Multi-virulence-locus Sequence Typing Research Articles

Frozen Vegetable Processing Plants Can Harbour Diverse Listeria monocytogenes Populations: Identification of Critical Operations by WGS

Frozen vegetables have emerged as a concern due to their association with foodborne outbreaks such as the multi-country outbreak of Listeria monocytogenes serogroup IVb linked to frozen corn. The capacity of L. monocytogenes to colonize food-processing environments is well-known, making the bacteria a real problem for consumers. However, the significance of the processing environment in the contamination of frozen foods is not well established. This study aimed to identify potential contamination niches of L. monocytogenes in a frozen processing plant and characterize the recovered isolates. A frozen vegetable processing plant was monitored before cleaning activities. A total of 78 points were sampled, including frozen vegetables. Environmental samples belonged to food-contact surfaces (FCS); and non-food-contact surfaces (n-FCS). Positive L. monocytogenes samples were found in FCS (n = 4), n-FCS (n = 9), and the final product (n = 1). A whole-genome sequencing (WGS) analysis revealed two clusters belonging to serotypes 1/2a-3a and 1/2b-3b). The genetic characterization revealed the presence of four different sequence types previously detected in the food industry. The isolate obtained from the final product was the same as one isolate found in n-FCS. A multi-virulence-locus sequence typing (MVLST) analysis showed four different virulence types (VT). The results obtained highlight the relevant role that n-FCS such as floors and drains can play in spreading L. monocytogenes contamination to the final product.

MLST/MVLST Analysis and Antibiotic Resistance of Vibrio cholerae in Shandong Province of China.

Background:Vibrio cholerae is an important bacterium causing profuse watery diarrhea. Cholera had swept the whole Shandong province from 1975 to 2013.Methods:From epidemiological data and pulsed-field gel electrophoresis data, we selected 86 V. cholerae isolates appearing in Shandong Province in China from 1975 to 2013 and characterized them by multilocus sequence typing (MLST)/multi-virulence locus sequence typing (MVLST), antibiogram and analysis of genes related to antibiotic resistance.Results:Combined MLST/MVLST data revealed 33 sequence types and a major group. Within the group, 3 subgroups (ST1, ST24 and ST29) were revealed, prevalent in the strains isolated during the 1980s, 1990s and 21st century, respectively. All the O1 isolates after 1990 were found to be El Tor variants harboring the classical ctxB gene. The tcpA gene of O139 strains had a mutation at amino acid position 62 (N→D). Antibiotic resistance of V. cholerae increased over time. Most El Tor variants between 1998 and 1999 were resistant to trimethoprim/sulfamethoxazole. The O139 strain, since its appearance in 1997, had significantly broader spectrum of antibiotic resistance than O1 variants. The presence of the SXT element corresponds to the trend of growing drug resistance.Conclusion:The analysis of genotypic polymorphism and enhanced resistance of V. cholerae indicated continuous variation and evolution of this pathogenic agent in Shandong Province.

Analysis of Genetic Diversity and Antibiotic Options for Clinical Listeria monocytogenes Infections in China.

BackgroundThe aim of this study was to investigate the mechanism of Listeria monocytogenes (Lm) pathogenicity and resistance. In addition, the effect of existing treatment options against Lm were systematically evaluated.MethodsSix Lm isolates were collected and antimicrobial susceptibility testing of 15 antibiotics were done. Subsequently, whole genome sequencing and bioinformatics analysis were performed. Biofilm formation was evaluated by crystal violet staining. Furthermore, the effect of meropenem, linezolid, penicillin, vancomycin, and trimethoprim/sulfamethoxazole were determined using the time-kill assay.ResultsFour sequence types (STs) were identified (ST1, ST3, ST87, ST451). Multivirulence-locus sequence typing results classified ST87 isolates into cluster. All isolates were resistant to fosfomycin and daptomycin with fosX and mprF. In addition, a total of 80 virulence genes were detected and 72 genes were found in all 6 isolates. Seven genes associated with hemolysin were found in 26530 and 115423. However, due to lack of one genomic island including virulence genes related to flagellar synthesis, isolate 115423 produced less biofilm than 5 other isolates. Although all isolates were susceptible to vancomycin, the in vitro time-kill assay showed that vancomycin monotherapy resulted in less than 2 log10 cerebrospinal fluid (CFU)/mL compared with the initial count. Trimethoprim/sulfamethoxazole at serum or CFU concentrations had bactericidal effect against tested Lm strains at 24 hours.ConclusionsST87 clone was a typical prevalent ST in clinical Lm isolates in China. Trimethoprim/sulfamethoxazole might be greater potential therapeutic option against Lm infections.

