ABSTRACTA total of 442Listeriaisolates, including 234Listeria seeligeri, 80L. monocytogenes, 74L. welshimeri, 50L. innocua, and 4L. marthiiisolates, were obtained from 1,805 soil, water, and other environmental samples collected over 2 years from four urban areas and four areas representing natural environments.Listeriaspp. showed similar prevalences in samples from natural (23.4%) and urban (22.3%) environments. WhileL. seeligeriandL. welshimeriwere significantly associated with natural environments (P≤ 0.0001),L. innocuaandL. monocytogeneswere significantly associated with urban environments (P≤ 0.0001). Sequencing ofsigBfor all isolates revealed 67 allelic types with a higher level of allelic diversity among isolates from urban environments. SomeListeriaspp. andsigBallelic types showed significant associations with specific urban and natural areas. Nearest-neighbor analyses also showed that certainListeriaspp. andsigBallelic types were spatially clustered within both natural and urban environments, and there was evidence that these species and allelic types persisted over time in specific areas. Our data show that members of the genusListerianot only are common in urban and natural environments but also show species- and subtype-specific associations with different environments and areas. This indicates thatListeriaspecies and subtypes within these species may show distinct ecological preferences, which suggests (i) that molecular source-tracking approaches can be developed forListeriaand (ii) that detection of someListeriaspecies may not be a good indicator forL. monocytogenes.

Four isolates (FSL S4-120 T , FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-spore-forming bacilli that were phenotypically similar to species of the genus Listeria were isolated from soil, standing water and flowing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined to be the same species by whole genome DNA-DNA hybridization studies (.82 % relatedness at 55 6C and .76 % relatedness at 70 6C with 0.0-0.5 % divergence). 16S rRNA gene sequence analysis confirmed their close phylogenetic relatedness to Listeria monocytogenes and Listeria innocua and more distant relatedness to Listeria welshimeri, L. seeligeri, L. ivanovii and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs showed that these isolates form a wellsupported sistergroup to L. monocytogenes. The four isolates were sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to warrant their designation as a new species of the genus Listeria. The four isolates yielded positive reactions in the AccuProbe test that is purported to be specific for L. monocytogenes, did not ferment L-rhamnose, were nonhaemolytic on blood agar media, and did not contain a homologue of the L. monocytogenes virulence gene island. On the basis of their phenotypic characteristics and their genotypic distinctiveness from L. monocytogenes and L. innocua, the four isolates should be classified as a new species within the genus Listeria, for which the name Listeria marthii sp. nov. is proposed. The type strain of L. marthii is FSL S4-120 T (5ATCC BAA-1595 T 5BEIR NR 9579 T 5CCUG 56148 T ). L. marthii has not been associated with human or animal disease at this time.

We used molecular subtyping to investigate an outbreak of listeriosis involving residents of 24 US states. We defined a case as infection with Listeria monocytogenes serotype 4b yielding one of several closely related patterns when subtyped by pulsed-field gel electrophoresis. Patients infected with strains yielding different patterns were used as controls. A total of 108 cases were identified with 14 associated deaths and four miscarriages or stillbirths. A case-control study implicated meat frankfurters as the likely source of infection (OR 17.3, 95% CI 2.4-160). The outbreak ended abruptly following a manufacturer-issued recall, and the outbreak strain was later detected in low levels in the recalled product. A second strain was recovered at higher levels but was not associated with human illness. Our findings suggest that L. monocytogenes strains vary widely in virulence and confirm that large outbreaks can occur even when only low levels of contamination are detected in sampled food. Standardized molecular subtyping and coordinated, multi-jurisdiction investigations can greatly facilitate detection and control of listeriosis outbreaks.

Characterization of 80 Listeria monocytogenes isolates from urban and natural environments differentiated 7 and 26 EcoRI ribotypes, respectively. Whereas the majority of isolates from the natural environment represented L. monocytogenes lineage II (12 of 13 isolates), urban isolates grouped evenly into lineages I and II (32 and 33 isolates, respectively) and included two lineage III isolates. Multilocus sequence typing of all natural isolates and a randomly selected subset of 30 urban isolates showed a higher overall diversity (Simpson index of discrimination [D] of 0.987 and 0.920, respectively) than did EcoRI ribotyping (D = 0.872 and 0.911, respectively). Combined analysis with ribotype and lineage data for 414 isolates from farm sources, 165 isolates from foods and food-processing environments, and 342 human clinical isolates revealed that lineage I was significantly more common among human (P < 0.0001) isolates, whereas lineage II was more common among isolates from the natural environment, farms, and foods (P < or = 0.05). Among a total of 92 ribotypes, 31 showed significant associations with specific isolate sources. One ribotype (DUP-1039C) was significantly associated with both natural environments and farms. A spatial analysis showed a marginal association between locations in the natural environment positive for L. monocytogenes and a proximity to farms. Our data indicate that (i) L. monocytogenes strains from different sources show a high level of diversity; (ii) L. monocytogenes subtypes differ significantly in their associations with different environments, even though populations overlap; and (iii) a higher proportion of isolates from environmental sources than from human clinical cases can be classified into L. monocytogenes lineage II, which supports the classification of this lineage as an environmentally adapted subgroup.