Molecular detection and quantification of viable probiotic strains in animal feedstuffs using the commercial direct fed microbial Lactobacillus animalis NP51 as a model.

Lactobacillus animalis NP51 is a direct-fed microbial strain (DFM) extensively used as a pre-harvest food safety mitigation in feedlot cattle due to its antagonistic effects against human foodborne pathogens such as Salmonella and Escherichia coli O157:H7. NP51 not only promotes overall gut health but interferes with the ability of these pathogens to colonize the gastrointestinal tract of cattle. As a result, NP51 reduces fecal shedding of Salmonella and E. coli O157:H7 in cattle presented for harvest and the load of these pathogens that enter the human food chain. Cattle are administered a high dose (1 × 109 CFU/head/day) of NP51 to reduce fecal shedding of foodborne pathogens. Ensiled animal feedstuffs naturally contain a high load of lactic acid bacteria (LAB) and it is not possible to detect and quantify the level of a specific LAB strain (e.g., NP51) in this matrix using traditional microbiological culture. The purpose of this study was to develop a molecular method to detect and quantify viable populations of a specific LAB strain (e.g., NP51) in cattle feedstuffs. The NP51 whole genome sequence was aligned with closely related LAB clustering within the same well-supported clade in a LAB phylogeny derived from 30 conserved amino acid encoding sequence to identify orthologs. A sequence encoding recombinational DNA repair protein RecT was found to be unique to NP51 and used to design primers and a probe for molecular detection and quantification of NP51. The primers and probe were confirmed to be specific to NP51 in vitro. Total RNA was extracted from silage samples, including samples naturally inoculated in the field and control samples that were artificially spiked with a range of NP51 concentrations in the laboratory. Reverse-transcriptase quantitative real-time (RT-qRTi) PCR was used to quantify cDNA copies in samples and cycle threshold (Ct) values were compared to a standard curve to estimate NP51 concentrations. Our results indicate this novel molecular method is suitable to confirm the presence and estimate the concentration of a specific LAB strain in animal feedstuffs containing high background levels of LAB.

Molecular ecology of Listeria spp., Salmonella, Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in pristine natural environments in Northern Colorado.

AIMS: Molecular subtyping is commonly used in foodborne disease surveillance and microbial source tracking. There is a knowledge gap regarding the molecular ecology of foodborne pathogens in non-food-associated environments. The objective of this study was to isolate and subtype foodborne pathogens from pristine natural environments with minimal anthropogenic inputs. MATERIALS AND RESULTS: Five locations (wilderness areas) in Northern Colorado were sampled during the spring, summer and fall over a 2-year period. Soil, water, sediment, surface soil and wildlife faecal samples were microbiologically analysed to detect Listeria, Salmonella and Shiga toxin-producing Escherichia coli (STEC), and resultant isolates were subtyped. Three samples tested positive for Listeria monocytogenes and 19 samples contained other Listeria spp. Salmonella was isolated from two samples, five samples contained non-O157 STEC, and E. coli O157:H7 was not detected. Two L. monocytogenes isolates from faecal samples collected from the same wilderness area over a year apart shared the same PFGE pattern, while all other isolates had a unique type. CONCLUSIONS: Our data indicate that (i) there was a rare presence of human foodborne pathogens in pristine natural environments in Northern Colorado, (ii) there was genetic diversity between organisms isolated within a given wilderness area, and (iii) the Northern Colorado climate and topography may contribute to the low occurrence of these organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Relatively little is known about the molecular ecology of foodborne pathogens in pristine natural environments. While foodborne pathogens were rarely detected in wildlife faecal and environmental samples from the wilderness areas in this study, some isolates shared DNA fingerprint types with human clinical isolates from same region during the same time frame, highlighting the need for environmental isolate subtype data. The availability of molecular subtyping data for non-food-associated foodborne pathogen isolates can facilitate epidemiological and microbial source tracking investigations.

