observed swarming efficiencies ranging from 45.4% for strain CSUFSL N1-081 (a human clinical strain from the 1988–1989 United Kingdom outbreak) to 110.6% for strain CSUFSL N1-008 (a human

5656

ROBERTS ET AL.


FIG. 4. Normalized inlA transcript levels for six L. monocytogenes strains clustering in a single inlA phylogenetic clade that includes strains demonstrating 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 number of the housekeeping gene rpoB (log10 copy number inlA ⫺ log10 copy number rpoB). Each strain was assayed in duplicate in each independent experiment, and at least two independent RNA isolations, cDNA syntheses, and qRT-PCR experiments were conducted to characterize each strain. The bars represent mean transcript levels for each strain, and the error bars represent the standard deviations around the mean transcript levels. Different letters in the bars indicate statistical significance (P ⬍ 0.05) based on one-way analysis of variance and comparison of strain mean normalized transcript levels with Tukey’s correction for multiple comparisons.

clinical strain from the 1981 Nova Scotia outbreak) normalized to the swarming efficiency for control strain 10403S, which was set at 100% (Fig. 5). Three of the four invasion-attenuated strains, CSUFSL N1-009, CSUFSL N1-044, and CSUFSL N1081, 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 transcript levels. Strain CSUFSL N1-081 was particularly attenuated in swarming ability compared to the fully invasive strains CSUFSL N1-035 (P ⫽ 0.002) and CSUFSL N1-072 (P ⫽ 0.002), suggesting that the attenuated invasion efficiencies in these strains might be at least partially attributable to defects

FIG. 5. Swarming abilities of six L. monocytogenes strains clustering within the same inlA phylogenetic clade, which includes strains demonstrating either 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. Each strain was assayed in at least three independent swarming assays. The bars represent the mean normalized swarming ability for each strain, and the error bars represent the standard deviations around the mean transcript levels. Different letters in the bars indicate statistical significance (P ⬍ 0.05) based on one-way analysis of variance and comparison of strain mean normalized transcript levels with Tukey’s correction for multiple comparisons.

VOL. 75, 2009


in flagellar motility. Future studies involving sequence and expression analyses of flagellin structural and regulatory genes in invasion-attenuated and genetically similar fully invasive outbreak strains are needed to provide additional insight into the mechanisms of reduced flagellar motility and to determine to what extent reduced flagellar motility contributes to the invasion-attenuated phenotype observed in this study. Conclusions. It has been well documented that considerable diversity in virulence-associated phenotypes exists among L. monocytogenes strains representing different serotypes and molecular subtypes (18, 24, 29, 35, 57, 62). Our data demonstrate that this diversity in virulence-associated phenotypes (i.e., invasion, inlA transcript levels, and swarming motility) also extends to strains belonging to highly clonal groups frequently linked to listeriosis outbreaks, such as ECI and ECIa. Specifically, L. monocytogenes epidemic clone strains examined in the current study that demonstrated reduced in vitro invasion in both Caco-2 and HepG2 cell lines also showed reduced inlA transcript levels, reduced swarming, or both reduced inlA transcript levels and reduced swarming. These findings suggest that invasion efficiency may depend, at least in part, on inlA transcript levels and swarming ability; however, future experiments are required to more precisely elucidate the association between inlA transcript levels, swarming motility, and invasion efficiency. Although strains that demonstrated attenuated in vitro virulence phenotypes were not characterized by notably reduced morbidity rates for their respective listeriosis outbreaks, these findings suggest that the association of epidemic clone strains with a majority of the human listeriosis outbreaks that have occurred worldwide may include factors other than enhanced virulence, such as survival and growth characteristics in food-associated environments and host immune status. Importantly, the outcomes of this study also support the idea that outbreak-associated L. monocytogenes epidemic clone strains cannot, a priori, be considered to demonstrate highly virulent in vitro phenotypes for molecular genetics and pathogenesis studies.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18. 19.

20.

21.

