Sampling of agricultural and natural environments in two US states (Colorado and Florida) yielded 18 Listeria-like isolates that could not be assigned to previously described species using traditional methods. Using whole-genome sequencing and traditional phenotypic methods, we identified five novel species, each with a genome-wide average blast nucleotide identity (ANIb) of less than 85 % to currently described species. Phylogenetic analysis based on 16S rRNA gene sequences and amino acid sequences of 31 conserved loci showed the existence of four well-supported clades within the genus Listeria ; (i) a clade representing Listeria monocytogenes , L. marthii , L. innocua , L. welshimeri , L. seeligeri and L. ivanovii , which we refer to as Listeria sensu stricto, (ii) a clade consisting of Listeria fleischmannii and two newly described species, Listeria aquatica sp. nov. (type strain FSL S10-1188T = DSM 26686T = LMG 28120T = BEI NR-42633T) and Listeria floridensis sp. nov. (type strain FSL S10-1187T = DSM 26687T = LMG 28121T = BEI NR-42632T), (iii) a clade consisting of Listeria rocourtiae , L. weihenstephanensis and three novel species, Listeria cornellensis sp. nov. (type strain TTU A1-0210T = FSL F6-0969T = DSM 26689T = LMG 28123T = BEI NR-42630T), Listeria grandensis sp. nov. (type strain TTU A1-0212T = FSL F6-0971T = DSM 26688T = LMG 28122T = BEI NR-42631T) and Listeria riparia sp. nov. (type strain FSL S10-1204T = DSM 26685T = LMG 28119T = BEI NR- 42634T) and (iv) a clade containing Listeria grayi . Genomic and phenotypic data suggest that the novel species are non-pathogenic.

Human norovirus (HuNoV) represents a significant public health burden worldwide and can be environmentally transmitted. Copper surfaces have been shown to inactivate the cultivable surrogate murine norovirus, but no such data exist for HuNoV. The purpose of this study was to characterize the destruction of GII.4 HuNoV and virus-like particles (VLPs) during exposure to copper alloy surfaces. Fecal suspensions positive for a GII.4 HuNoV outbreak strain or GII.4 VLPs were exposed to copper alloys or stainless steel for 0 to 240 min and recovered by elution. HuNoV genome integrity was assessed by reverse transcriptionquantitative PCR (RT-qPCR) (without RNase treatment), and capsid integrity was assessed by RT-qPCR (with RNase treatment), transmission electron microscopy (TEM), SDS-PAGE/Western blot analysis, and a histo-blood group antigen (HBGA) binding assay. Exposure of fecal suspensions to pure copper for 60 min reduced the GII.4 HuNoV RNA copy number by ϳ3 log 10 units when analyzed by RT-qPCR without RNase treatment and by 4 log 10 units when a prior RNase treatment was used. The rate of reduction of the HuNoV RNA copy number was approximately proportional to the percentage of copper in each alloy. Exposure of GII.4 HuNoV VLPs to pure-copper surfaces resulted in noticeable aggregation and destruction within 240 min, an 80% reduction in the VP1 major capsid protein band intensity in 15 min, and a near-complete loss of HBGA receptor binding within 8 min.In all experiments, HuNoV remained stable on stainless steel. These results suggest that copper surfaces destroy HuNoV and may be useful in preventing environmental transmission of the virus in at-risk settings.H uman norovirus (HuNoV) is the leading cause of viral gastroenteritis worldwide (1, 2). In the United States alone, the virus is responsible for 19 million to 21 million illnesses annually, contributing to nearly $500 million in hospital-associated costs each year (3, 4). The illness is characterized by nausea, vomiting, and diarrhea and typically lasts 24 to 48 h. While the disease is usually self-limiting, in individuals belonging to sensitive populations, symptoms can become life threatening if rehydration therapy is ignored.HuNoV transmission occurs via the fecal-oral route, usually through ingestion of contaminated food or water, or by direct contact with an infected individual. Environmental transmission also occurs, and episodes of vomiting or diarrhea can contaminate surfaces with infectious virus particles that may persist for weeks (5). Environmental persistence of HuNoV is enhanced by its resistance to a variety of commonly used sanitizers and disinfectants, for example, alcohol-based hand sanitizers and hypochlorite at regulated concentrations (6, 7). These unique traits of HuNoV, combined with the low infectious dose and the fact that infected individuals shed a large amount of virus in both fecal material and vomitus, contribute to the high number of outbreaks observed annually in environments with close quarters such as cruise ships, restau...