Genetic diversity of Listeria monocytogenes serotype 1/2a strains collected in Brazil by Multi-Virulence-Locus Sequence Typing.

Listeria monocytogenes is an opportunistic pathogen with the ability to adapt to different environmental conditions, resulting in safety issues for food producers. Foods contaminated by L. monocytogenes can represent a risk if consumed by susceptible individuals such as elderly, pregnant women and the immunocompromised. The aim of this study was to evaluate the genetic diversity of a collection of L. monocytogenes isolated from different matrices in Brazil during the period of 1979-2015. A total of 51 L. monocytogenes serotype 1/2a strains isolated from clinical samples (n=3) and food samples (n=48) were characterized by Multi-Virulence-Locus Sequence Typing (MVLST). The strains were assigned to nine virulence types (VT): VT-11 (n=3, 5·9%), VT-45 (n=27, 52·9%), VT-59 (n=11, 21·6%), VT-68 (n=3, 5·9%), VT-94 (n=2, 3·9%), VT-107 (n=2, 3·9%), VT-184 (n=1, 1·9%), VT-185 (n=1, 1·9%) and VT-186 (n=1, 1·9%); and four of them (VT-11, VT-45, VT-59 and VT-68) have already been associated with cases of listeriosis worldwide. The VT-11, VT-59 (Epidemic Clone V) and VT-186 were identified in blood culture samples, as well as in different classes of foods. It is recommended that the epidemiological surveillance agencies evaluate the risk that foods contaminated with L. monocytogenes VTs pose to susceptible populations.

Characterization of Virulence and Persistence Abilities of Listeria monocytogenes Strains Isolated from Food Processing Premises

We report the characterization of 15 Listeria monocytogenes strains isolated from various food processing plants by multivirulence locus sequence typing to determine virulence types (VTs) and epidemic clones. Molecular mechanisms involved in adaptation to food processing environments and related to virulence were also studied. Phenotypic behaviors associated with various antimicrobials, biofilm formations, and invasiveness were assessed. There were 11 VTs among the 15 L. monocytogenes strains. Strains belonging to six VTs were stress survival islet 1 (SSI-1) and one strain of VT94 was SSI-2. Tn6188 was found in VT6 and VT94 strains, and bcrABC cassette genes were identified in VT21, VT60, and VT63 strains. Only one strain, in VT20, showed llxS, whereas a full-size inlA was detected in strains belonging to VT8, VT20, VT21, and VT63. VT10, VT20, VT21, VT60, and VT63 strains were the most tolerant to studied disinfectants. A VT6 strain showed the strongest biofilm formation ability in polyvinyl chloride, and strains belonging to VT10, VT11, VT20, and VT94 had moderate abilities. Antimicrobial sensitivity tests showed that all the L. monocytogenes strains were multidrug resistant. F tests revealed that only strains of VT10, VT60, and VT94 were significantly noninvasive (P < 0.05) in Caco-2 cells. Our findings illustrate how L. monocytogenes isolates exploit diverse mechanisms to adapt to adverse conditions. Consequently, detailed characterization of L. monocytogenes isolates is required for comprehensive elimination of this pathogenic bacterium in food processing environments.

Retrospective Study of Listeria Monocytogenes Isolated in the Territory of Inner Eurasia from 1947 to 1999.