Applications and safety considerations of Lactobacillus salivarius as a probiotic in animal and human health.

The goals of this review are to summarize the current knowledge on the application of Lactobacillus salivarius as a probiotic in animals and humans, and to address safety concerns with its use on live hosts. Overall, several strains of L. salivarius are well established probiotics with multiple applications in animal health, particularly to reduce colonization by gastrointestinal pathogens, and to a lesser extent, as a production and quality aid. In humans, L. salivarius has been used to prevent and treat a variety of chronic diseases, including asthma, cancer, atopic dermatitis and halitosis, and to a much limited extent, to prevent or treat infections. Based on the results from primary research evidence, it seems that L. salivarius does not pose a health risk to animals or humans in the doses currently used for a variety of applications; however, there is a systematic lack of studies assuring the safety of many of the strains intended for clinical use. This review provides researchers in the field with up-to-date information regarding applications and safety of L. salivarius. Furthermore, it helps researchers identify knowledge gaps and potential opportunities for microbiological and clinical research.

Molecular detection assay of five Salmonella serotypes of public interest: Typhimurium, Enteritidis, Newport, Heidelberg, and Hadar.

Foodborne illnesses due to Salmonella represent an important public-health concern worldwide. In the United States, a majority of Salmonella infections are associated with a small number of serotypes. Furthermore, some serotypes that are overrepresented among human disease are also associated with multi-drug resistance phenotypes. Rapid detection of serotypes of public-health concern might help reduce the burden of salmonellosis cases and limit exposure to multi-drug resistant Salmonella. We developed a two-step real-time PCR-based rapid method for the identification and detection of five Salmonella serotypes that are either overrepresented in human disease or frequently associated with multi-drug resistance, including serotypes Enteritidis, Typhimurium, Newport, Hadar, and Heidelberg. Two sets of four markers were developed to detect and differentiate the five serotypes. The first set of markers was developed as a screening step to detect the five serotypes; whereas, the second set was used to further distinguish serotypes Heidelberg, Newport and Hadar. The utilization of these markers on a two-step investigation strategy provides a diagnostic specificity of 97% for the detection of Typhimurium, Enteritidis, Heidelberg, Infantis, Newport and Hadar. The diagnostic sensitivity of the detection makers is >96%. The availability of this two-step rapid method will facilitate specific detection of Salmonella serotypes that contribute to a significant proportion of human disease and carry antimicrobial resistance.

Interrogation of single nucleotide polymorphisms in gnd provides a novel method for molecular serogrouping of clinically important Shiga toxin producing Escherichia coli (STEC) targeted by regulation in the United States, including the "big six" non-O157 STEC and STEC O157.

Escherichia coli O157:H7 has frequently been associated with foodborne infections and is considered an adulterant in raw non-intact beef in the U.S. Shiga toxin-producing E. coli (STEC) belonging to serogroups O26, O45, O103, O111, O121, and O145 (known as the "big six" non-O157) were estimated to cause >70% of foodborne infections attributed to non-O157 serogroups in the U.S., as a result, these six serogroups have also been targeted by regulation in the U.S. The purpose of this study was to develop a rapid and high-throughput molecular method to group STEC isolates into seven clinically important serogroups (i.e., O157 and the "big six" non-O157 serogroups) targeted by regulation in the U.S. by interrogating single nucleotide polymorphisms (SNPs) in gnd. A collection of 195 STEC isolates, including isolates belonging to O157:H7 (n=18), O26 (n=21), O45 (n=19), O103 (n=24), O111 (n=24), O121 (n=23), O145 (n=21), and ten other STEC serogroups (n=45), was assembled and characterized by full gnd sequencing to identify informative SNPs for molecular serogrouping. A multiplex SNP typing assay was developed to interrogate twelve informative gnd SNPs by single base pair extension chemistry and used to characterize the STEC isolate collection assembled here. SNP types were assigned to each isolate by the assay and polymorphisms were confirmed with gnd sequence data. O-serogroup-specific SNP types were identified for each of the seven clinically important STEC serogroups, which allowed the differentiation of these seven STEC serogroups from other non-O157 STEC serogroups. Although serogroups of the "big six" non-O157 STEC and O157:H7 contained multiple SNP types per O-serogroup, there were no overlapping SNP types between serogroups. Our results demonstrate that molecular serogrouping of STEC isolates by interrogation of informative SNPs in gnd represents an alternative to traditional serogrouping by agglutination for rapid and high-throughput identification of clinically important STEC serogroups targeted by regulation for surveillance and epidemiological investigations.