ACKNOWLEDGMENTS This project was supported by USDA Special Research Grants 200534459-15625 and 2006-34459-16952 (both to M.W.), USDA-CSREES Hatch Funds (NYC-143451; to M.W.), and the Colorado State University Agriculture Experiment Station. We thank Jessica Corron for assistance with growth curve experiments and Julie Simpson and Anna Van Stelten for assistance with DNA sequencing. REFERENCES 1. Bigot, A., H. Pagniez, E. Botton, C. Frehel, I. Dubail, C. Jacquet, A. Charbit, and C. Raynaud. 2005. Role of FliF and FliI of Listeria monocytogenes in flagellar assembly and pathogenicity. Infect. Immun. 73:5530–5539. 2. Bille, J. 1990. Epidemiology of human listeriosis in Europe, with special reference to the Swiss outbreak, p. 71–74. In A. J. Miller, J. L. Smith, and G. A. Somkuti (ed.), Foodborne listeriosis. Elsevier, Amsterdam, The Netherlands. 3. Bishop, D. K., and D. J. Hinrichs. 1987. Adoptive transfer of immunity to Listeria monocytogenes. The influence of in vitro stimulation on lymphocyte subset requirements. J. Immunol. 139:2005–2009. 4. Carbonnelle, B., J. Cottin, F. Parvery, G. Chambreuil, S. Kouyoumdjian, M. Le Lirzin, G. Cordier, and F. Vincent. 1979. Epidemic of listeriosis in Western France (1975–1976). Rev. Epidemiol. Sante Publique. 26:451–467. 5. Centers for Disease Control and Prevention. 1989. Listeriosis associated with consumption of turkey franks. MMWR Morb. Mortal. Wkly. Rep. 38:267–268. 6. Centers for Disease Control and Prevention. 1998. Multistate outbreak of

22.

23.

24.

25.

26.

27.

28.

29. 30.