Listeriosis is one of the most significant humans and animals foodborne infectious diseases. Here, we characterized 48 Listeria monocytogenes strains isolated in the territory of inner Eurasia during the second half of the 20th century. A total of 23 strains (52.3%) were susceptible to the nine antibiotics tested, 30.43%, 15.22%, and 8.7% were resistant penicillin G, ampicillin, and enrofloxacin, respectively. We applied the multilocus sequence typing (MLST) scheme to determine the phylogenetic positions of the strains. All but one strain belonged to the II phylogenetic lineage, and the majority of the strains belonged to one of the previously described clonal complexes (CCs). More than 60% of the strains belonged to the clonal complex CC7 that prevailed among all sources, including cattle (58%), small ruminants (64%), rodents (71%), and humans (50%). Further, CC7, CC101, and CC124 were found among human isolates. The MLST scheme was supplemented with virulence gene analysis. In total, eight inlA, six inlB, and six inlC allelic variants were found, and all but one strain carried one of the two inlE alleles. Most strains (62.5%) belonged to the same multivirulence locus sequence typing (MvLST) type, which includes CC7, inlA allele 4, inlB allele 14, inlC allele 6, and inlE allele 8.

Prevalence, Genotypic Characteristics and Antibiotic Resistance of Listeria monocytogenes From Retail Foods in Bulk in Zhejiang Province, China.

Listeria monocytogenes is an important foodborne pathogen causing public concern. A total of 3354 retail foods in bulk were sampled and screened for L. monocytogenes. Seventy-three (2.2%) samples including 21 ready-to-eat (RTE) foods and 52 raw foods were confirmed positive for L. monocytogenes. Sushi and salmon sashimi occupied the top two slots in RTE foods with relatively high presence rate of 12.9 and 6.9%, respectively. Meanwhile, L. monocytogenes was found to be distributed unequally in raw foods; the presence rates in raw meat (3.5%) and poultry (3.8%) were significantly higher than that in raw seafood (1.3%). Notably, L. monocytogenes was not detected in raw freshwater food. The L. monocytogenes isolates belonged to four serotypes, 1/2a, 1/2b, 1/2c, and 4b, with the most prevalent serotype being 1/2a (47.9%). Eighteen sequence types (STs) and eighteen virulence types (VTs) containing four newly assigned VTs (VT180, VT181, VT182, and VT183) were determined via multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MVLST). Among the 73 L. monocytogenes isolates, 23 (31.5%) belonged to epidemic clones (ECs) including ECI, ECIV, ECV, ECVI, ECVIII and ECXI among which ECV was predominant. Antibiotic susceptibility tests revealed a high resistance rate (11.0%) to tetracycline. Moreover, we identified the distribution patterns of virulence genes of four Listeria pathogenicity islands (LIPI) in L. monocytogenes isolates. prfA, hly, plcA, plcB, mpl, actA genes in LIPI-1 and inlA, inlB, inlC, inlJ genes in LIPI-2 were detected in approximately all L. monocytogenes isolates. The distribution of both LIPI-3 genes and LIPI-4 genes exhibited association with lineage and ST. LIPI-4 genes were present exclusively in ST87 isolates. Relatedness analysis revealed the absence of distinct association between STs, ECs, LIPI-3 and LIPI-4 distribution and specific food groups. This study provided fundamental data for Chinese food safety authorities to grasp the contamination status of L. monocytogenes in foods, assess the potential risk of this pathogen and further address the safety issue of retail foods in bulk in China.

Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme

To our knowledge, the phylodistribution of M. hominis clinical strains associated with various pathological conditions of the urogenital tract has not been explored hitherto. Here we analyzed the genetic diversity and phylogenetic relationships among 59 M. hominis Tunisian clinical isolates, categorized as gynecological infections- or infertility-associated pathotypes. For this purpose, we developed an expanded multilocus sequence typing (eMLST) scheme, combining the previously reported multilocus sequence typing (MLST) loci (gyrB, tuf, ftsY, uvrA, gap) with a new selected set of putative virulence genes (p120’, vaa, lmp1, lmp3, p60), referred herein to as multi-virulence-locus sequence typing (MVLST) loci. In doing so, M. hominis population was segregated into two distinct genetic lineages, which were differentially associated with each pathotype. Such a clear dichotomy was supported by several phylogenetic and population genetic analysis tools. Recombination was found to take place, but not sufficient enough to break down the overall clonal population structure of M. hominis, most likely as a result of purifying selection, which accommodated the most fit clones. In sum, and owing to the eMLST scheme described herein, we provide insightful data on the phylogenetics of M. hominis, arguing for the existence of genetically differentiable urogenital pathotypes.

Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) and Tandem Repeat Sequence Typing (TRST), helpful tools for subtyping Staphylococcus lugdunensis

Staphylococcus lugdunensis is an emergent virulent coagulase-negative Staphylococcus that is increasingly responsible for severe infections. In an attempt to generate informative sequence data for subtyping S. lugdunensis, we selected and sequenced seven polymorphic variable number of tandem repeats (VNTRs) to develop two new methods: a classic length-based multiple-locus VNTR analysis (MLVA) method and a tandem repeat sequence typing (TRST) method. We assessed their performances compared to two existing methods, multilocus sequence typing (MLST) and multivirulence-locus sequence typing (MVLST) for 128 isolates from diverse clinical settings and geographical origins. The clustering achieved by the four methods was highly congruent, with MLVA discriminating within clonal complexes as defined by MLST. Indeed, MLVA was highly discriminant compared to MLST and MVLST in terms of number of genotypes as well as diversity indexes. Sequencing of the seven VNTRs showed that they were stable, and analysis of sequence polymorphisms provided superior discriminatory power. The typeability, reproducibility, and epidemiological concordance of these new methods were excellent. Of note, no link between clustering and clinical settings was identified. This study demonstrates that MLVA and TRST provide valuable information for molecular epidemiological study of S. lugdunensis, and represent promising tools to distinguish between strains of homogenous lineages in this clonal species.

Genetic diversity and virulence profiles of Listeria monocytogenes recovered from bulk tank milk, milk filters, and milking equipment from dairies in the United States (2002 to 2014).

Unpasteurized dairy products are known to occasionally harbor Listeria monocytogenes and have been implicated in recent listeriosis outbreaks and numerous sporadic cases of listeriosis. However, the diversity and virulence profiles of L. monocytogenes isolates recovered from these products have not been fully described. Here we report a genomic analysis of 121 L. monocytogenes isolates recovered from milk, milk filters, and milking equipment collected from bovine dairy farms in 19 states over a 12-year period. In a multi-virulence-locus sequence typing (MVLST) analysis, 59 Virulence Types (VT) were identified, of which 25% were Epidemic Clones I, II, V, VI, VII, VIII, IX, or X, and 31 were novel VT. In a multi-locus sequence typing (MLST) analysis, 60 Sequence Types (ST) of 56 Clonal Complexes (CC) were identified. Within lineage I, CC5 and CC1 were among the most abundant, and within lineage II, CC7 and CC37 were the most abundant. Multiple CCs previously associated with central nervous system and maternal-neonatal infections were identified. A genomic analysis identified variable distribution of virulence markers, Listeria pathogenicity islands (LIPI) -1, -3, and -4, and stress survival island-1 (SSI-1). Of these, 14 virulence markers, including LIPI-3 and -4 were more frequently detected in one lineage (I or II) than the other. LIPI-3 and LIPI-4 were identified in 68% and 28% of lineage I CCs, respectively. Results of this analysis indicate that there is a high level of genetic diversity among the L. monocytogenes present in bulk tank milk in the United States with some strains being more frequently detected than others, and some being similar to those that have been isolated from previous non-dairy related outbreaks. Results of this study also demonstrate significant number of strains isolated from dairy farms encode virulence markers associated with severe human disease.

CRISPR Typing and Antibiotic Resistance Correlates with Polyphyletic Distribution in Human Isolates of Salmonella Kentucky.

Although infrequently associated with reported salmonellosis in humans, Salmonella enterica, subsp. enterica serovar Kentucky (ser. Kentucky) is the most common nonclinical, nonhuman serovar reported in the United States. The goal of this study was to use Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-multi-virulence-locus sequence typing (MVLST) to subtype a collection of human clinical isolates of ser. Kentucky submitted to the Pennsylvania Department of Health and to determine the extent of antibiotic resistance in these strains. This analysis highlighted the polyphyletic nature of ser. Kentucky, and separated our isolates into two groups, Group I and Group II, which were equally represented in our collection. Furthermore, antimicrobial susceptibility testing on all isolates using a National Antimicrobial Resistance Monitoring System (NARMS) panel of antibiotics demonstrated that resistance profiles could be divided into two groups. Group I isolates were resistant to cephems and penicillins, whereas Group II isolates were resistant to quinolones, gentamicin, and sulfisoxazole. Collectively, 50% of isolates were resistant to three or more classes of antibiotics and 30% were resistant to five or more classes. The correlation of antibiotic resistance with the two different lineages may reflect adaptation within two distinct reservoirs of ser. Kentucky, with differential exposure to antimicrobials.