Listeria monocytogenes Isolates Carrying Virulence-Attenuating Mutations in Internalin A Are Commonly Isolated from Ready-to-Eat Food Processing Plant and Retail Environments.

Listeria monocytogenes is a human foodborne pathogen that may cause an invasive disease known as listeriosis in susceptible individuals. Internalin A (InlA; encoded by inlA) is a virulence factor that facilitates crossing of host cell barriers by L. monocytogenes . At least 19 single nucleotide polymorphisms (SNPs) in inlA that result in a premature stop codon (PMSC) have been described worldwide. SNPs leading to a PMSC in inlA have been shown to be causally associated with attenuated virulence. L. monocytogenes pathogens carrying virulence-attenuating (VA) mutations in inlA have been commonly isolated from ready-to-eat (RTE) foods but rarely have been associated with human disease. This study was conducted to determine the prevalence of VA SNPs in inlA among L. monocytogenes from environments associated with RTE food production and handling. More than 700 L. monocytogenes isolates from RTE food processing plant (n = 409) and retail (n = 319) environments were screened for the presence of VA SNPs in inlA. Overall, 26.4% of isolates from RTE food processing plant and 32.6% of isolates from retail environments carried a VA mutation in inlA. Food contact surfaces sampled at retail establishments were significantly (P < 0.0001) more likely to be contaminated by a L. monocytogenes isolate carrying a VA mutation in inlA (56% of 55 isolates) compared with nonfood contact surfaces (28% of 264 isolates). Overall, a significant proportion of L. monocytogenes isolated from RTE food production and handling environments have reduced virulence. These data will be useful in the revision of current and the development of future risk assessments that incorporate strain-specific virulence parameters.

Evaluation of lactic acid as an initial and secondary subprimal intervention for Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli, and a nonpathogenic E. coli surrogate for E. coli O157:H7.

Lactic acid can reduce microbial contamination on beef carcass surfaces when used as a food safety intervention, but effectiveness when applied to the surface of chilled beef subprimal sections is not well documented. Studies characterizing bacterial reduction on subprimals after lactic acid treatment would be useful for validations of hazard analysis critical control point (HACCP) systems. The objective of this study was to validate initial use of lactic acid as a subprimal intervention during beef fabrication followed by a secondary application to vacuum-packaged product that was applied at industry operating parameters. Chilled beef subprimal sections (100 cm(2)) were either left uninoculated or were inoculated with 6 log CFU/cm(2) of a 5-strain mixture of Escherichia coli O157:H7, a 12-strain mixture of non-O157 Shiga toxin-producing E. coli (STEC), or a 5-strain mixture of nonpathogenic (biotype I) E. coli that are considered surrogates for E. coli O157:H7. Uninoculated and inoculated subprimal sections received only an initial or an initial and a second "rework" application of lactic acid in a custombuilt spray cabinet at 1 of 16 application parameters. After the initial spray, total inoculum counts were reduced from 6.0 log CFU/cm(2) to 3.6, 4.4, and 4.4 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. After the second (rework) application, total inoculum counts were 2.6, 3.2, and 3.6 log CFU/cm(2) for the E. coli surrogates, E. coli O157:H7, and non-O157 STEC inoculation groups, respectively. Both the initial and secondary lactic acid treatments effectively reduced counts of pathogenic and nonpathogenic strains of E. coli and natural microflora on beef subprimals. These data will be useful to the meat industry as part of the HACCP validation process.