5657

listeriosis—United States, 1998. MMWR Morb. Mortal. Wkly. Rep. 47: 1085–1086. Centers for Disease Control and Prevention. 2000. Multistate outbreak of listeriosis—United States, 2000. MMWR Morb. Mortal. Wkly. Rep. 49: 1129–1130. Centers for Disease Control and Prevention. 2001. Outbreak of listeriosis associated with homemade Mexican-style cheese—North Carolina, October 2000-January 2001. MMWR Morb. Mortal. Wkly. Rep. 50:560–562. Centers for Disease Control and Prevention. 2002. Outbreak of listeriosis— northeastern United States, 2002. MMWR Morb. Mortal. Wkly. Rep. 51: 950–951. Centers for Disease Control and Prevention. 1999. Update: multistate outbreak of listeriosis—United States, 1998–1999. MMWR Morb. Mortal. Wkly. Rep. 47:1117–1118. Chatterjee, S. S., S. Otten, T. Hain, A. Lingnau, U. D. Carl, J. Wehland, E. Domann, and T. Chakraborty. 2006. Invasiveness is a variable and heterogeneous phenotype in Listeria monocytogenes serotype strains. Int. J. Med. Microbiol. 296:277–286. Chen, Y., W. Zhang, and S. J. Knabel. 2005. Multi-virulence-locus sequence typing clarifies epidemiology of recent listeriosis outbreaks in the United States. J. Clin. Microbiol. 43:5291–5294. Chen, Y., W. Zhang, and S. J. Knabel. 2007. Multi-virulence-locus sequence typing identifies single nucleotide polymorphisms which differentiate epidemic clones and outbreak strains of Listeria monocytogenes. J. Clin. Microbiol. 45:835–846. Dalton, C. B., C. C. Austin, J. Sobel, P. S. Hayes, W. F. Bibb, L. M. Graves, B. Swaminathan, M. E. Proctor, and P. M. Griffin. 1997. An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk. N. Engl. J. Med. 336:100–105. Donaldson, J. R., B. Nanduri, S. C. Burgess, and M. L. Lawrence. 2008. Comparative proteomic analysis of Listeria monocytogenes strains F2365 and EGD. Appl. Environ. Microbiol. 75:366–373. Dons, L., E. Eriksson, Y. Jin, M. E. Rottenberg, K. Kristensson, C. N. Larsen, J. Bresciani, and J. E. Olsen. 2004. Role of flagellin and the twocomponent CheA/CheY system of Listeria monocytogenes in host cell invasion and virulence. Infect. Immun. 72:3237–3244. Dramsi, S., I. Biswas, E. Maguin, L. Braun, P. Mastroeni, and P. Cossart. 1995. Entry of Listeria monocytogenes into hepatocytes requires expression of inlB, a surface protein of the internalin multigene family. Mol. Microbiol. 16:251–261. Farber, J. M., and P. I. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476–511. FDA, USDA, and Centers for Disease Control and Prevention. 2003. Quantitative assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. FDA, Washington, DC. http://www.foodsafety.gov/⬃dms/lmr2-toc.html. Felicio, M. T., T. Hogg, P. Gibbs, P. Teixeira, and M. Wiedmann. 2007. Recurrent and sporadic Listeria monocytogenes contamination in alheiras represents considerable diversity, including virulence-attenuated isolates. Appl. Environ. Microbiol. 73:3887–3895. Fleming, D. W., S. L. Cochi, K. L. MacDonald, J. Brondum, P. S. Hayes, B. D. Plikaytis, M. B. Holmes, A. Audurier, C. V. Broome, and A. L. Reingold. 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. N. Engl. J. Med. 312:404–407. Garner, M. R., B. L. Njaa, M. Wiedmann, and K. J. Boor. 2006. Sigma B contributes to Listeria monocytogenes gastrointestinal infection but not to systemic spread in the guinea pig infection model. Infect. Immun. 74:876–886. Gianfranceschi, M. V., A. Gattuso, M. C. D’Ottavio, S. Fokas, and P. Aureli. 2007. Results of a 12-month long enhanced surveillance of listeriosis in Italy. Euro. Surveill. 12:E7–E8. Gray, M. J., R. N. Zadoks, E. D. Fortes, B. Dogan, S. Cai, Y. Chen, V. N. Scott, D. E. Gombas, K. J. Boor, and M. Wiedmann. 2004. Listeria monocytogenes isolates from foods and humans form distinct but overlapping populations. Appl. Environ. Microbiol. 70:5833–5841. Ho, J. L., K. N. Shands, G. Friedland, P. Eckind, and D. W. Fraser. 1986. An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Intern. Med. 146:520–524. Ireton, K., B. Payrastre, H. Chap, W. Ogawa, H. Sakaue, M. Kasuga, and P. Cossart. 1996. A role for phosphoinositide 3-kinase in bacterial invasion. Science 274:780–782. Jacquet, C., M. Doumith, J. I. Gordon, P. M. Martin, P. Cossart, and M. Lecuit. 2004. A molecular marker for evaluating the pathogenic potential of foodborne Listeria monocytogenes. J. Infect. Dis. 189:2094–2100. Kathariou, S. 2003. Foodborne outbreaks of listeriosis and epidemic-associated lineages of Listeria monocytogenes, p. 243–256. In M. E. Torrence and R. E. Isaacson (ed.), Microbial food safety in animal agriculture: current topics. Iowa State Press, Ames. Kathariou, S. 2002. Listeria monocytogenes virulence and pathogenicity, a food safety perspective. J. Food Prot. 65:1811–1829. Kim, H., K. J. Boor, and H. Marquis. 2004. Listeria monocytogenes ␴B contributes to invasion of human intestinal epithelial cells. Infect. Immun. 72:7374–7378.

5658

ROBERTS ET AL.