Whole genome sequencing for typing and characterisation of Listeria monocytogenes isolated in a rabbit meat processing plant.

Listeria monocytogenes is a food-borne pathogen able to survive and grow in different environments including food processing plants where it can persist for month or years. In the present study the discriminatory power of Whole Genome Sequencing (WGS)-based analysis (cgMLST) was compared to that of molecular typing methods on 34 L. monocytogenes isolates collected over one year in the same rabbit meat processing plant and belonging to three genotypes (ST14, ST121, ST224). Each genotype included isolates indistinguishable by standard molecular typing methods. The virulence potential of all isolates was assessed by Multi Virulence-Locus Sequence Typing (MVLST) and the investigation of a representative database of virulence determinant genes. The whole genome of each isolate was sequenced on a MiSeq platform. The cgMLST, MVLST, and in silico identification of virulence genes were performed using publicly available tools. Draft genomes included a number of contigs ranging from 13 to 28 and N50 ranging from 456298 to 580604. The coverage ranged from 41 to 187X. The cgMLST showed a significantly superior discriminatory power only in comparison to ribotyping, nevertheless it allows the detection of two singletons belonging to ST14 that were not observed by other molecular methods. All ST14 isolates belonged to VT107, which 7-loci concatenated sequence differs for only 4 nucleotides to VT1 (Epidemic clone III). Analysis of virulence genes showed the presence of a fulllength inlA version in all ST14 isolates and of a mutated version including a premature stop codon (PMSC) associated to attenuated virulence in all ST121 isolates.

A Comprehensive Evaluation of the Genetic Relatedness of Listeria monocytogenes Serotype 4b Variant Strains.

Recently, we have identified a link between four listeriosis incidents/outbreaks to a variant of Listeria monocytogenes (Lm) serotype 4b strains, 4bV. Although 4bV strains have been reported from clinical specimens as well as from foods, listeriosis outbreaks occurring in 2014–2016 were the first reported outbreaks involving 4bV in the USA. Since traditional typing methods do not detect members of this group, we undertook a systematic and retrospective analysis of all Lm in the NCBI WGS Sequence Read Archive database to investigate the burden of 4bV strains among all listeriosis cases. This analysis identified the presence of isolates causing sporadic cases as well as those associated with the aforementioned outbreaks, as determined by WGS and traditional epidemiology. In total, approximately 350 Lm 4bV strains were identified from multiple parts of the USA as well as from Australia and Chile, dating back to 2001. The genomic relatedness of these strains was compared using the CFSAN SNP Pipeline and multi-virulence-locus sequence typing (MVLST). Using the CFSAN Pipeline tool, the 4bV strains were found to group into seven clusters that were separate from 4b strains. All seven clades appeared to contain isolates from both clinical and non-clinical sources. Conversely, the MVLST analysis revealed that practically all of the strains belonged to a single clade, suggesting that 4bV strains from disparate geographic regions and sources are under varied selective pressure, restricting diversity across these six virulence loci while allowing more variability across the genome as a whole. Further evaluation of these 4bV strains identified genes potentially acquired from a lineage II source external to the lmo0733–lmo0739 region, as well as highly conserved SNPs unique to the 4bV strains when compared to those from other lineages. Taken together, these data suggest that 4bV strains have undergone adaptive responses to selective pressures that may enhance survival in the environment while maintaining the pathogenic potential of serotype 4b strains.

Identification of a major Listeria monocytogenes outbreak clone linked to soft cheese in Northern Italy \u2013 2009-2011