High-throughput small molecule screening reveals structurally diverse compounds that inhibit the growth of Escherichia coli O157:H7 in vitro.

Escherichia coli O157:H7 colonizes the gastrointestinal tract of ruminants asymptomatically and may enter the human food supply through fecal contamination. A fraction of individuals infected by E. coli O157:H7 develop hemolytic uremic syndrome, a life-threatening condition. When individuals infected by E. coli O157:H7 are treated with certain antibiotics, an increased incidence of hemolytic uremic syndrome may result. This finding supports the need to identify novel compounds that can either reduce the load of E. coli O157:H7 entering the human food supply or serve as alternative therapeutic treatments for infected individuals. We developed a high-throughput turbidometric assay to identify novel compounds that inhibit E. coli O157:H7 growth. Pin transfers were performed to introduce small molecule libraries into 384-well plates, where each well contained approximately 5.0 log CFU of E. coli O157:H7. Plates were incubated at 37°C for 18 h, and the optical density was measured to determine the effect of each small molecule. A total of 64,562 compounds were screened in duplicate, and 43 unique compounds inhibited E. coli O157:H7 growth. Thirty-eight of the 43 inhibitory compounds belonged to known bioactive libraries, and the other 5 compounds were from commercial libraries derived from splitting and pooling. Inhibitory compounds from known bioactive libraries were most frequently therapeutic antibiotics (n = 34) but also included an antiviral compound, a compound that disrupts the citric acid cycle, and two biguanide compounds, which have been used for various nonclinical applications. We identified two novel compounds (i.e., biguanides) that should be studied further for their ability to reduce pathogen populations in foods.

Naturally colonized beef cattle populations fed combinations of yeast culture and an ionophore in finishing diets containing dried distiller's grains with solubles had similar fecal shedding of Escherichia coli O157:H7.

Beef steers (n = 252) were used to evaluate the effects of dietary supplement on fecal shedding of Escherichia coli O157:H7. Seven pens of 9 steers (63 steers per treatment) were fed diets supplemented with or without yeast culture (YC) or monensin (MON) and their combination (YC × MON). YC and MON were offered at 2.8 g/kg and 33 mg/kg of dry matter intake, respectively. Environmental sponge samples (from each pen floor, feed bunk, and water trough) were collected on day 0. Rectal fecal grab samples were collected on days 0, 28, 56, 84, 110, and 125. Samples were collected and pooled by pen and analyzed for presumptive E. coli O157:H7 colonies, which were confirmed by a multiplex PCR assay and characterized by pulsed-field gel electrophoresis (PFGE) typing. On day 0, E. coli O157:H7 was detected in 7.0% of feed bunk samples and 14.3% of pen floor samples but in none of the water trough samples. The 71.4% prevalence of E. coli O157:H7 in fecal samples on day 0 decreased significantly (P < 0.05) over time. E. coli O157:H7 fecal shedding was not associated with dietary treatment (P > 0.05); however, in cattle fed YC and YC × MON fecal shedding was 0% by day 28. Eight Xba I PFGE subtypes were identified, and a predominant subtype and three closely related subtypes (differing by three or fewer bands) accounted for 78.7% of environmental and fecal isolates characterized. Results from this study indicate that feeding YC to cattle may numerically decrease but not eliminate fecal shedding of E. coli O157:H7 at the onset of treatment and that certain E. coli O157 subtypes found in the feedlot environment may persist in feedlot cattle.

Significant shift in median guinea pig infectious dose shown by an outbreak-associated Listeria monocytogenes epidemic clone strain and a strain carrying a premature stop codon mutation in inlA.