31. Kim, H., H. Marquis, and K. J. Boor. 2005. ␴B contributes to Listeria monocytogenes invasion by controlling expression of inlA and inlB. Microbiology 151:3215–3222. 32. Lecuit, M., S. Vandormael-Pournin, J. Lefort, M. Huerre, P. Gounon, C. Dupuy, C. Babinet, and P. Cossart. 2001. A transgenic model for listeriosis: role of internalin in crossing the intestinal barrier. Science 292:1722–1725. 33. Lianou, A., and J. N. Sofos. 2007. A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. J. Food Prot. 70:2172–2198. 34. Linnan, M. J., L. Mascola, X. D. Lou, V. Goulet, S. May, C. Salminen, D. W. Hird, M. L. Yonekura, P. Hayes, R. Weaver, et al. 1988. Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med. 319:823–828. 35. McLauchlin, J. 1990. Distribution of serovars of Listeria monocytogenes isolated from different categories of patients with listeriosis. Eur. J. Clin. Microbiol. Infect. Dis. 9:210–213. 36. McLauchlin, J., S. M. Hall, S. K. Velani, and R. J. Gilbert. 1991. Human listeriosis and pate: a possible association. BMJ 303:773–775. 37. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, and R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5:607–625. 38. Mereghetti, L., P. Lanotte, V. Savoye-Marczuk, N. Marquet-Van Der Mee, A. Audurier, and R. Quentin. 2002. Combined ribotyping and random multiprimer DNA analysis to probe the population structure of Listeria monocytogenes. Appl. Environ. Microbiol. 68:2849–2857. 39. Nadon, C. A., D. L. Woodward, C. Young, F. G. Rodgers, and M. Wiedmann. 2001. Correlations between molecular subtyping and serotyping of Listeria monocytogenes. J. Clin. Microbiol. 39:2704–2707. 40. Nelson, K. E., D. E. Fouts, E. F. Mongodin, J. Ravel, R. T. DeBoy, J. F. Kolonay, D. A. Rasko, S. V. Angiuoli, S. R. Gill, I. T. Paulsen, J. Peterson, O. White, W. C. Nelson, W. Nierman, M. J. Beanan, L. M. Brinkac, S. C. Daugherty, R. J. Dodson, A. S. Durkin, R. Madupu, D. H. Haft, J. Selengut, S. Van Aken, H. Khouri, N. Fedorova, H. Forberger, B. Tran, S. Kathariou, L. D. Wonderling, G. A. Uhlich, D. O. Bayles, J. B. Luchansky, and C. M. Fraser. 2004. Whole genome comparisons of serotype 4b and 1/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res. 32:2386–2395. 41. Nightingale, K. K., R. A. Ivy, A. J. Ho, E. D. Fortes, B. L. Njaa, R. M. Peters, and M. Wiedmann. 2008. inlA premature stop codons are common among Listeria monocytogenes isolates from foods and yield virulence-attenuated strains that confer protection against fully virulent strains. Appl. Environ. Microbiol. 74:6570–6583. 42. Nightingale, K. K., S. R. Milillo, R. A. Ivy, A. J. Ho, H. F. Oliver, and M. Wiedmann. 2007. Listeria monocytogenes F2365 carries several authentic mutations potentially leading to truncated gene products, including inlB, and demonstrates atypical phenotypic characteristics. J. Food Prot. 70:482–488. 43. Nightingale, K. K., K. Windham, K. E. Martin, M. Yeung, and M. Wiedmann. 2005. Select Listeria monocytogenes subtypes commonly found in foods carry distinct nonsense mutations in inlA, leading to expression of truncated and secreted internalin A, and are associated with a reduced invasion phenotype for human intestinal epithelial cells. Appl. Environ. Microbiol. 71:8764–8772. 44. Olier, M., F. Pierre, J. P. Lemaitre, C. Divies, A. Rousset, and J. Guzzo. 2002. Assessment of the pathogenic potential of two Listeria monocytogenes human faecal carriage isolates. Microbiology 148:1855–1862. 45. O’Neil, H. S., and H. Marquis. 2006. Listeria monocytogenes flagella are used for motility, not as adhesins, to increase host cell invasion. Infect. Immun. 74:6675–6681. 46. Orsi, R. H., M. L. Borowsky, P. Lauer, S. K. Young, C. Nusbaum, J. E. Galagan, B. W. Birren, R. A. Ivy, Q. Sun, L. M. Graves, B. Swaminathan,


47.

48.

49.

50.

51. 52.

53.

54.

55.

56.

57. 58.

59.

60.

61.