BackgroundMolecular subtyping and enhanced surveillance in Lombardy region identified a cluster of possibly related listeriosis cases from 2006 to 2010. This cluster grouped 31 isolates that belonged to serotype 1/2a and Sequence Type 38 (ST38) as defined by Multilocus Sequence Typing (MLST).MethodsOur study expanded the previous investigation to include cases from 2011 to 2014 and used Multi-Virulence-Locus Sequence Typing (MVLST) on all ST38 isolates to better understand their epidemiology and possibly identify a common source outbreak.ResultsOut of 306 L. monocytogenes clinical isolates collected, 43 (14.1%) belonged to ST38 with cases occurring in nine out of twelve Lombardy provinces. The ST38 isolates were split by MVLST into two Virulence Types (VTs): VT80 (n = 12) and VT104 (n = 31). VT104 cases were concentrated between 2009 and 2011 in two provinces, Bergamo and Milan. An epidemiologic investigation was performed and in one case, a matching VT104 isolate was retrieved from a soft cheese sample from a patient’s refrigerator.ConclusionsOur findings revealed a major listeriosis outbreak in Northern Italy linked to soft cheese in 2009–2011, which went undetected by local health authorities. Our study shows that integrating subtyping methods with conventional epidemiology can help identify the source of L. monocytogenes outbreak clones.

Diversity and persistence of Listeria monocytogenes within the Gorgonzola PDO production chain and comparison with clinical isolates from the same area

Listeria monocytogenes causes invasive syndromes with high fatality rates in specific population groups. Cheeses have been commonly implicated in outbreaks worldwide. Gorgonzola is a cheese only produced in Northwestern Italy (it is the third Italian cheese in terms of production and export) and L. monocytogenes is frequently isolated from the production chain. The aims of this study were to assess the distribution of L. monocytogenes Virulence Types (VTs) in isolates collected in Gorgonzola processing plants and to determine the presence of Epidemic Clones (ECs). Fifty-Six L. monocytogenes strains collected between 2004 and 2016 from cheese and environmental samples were subtyped with Multi-Virulence-Locus Sequence Typing (MVLST) and compared to previously typed strains. Most isolates (n=50) belonged to two new VTs (VT113 and VT114). The remaining isolates belonged to previously identified VTs: VT14-ECVIII (milk chocolate outbreak, 1994, USA) and VT80 (ricotta salata outbreak, 2012, USA). VT14, VT80 and VT113 were shared with isolates from apparently sporadic human cases in the same geographical area and temporal period (Piedmont and Lombardy, 2005–2016). The overall L. monocytogenes population appears to be homogeneous and may be characteristic of Gorgonzola production. Nevertheless, the detection in cheese and environmental samples of VTs observed in clinical isolates or outbreak related strains (VT80, VT14) contributed to better describe the current scenario and pointed out the need for increased surveillance.

Whole genome sequence-based serogrouping of Listeria monocytogenes isolates

Whole genome sequencing (WGS) is currently becoming the method of choice for characterization of Listeria monocytogenes isolates in national reference laboratories (NRLs). WGS is superior with regards to accuracy, resolution and analysis speed in comparison to several other methods including serotyping, PCR, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable number tandem repeat analysis (MLVA), and multivirulence-locus sequence typing (MVLST), which have been used thus far for the characterization of bacterial isolates (and are still important tools in reference laboratories today) to control and prevent listeriosis, one of the major sources of foodborne diseases for humans. Backward compatibility of WGS to former methods can be maintained by extraction of the respective information from WGS data. Serotyping was the first subtyping method for L. monocytogenes capable of differentiating 12 serovars and national reference laboratories still perform serotyping and PCR-based serogrouping as a first level classification method for Listeria monocytogenes surveillance. Whole genome sequence based core genome MLST analysis of a L. monocytogenes collection comprising 172 isolates spanning all 12 serotypes was performed for serogroup determination. These isolates clustered according to their serotypes and it was possible to group them either into the IIa, IIc, IVb or IIb clusters, respectively, which were generated by minimum spanning tree (MST) and neighbor joining (NJ) tree data analysis, demonstrating the power of the new approach.

Far East Scarlet-Like Fever Caused by a Few Related Genotypes of Yersinia pseudotuberculosis, Russia.

We used multivirulence locus sequence typing to analyze 68 Yersinia pseudotuberculosis isolated in Russia during 1973–2014, including 41 isolates from patients with Far East scarlet-like fever. Four genotypes were found responsible, with 1 being especially prevalent. Evolutionary analysis suggests that epidemiologic advantages could cause this genotype’s dominance.

Diversity of Listeria monocytogenes within a U.S. dairy herd, 2004-2010.