Listeria monocytogenes contains (i) epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States, and (ii) strains commonly isolated from ready-to-eat foods that carry a mutation leading to a premature stop codon (PMSC) in inlA, which encodes the key virulence factor internalin A (InlA). Internalin A binds certain isoforms of the cellular receptor E-cadherin to facilitate crossing the intestinal barrier during the initial stages of an L. monocytogenes infection. Juvenile guinea pigs, which express the human isoform of E-cadherin that binds InlA, were intragastrically challenged with a range of doses of (i) an EC strain associated with a listeriosis outbreak or (ii) a strain carrying a PMSC mutation in inlA. Recovery of L. monocytogenes from tissues (i.e., liver, spleen, mesenteric lymph nodes, and ileum) was used to develop strain-specific dose-response curves on the basis of individual and combined organ data. Modeling of individual and combined organ data revealed an approximate 1.2 to 1.3 log(10) increase in the median infectious dose for the strain carrying a PMSC in inlA relative to that for the EC strain. Inclusion of the strain parameter significantly improved the goodness of fit for individual and combined organ models, indicating a significant shift in median infectious dose for guinea pigs challenged with an inlA PMSC strain compared to that for guinea pigs challenged with an EC strain. Results from this work provide evidence that the L. monocytogenes dose-response relationship is strain specific and will provide critical data for enhancement of current risk assessments and development of future risk assessments.

Revelation by single-nucleotide polymorphism genotyping that mutations leading to a premature stop codon in inlA are common among Listeria monocytogenes isolates from ready-to-eat foods but not human listeriosis cases.

Listeria monocytogenes utilizes internalin A (InlA; encoded by inlA) to cross the intestinal barrier to establish a systemic infection. Multiple naturally occurring mutations leading to a premature stop codon (PMSC) in inlA have been reported worldwide, and these mutations are causally associated with attenuated virulence. Five inlA PMSC mutations recently discovered among isolates from France and the United States were included as additional markers in our previously described inlA single-nucleotide polymorphism (SNP) genotyping assay. This assay was used to screen >1,000 L. monocytogenes isolates from ready-to-eat (RTE) foods (n = 502) and human listeriosis cases (n = 507) for 18 inlA PMSC mutations. A significantly (P < 0.0001) greater proportion of RTE food isolates (45.0%) carried a PMSC mutation in inlA compared to human clinical isolates (5.1%). The proportion of L. monocytogenes with or without PMSC mutations in inlA was similar among isolates from different RTE food categories except for deli meats, which included a marginally higher proportion (P = 0.12) of isolates carrying a PMSC in inlA. We also analyzed the distribution of epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States. We observed a significant (P < 0.05) overrepresentation of EC strains in deli and seafood salads and a significant (P < 0.05) underrepresentation of EC strains in smoked seafood. These results provide important data to predict the human health risk of exposure to L. monocytogenes strains that differ in pathogenic potential through consumption of contaminated RTE foods.

Some Listeria monocytogenes outbreak strains demonstrate significantly reduced invasion, inlA transcript levels, and swarming motility in vitro.

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 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.

Development and implementation of a multiplex single-nucleotide polymorphism genotyping assay for detection of virulence-attenuating mutations in the Listeria monocytogenes virulence-associated gene inlA.

The virulence factor internalin A (InlA) facilitates the uptake of Listeria monocytogenes by epithelial cells that express the human isoform of E-cadherin. Previous studies identified naturally occurring premature stop codon (PMSC) mutations in inlA and demonstrated that these mutations are responsible for virulence attenuation. We assembled >1,700 L. monocytogenes isolates from diverse sources representing 90 EcoRI ribotypes. A subset of this isolate collection was selected based on ribotype frequency and characterized by a Caco-2 cell invasion assay. The sequencing of inlA genes from isolates with attenuated invasion capacities revealed three novel inlA PMSCs which had not been identified previously among U.S. isolates. Since ribotypes include isolates with and without inlA PMSCs, we developed a multiplex single-nucleotide polymorphism (SNP) genotyping assay to detect isolates with virulence-attenuating PMSC mutations in inlA. The SNP genotyping assay detects all inlA PMSC mutations that have been reported worldwide and verified in this study to date by the extension of unlabeled primers with fluorescently labeled dideoxynucleoside triphosphates. We implemented the SNP genotyping assay to characterize human clinical and food isolates representing common ribotypes associated with novel inlA PMSC mutations. PMSCs in inlA were significantly (ribotypes DUP-1039C and DUP-1045B; P < 0.001) or marginally (ribotype DUP-1062D; P = 0.11) more common among food isolates than human clinical isolates. SNP genotyping revealed a fourth novel PMSC mutation among U.S. L. monocytogenes isolates, which was observed previously among isolates from France and Portugal. This SNP genotyping assay may be implemented by regulatory agencies and the food industry to differentiate L. monocytogenes isolates carrying virulence-attenuating PMSC mutations in inlA from strains representing the most significant health risk.