62.

and M. Wiedmann. 2008. Short-term genome evolution of Listeria monocytogenes in a non-controlled environment. BMC Genomics 9:539. Orsi, R. H., D. R. Ripoll, M. Yeung, K. K. Nightingale, and M. Wiedmann. 2007. Recombination and positive selection contribute to evolution of Listeria monocytogenes inlA. Microbiology 153:2666–2678. Parida, S. K., E. Domann, M. Rohde, S. Muller, A. Darji, T. Hain, J. Wehland, and T. Chakraborty. 1998. Internalin B is essential for adhesion and mediates the invasion of Listeria monocytogenes into human endothelial cells. Mol. Microbiol. 28:81–93. Peterson, L. D., N. G. Faith, and C. J. Czuprynski. 2008. Growth of L. monocytogenes strain F2365 on ready-to-eat turkey meat does not enhance gastrointestinal listeriosis in intragastrically inoculated A/J mice. Int. J. Food Microbiol. 126:112–115. Piffaretti, J. C., H. Kressebuch, M. Aeschbacher, J. Bille, E. Bannerman, J. M. Musser, R. K. Selander, and J. Rocourt. 1989. Genetic characterization of clones of the bacterium Listeria monocytogenes causing epidemic disease. Proc. Natl. Acad. Sci. USA 86:3818–3822. Posada, D., and K. A. Crandall. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818. Raengpradub, S., M. Wiedmann, and K. J. Boor. 2008. Comparative analysis of the sigma B-dependent stress responses in Listeria monocytogenes and Listeria innocua strains exposed to selected stress conditions. Appl. Environ. Microbiol. 74:158–171. Rocourt, J., P. BenEmbarek, H. Toyofuku, and J. Schlundt. 2003. Quantitative risk assessment of Listeria monocytogenes in ready-to-eat foods: the FAO/WHO approach. FEMS Immunol. Med. Microbiol. 35:263–267. Rogers, J. S., and D. L. Swofford. 1998. A fast method for approximating maximum likelihoods of phylogenetic trees from nucleotide sequences. Syst. Biol. 47:77–89. Ryser, E. T. 1999. Foodborne listeriosis, p. 299–358. In E. T. Ryser and E. H. Marth (ed.), Listeria, listeriosis and food safety, vol. 2. Marcel Dekker, New York, NY. Schlech, W. F., III, P. M. Lavigne, R. A. Bortolussi, A. C. Allen, E. V. Haldane, A. J. Wort, A. W. Hightower, S. E. Johnson, S. H. King, E. S. Nicholls, and C. V. Broome. 1983. Epidemic listeriosis—evidence for transmission by food. N. Engl. J. Med. 308:203–206. Schuchat, A., B. Swaminathan, and C. V. Broome. 1991. Epidemiology of human listeriosis. Clin. Microbiol. Rev. 4:169–183. Schwartz, B., D. Hexter, C. V. Broome, A. W. Hightower, R. B. Hirschhorn, J. D. Porter, P. S. Hayes, W. F. Bibb, B. Lorber, and D. G. Faris. 1989. Investigation of an outbreak of listeriosis: new hypotheses for the etiology of epidemic Listeria monocytogenes infections. J. Infect. Dis. 159:680–685. Sue, D., D. Fink, M. Wiedmann, and K. J. Boor. 2004. ␴B-dependent gene induction and expression in Listeria monocytogenes during osmotic and acid stress conditions simulating the intestinal environment. Microbiology 150: 3843–3855. Ward, T. J., L. Gorski, M. K. Borucki, R. E. Mandrell, J. Hutchins, and K. Pupedis. 2004. Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes. J. Bacteriol. 186:4994–5002. Werbrouck, H., K. Grijspeerdt, N. Botteldoorn, E. Van Pamel, N. Rijpens, J. Van Damme, M. Uyttendaele, L. Herman, and E. Van Coillie. 2006. Differential inlA and inlB expression and interaction with human intestinal and liver cells by Listeria monocytogenes strains of different origins. Appl. Environ. Microbiol. 72:3862–3871. Wiedmann, M., J. L. Bruce, C. Keating, A. E. Johnson, P. L. McDonough, and C. A. Batt. 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65:2707–2716.