Listeria monocytogenes, the causative agent of listeriosis, is frequently isolated from the environment. Dairy cows and dairy farm environments are reservoirs of this pathogen, where fecal shedding contributes to its environmental dispersal and contamination of milk, dairy products, and meat. The molecular diversity of 40 L. monocytogenes isolates representing 3 serogroups (1/2a, 1/2b, and 4b) collected between 2004 and 2010 from the feces of dairy cattle on a single dairy farm was assessed using a multivirulence locus sequence typing (MVLST) assay. The dairy farm L. monocytogenes MVLST patterns were compared to those from 138 strains isolated globally from clinical cases, foods, and the environment. Results of the study demonstrated that several distantly related L. monocytogenes strains persisted among members of the herd over the course of the study while other strains were transient. Furthermore, some strains isolated during this study appear to be distantly related to previously isolated L. monocytogenes while others are closely related to Epidemic Clones associated with human illness. This work demonstrates that dairy cows can be reservoirs of a diverse population of potentially human pathogenic L. monocytogenes that represents a risk to consumers of milk, dairy products, and meat.

Prevalence and Spatial Distribution of Salmonella Infections in the Pennsylvania Raccoon (Procyon lotor).

A study was conducted to determine the prevalence and spatial distribution of Salmonella infection in Pennsylvania raccoons (Procyon lotor), common wildlife mammals known to occupy overlapping habitats with humans and domestic food animals. The Pennsylvania Game Commission provided a total of 371 raccoon intestinal samples from trapped and road-killed raccoons collected between May and November 2011. Salmonella was isolated from the faeces of 56 (15.1%) of 371 raccoons in 35 (54%) of 65 counties across Pennsylvania. The five most frequently isolated serotypes were Newport (28.6%), Enteritidis (19.6%), Typhimurium (10.7%), Braenderup (8.9%) and Bareilly (7.1%). Pulsed-field gel electrophoresis (PFGE) analysis of the Salmonella isolates and subsequent comparison to the Pennsylvania Department of Health human Salmonella PFGE database revealed 16 different pulsetypes in Salmonella isolates recovered from raccoons that were indistinguishable from pulsetypes of Salmonella collected from clinically ill humans during the study period. The pulsetypes of seven raccoon Salmonella isolates matched those of 56 human Salmonella isolates by month and geographical region of sample collection. Results from Clustered Regularly Interspaced Short Palindromic Repeats and Multi-Virulence Locus Sequence Typing (CRISPR-MVLST) analysis corroborated the PFGE and serotyping data. The findings of this study show that several PFGE pulsetypes of Salmonella were shared between humans and raccoons in Pennsylvania, indicating that raccoons and humans might share the same source of Salmonella.

Geographical and longitudinal analysis of Listeria monocytogenes genetic diversity reveals its correlation with virulence and unique evolution

Listeria monocytogenes is one of the most important foodborne pathogens causing severe diseases with a mortality rate of 24%. However, the genetic diversity and evolution of L. monocytogenes, particularly at the worldwide level, are poorly defined. In this study, we performed multilocus sequence typing (MLST) and multi virulence locus sequence typing (MVLST) for 86 L. monocytogenes strains derived from 8 countries from 1926 to 2012 in order to better understand the molecular evolution and genetic characteristics of this pathogen. A total of 13 clonal complexes (CCs) were detected, of which CC1, CC2, CC3, CC7, CC9, CC4 are the most prevalent. Notably, polymorphism of housekeeping genes of isolates belong to CC1 (STs=47) increased more rapidly over the time. MLST-based phylogenetic analysis showed that serotype 1/2b and 4b strains had an “interval-type” evolution pattern, while serotype 1/2a and 1/2c strains had a “progressive-type” evolution pattern. Furthermore, strains from temporally and geographically unrelated outbreaks in differernt countries were clustered in the same subgroup of phylogenetic tree, indicating that that L. monocytogenes developed highly similar virulence genes and genetic characteristics to adaptation in a special ecological niche. Interestingly, there was a high correlation between the population structure of MVLST and MLST among the isolates of cluster IA corresponding to CC1, CC2, CC4 and CC6 that had the highest potential to cause listeriosis outbreaks, strengthening that surveillance of these CCs is important for prevention of listeriosis. The present study offers insights into the internal relationships between the population structure, distribution and pathogenicity of L. monocytogenes.