inlA premature stop codons are common among Listeria monocytogenes isolates from foods and yield virulence-attenuated strains that confer protection against fully virulent strains.

Previous studies showed that a considerable proportion of Listeria monocytogenes isolates obtained from foods carry a premature stop codon (PMSC) mutation in inlA that leads to production of a truncated and secreted InlA. To further elucidate the role these mutations play in virulence of L. monocytogenes, we created isogenic mutants, including (i) natural isolates where an inlA PMSC was reverted to a wild-type inlA allele (without a PMSC) and (ii) natural isolates where a PMSC mutation was introduced into a wild-type inlA allele; isogenic mutant sets were constructed to represent two distinct inlA PMSC mutations. Phenotypical and transcriptional analysis data showed that inlA PMSC mutations do not have a polar effect on the downstream inlB. Isogenic and natural strains carrying an inlA PMSC showed significantly reduced invasion efficiencies in Caco-2 and HepG2 cell lines as well as reduced virulence in oral guinea pig infections. Guinea pigs were also orally infected with a natural strain carrying the most common inlA PMSC mutation (vaccinated group), followed by challenge with a fully virulent L. monocytogenes strain 15 days postvaccination to probe potentially immunizing effects of exposure to L. monocytogenes with inlA PMSC mutations. Vaccinated guinea pigs showed reduced bacterial loads in internal organs and improved weight gain postchallenge, indicating reduced severity of infections in guinea pigs exposed to natural strains with inlA PMSC mutations. Our data support that (i) inlA PMSC mutations are causally associated with attenuated virulence in mammalian hosts and (ii) naturally occurring virulence-attenuated L. monocytogenes strains commonly found in food confer protective immunity.

Central nervous system tissue in meat products: an evaluation of risk, prevention strategies, and testing procedures.

Since the outbreak of bovine spongiform encephalopathy (BSE) in the United Kingdom in 1986 and its subsequent link to the human neurological disorder variant Creutzfeldt-Jakob disease (vCJD), presence of tissues from the central nervous system (CNS) in meat products has been considered a public health concern and, thus, has been banned from entering the human food chain in many countries. Despite this, potential can exist during harvesting to contaminate or cross-contaminate edible meat products with CNS tissue that is designated as a specified risk material (SRM) in many countries. Methods used to detect CNS tissue in meat products vary greatly in their sensitivity, specificity, cost, labor and expertise needed, ease of completion, and type of results given (qualitative vs quantitative) and, within these constraints, appropriate testing methods must be selected to monitor or verify that meat products system controls are effective in removing CNS tissue from the human food chain. The extent to which monitoring procedures are needed should be based on the public health risk of CNS tissue in meat products as determined by each sovereign nation and/or third-party international organizations such as the World Organization for Animal Health (OIE). Risk associated with consumption of CNS tissue should be estimated by sovereign nations by establishing prevalence of BSE within their borders. Using this information, science-based decisions may guide international policy and trade. Using available scientific information, appropriate testing methods for monitoring or verification, and prevalence information, nations can estimate and reduce, to the extent deemed necessary, the public health risk of vCJD.