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cIn Listeria monocytogenes, 18 mutations leading to premature stop codons (PMSCs) in the virulence gene inlA have been identified to date. While most of these mutations represent nucleotide substitutions, a frameshift deletion in a 5= seven-adenine homopolymeric tract (HT) in inlA has also been reported. This HT may play a role in phase variation and was first identified among L. monocytogenes lineage II ribotype DUP-1039C isolates. In order to better understand the distribution of different inlA mutations in this ribotype, a newly developed multiplex real-time PCR assay was used to screen 368 DUP-1039C isolates from human, animal, and food-associated sources for three known 5= inlA HT alleles: (i) wild-type (WT) (A 7 ), (ii) frameshift (FS) (A 6 ), and (iii) guanine interruption (A 2 GA 4 ) alleles. Additionally, 228 DUP-1039C isolates were screened for all inlA PMSCs; data on the presence of all inlA PMSCs for the other 140 isolates were obtained from previous studies. The statistical analysis based on 191 epidemiologically unrelated strains showed that strains with inlA PMSC mutations (n ‫؍‬ 41) were overrepresented among food-associated isolates, while strains encoding full-length InlA (n ‫؍‬ 150) were overrepresented among isolates from farm animals and their environments. Furthermore, the A 6 allele was overrepresented and the A 7 allele was underrepresented among food isolates, while the A 6 allele was underrepresented among farm and animal isolates. Our results indicate that genetic variation in inlA contributes to niche adaptation within the lineage II subtype DUP-1039C. Listeria monocytogenes is a food-borne pathogen and the etiological agent of listeriosis, a severe invasive disease that can affect both humans and animals (1). More than 99% of human listeriosis cases are estimated to be transmitted through food (2). Despite its presence in a wide range of environments and foods, the majority of human listeriosis infections appear to be linked to consumption of contaminated ready-to-eat (RTE) foods (3) that support L. monocytogenes growth. Numerous studies have indicated that not all L. monocytogenes strains are equally associated with invasive disease. For example, McLauchlin reported that three (1/2a, 1/2b, and 4b) of the 13 serotypes of L. monocytogenes were responsible for 90% of 1,363 listeriosis cases from the United Kingdom (4). Additionally, multiple studies using both DNA band-based and sequence-based subtyping methods have shown that L. monocytogenes forms a structured population composed of at least four divergent lineages (I, II, III, and IV), which in a number of studies have been suggested to differ in their associations with different sources and in their pathogenic potentials (5-9). The majority of L. monocytogenes isolates belong to lineages I and II, which contain the serotypes most commonly associated with human clinical cases; serotypes 1/2b and 4b group into lineage I, while serotypes1/2a and 1/2c group into lineage II. Lineage III and IV strains are rare and usually isolated from ...

Twenty Listeria -like isolates were obtained from environmental samples collected on a cattle ranch in northern Colorado; all of these isolates were found to share an identical partial sigB sequence, suggesting close relatedness. The isolates were similar to members of the genus Listeria in that they were Gram-stain-positive, short rods, oxidase-negative and catalase-positive; the isolates were similar to Listeria fleischmannii because they were non-motile at 25 °C. 16S rRNA gene sequencing for representative isolates and whole genome sequencing for one isolate was performed. The genome of the type strain of Listeria fleischmannii (strain LU2006-1T) was also sequenced. The draft genomes were very similar in size and the average MUMmer nucleotide identity across 91 % of the genomes was 95.16 %. Genome sequence data were used to design primers for a six-gene multi-locus sequence analysis (MLSA) scheme. Phylogenies based on (i) the near-complete 16S rRNA gene, (ii) 31 core genes and (iii) six housekeeping genes illustrated the close relationship of these Listeria -like isolates to Listeria fleischmannii LU2006-1T. Sufficient genetic divergence of the Listeria -like isolates from the type strain of Listeria fleischmannii and differing phenotypic characteristics warrant these isolates to be classified as members of a distinct infraspecific taxon, for which the name Listeria fleischmannii subsp. coloradonensis subsp. nov. is proposed. The type strain is TTU M1-001T ( = BAA-2414T = DSM 25391T). The isolates of Listeria fleischmannii subsp. coloradonensis subsp. nov. differ from the nominate subspecies by the inability to utilize melezitose, turanose and sucrose, and the ability to utilize inositol. The results also demonstrate the utility of whole genome sequencing to facilitate identification of novel taxa within a well-described genus. The genomes of both subspecies of Listeria fleischmannii contained putative enhancin genes; the Listeria fleischmannii subsp. coloradonensis subsp. nov. genome also encoded a putative mosquitocidal toxin. The presence of these genes suggests possible adaptation to an insect host, and further studies are needed to probe niche adaptation of Listeria fleischmannii .