Recombination and positive selection contribute to evolution of Listeria monocytogenes inlA.

The surface molecule InlA interacts with E-cadherin to promote invasion of Listeria monocytogenes into selected host cells. DNA sequencing of inlA for 40 L. monocytogenes isolates revealed 107 synonymous and 45 nonsynonymous substitutions. A frameshift mutation in a homopolymeric tract encoding part of the InlA signal peptide was identified in three lineage II isolates, which also showed reduced ability to invade human intestinal epithelial cells. Phylogenies showed clear separation of inlA sequences into lineages I and II. Thirteen inlA recombination events, predominantly involving lineage II strains as recipients (12 events), were detected and a number of amino acid residues were shown to be under positive selection. Four of the 45 non-synonymous changes were found to be under positive selection with posterior probabilities >95 %. Mapping of polymorphic and positively selected amino acid sites on the partial crystal structure for InlA showed that the internalin surface of the leucine-rich repeat (LRR) region that faces the InlA receptor E-cadherin does not include any polymorphic sites; all polymorphic and positively selected amino acids mapped to the outer face of the LRR region or to other InlA regions. The data show that (i) inlA is highly polymorphic and evolution of inlA involved a considerable number of recombination events in lineage II isolates; (ii) positive selection at specific amino acid sites appears to contribute to evolution of inlA, including fixation of recombinant events; and (iii) single-nucleotide deletions in a lineage II-specific 3' homopolymeric tract in inlA lead to complete loss of InlA or to production of truncated InlA, which conveys reduced invasiveness. In conclusion, inlA has a complex evolutionary history, which is consistent with L. monocytogenes' natural history as an environmental pathogen with broad host-range, including its adaptation to environments and hosts where different inlA alleles may provide a selective advantage or where inlA may not be required.

Listeria monocytogenes fecal shedding in dairy cattle shows high levels of day-to-day variation and includes outbreaks and sporadic cases of shedding of specific L. monocytogenes subtypes.

Fecal shedding of Listeria monocytogenes poses a risk for contamination of animal feed and agricultural environments and raw food at the pre-harvest stages of food production. To be able to reduce these risks it is critical to improve understanding of the epidemiology of L. monocytogenes shedding in feces. The objective of this study was to assess the daily variability of fecal shedding and its association with individual animal (lactation number and the day of current lactation) and environmental (feed) risk factors. That was achieved by application of longitudinal daily sample collection in a herd of dairy cattle and molecular characterization of isolated L. monocytogenes. Fecal samples (25) and silage samples (2) were collected daily during two 2-week periods and one 5-day period. L. monocytogenes was isolated from 255 out of 825 (31%) fecal samples on 24 out of 33 (73%) days, and from 25 out of 66 (38%) silage samples on 16 out of 33 (48%) days. Ninety-four percent of cows excreted L. monocytogenes in feces at least once during the study period. Our data analyses indicated that (i) the prevalence and incidence risk of L. monocytogenes fecal shedding in cattle vary considerably over time, from 0 to 100%, and both are associated with contamination of silage, (ii) L. monocytogenes fecal shedding in cattle could occur as part of an outbreak or as an isolated sporadic case, (iii) L. monocytogenes subtypes associated with human infections are commonly isolated from cattle feces and silage, and (iv) a single cow can harbor more than one L. monocytogenes subtype on any given day. Although limited to a single dairy cattle herd, these findings provide a significant advancement in the understanding of the epidemiology of L. monocytogenes fecal shedding in dairy cattle.

Listeria monocytogenes F2365 carries several authentic mutations potentially leading to truncated gene products, including inlB, and demonstrates atypical phenotypic characteristics.

Searches of the genome annotation of Listeria monocytogenes F2365, an isolate from the 1985 listeriosis epidemic in California, showed that this strain carries 20 authentic mutations resulting in premature stop codons, including a nonsense mutation in inlB. Here we showed that L. monocytogenes F2365 demonstrates atypical virulence-associated characteristics, including significantly (P < 0.05) reduced invasion efficiency in Caco-2 cells as compared with a closely related lineage I serotype 4b strain as well as significantly (P < 0.05) greater variation in invasiveness when grown under different conditions compared with standard laboratory control and other lineage I serotype 4b strains. In addition, L. monocytogenes F2365 demonstrated distinct growth characteristics, including a significantly (P < 0.05) reduced exponential growth rate when compared with laboratory control and other lineage I serotype 4b outbreak-associated strains as well as a significantly (P < 0.05) longer lag phase duration time compared with another lineage I serotype 4b strain. Our results support that L. monocytogenes F2365 is characterized by genotypic and phenotypic properties that are atypical of other L. monocytogenes strains.

Novel method to identify source-associated phylogenetic clustering shows that Listeria monocytogenes includes niche-adapted clonal groups with distinct ecological preferences.

While phylogenetic and cluster analyses are often used to define clonal groups within bacterial species, the identification of clonal groups that are associated with specific ecological niches or host species remains a challenge. We used Listeria monocytogenes, which causes invasive disease in humans and different animal species and which can be isolated from a number of environments including food, as a model organism to develop and implement a two-step statistical approach to the identification of phylogenetic clades that are significantly associated with different source populations, including humans, animals, and food. If the null hypothesis that the genetic distances for isolates within and between source populations are identical can be rejected (SourceCluster test), then particular clades in the phylogenetic tree with significant overrepresentation of sequences from a given source population are identified (TreeStats test). Analysis of sequence data for 120 L. monocytogenes isolates revealed evidence of clustering between isolates from the same source, based on the phylogenies inferred from actA and inlA (P = 0.02 and P = 0.07, respectively; SourceCluster test). Overall, the TreeStats test identified 10 clades with significant (P < 0.05) or marginally significant (P < 0.10) associations with defined sources, including human-, animal-, and food-associated clusters. Epidemiological and virulence phenotype data supported the fact that the source-associated clonal groups identified here are biologically valid. Overall, our data show that (i) the SourceCluster and TreeStats tests can identify biologically meaningful source-associated phylogenetic clusters and (ii) L. monocytogenes includes clonal groups that have adapted to infect specific host species or colonize nonhost environments.

Validation of a traditional Italian-style salami manufacturing process for control of Salmonella and Listeria monocytogenes.

Italian-style salami batter (formulated with pork shoulder) was inoculated with ca. 7.0 log CFU/g of either Salmonella or Listeria monocytogenes. Salami links (55-mm cellulose casings) were fermented at 30 degrees C for 24, 40, or 72 h and then dried to target moisture/protein ratios (MPRs) of 1.9:1 or 1.4:1. Links were sampled after fermentation (24, 40, and 72 h) and after combined fermentation-drying treatments (MPRs of 1.9:1 and 1.4:1 for all fermentation periods), and microbiological and proximate analyses were performed at each sampling. Pathogen populations were enumerated by direct plating on selective agar and by an injured-cell recovery method. When enumerated by the injured-cell recovery method, Salmonella populations were reduced by 1.2 to 2.1 log CFU/g after fermentation alone (24 to 72 h) and by 2.4 to 3.4 log CFU/g when fermentation was followed by drying. Drying to an MPR of 1.4:1 was no more effective than drying to an MPR of 1.9:1 (P > 0.05). When enumerated directly on selective media, Salmonella populations were reduced from 1.6 to 2.4 log CFU/g and from 3.6 to 4.5 log CFU/g for fermentation alone and fermentation followed by drying, respectively. L. monocytogenes populations were reduced by <1.0 log CFU/g following all fermentation and combined fermentation-drying treatments, regardless of the enumeration method. These results suggest that the Italian-style salami manufacturing process evaluated does not adequately reduce high pathogen loads. Processors may thus need to consider supplemental measures, such as raw material specifications and a final heating step, to enhance the lethality of the overall manufacturing process.