Sara Lomonaco

Novel Epidemic Clones of Listeria monocytogenes, United States, 2011

We identified a novel serotype 1/2a outbreak strain and 2 novel epidemic clones of Listeria monocytogenes while investigating a foodborne outbreak of listeriosis associated with consumption of cantaloupe during 2011 in the United States. Comparative analyses of strains worldwide are essential to identification of novel outbreak strains and epidemic clones.

Listeria monocytogenes in Gorgonzola: Subtypes, diversity and persistence over time

L. monocytogenes represents a primary concern in the production of Gorgonzola, a Protected Designation of Origin (PDO) Italian blue-veined cheese produced only in the Piedmont and Lombardy regions. L. monocytogenes isolates (N=95) obtained from Gorgonzola rinds, paste, and production/ripening environments were serotyped and then genotyped using Pulsed Field Gel Electrophoresis (PFGE). The goal of this study was to investigate the variability of L. monocytogenes PFGE-types across different PDO Gorgonzola manufacturers (N=22). The majority of the strains (88%) were serotyped as 1/2a. PFGE identified 2 major pulse-types grouping 62 strains, detected from different plants and years, suggesting the presence of persistent and niche-adapted L. monocytogenes. In 9 plants, environmental strains shared the same pulse-types with strains from rinds or paste, suggesting a possible transmission pathway. Encouragingly, L. monocytogenes was retrieved from only 1 paste, indicating that production processes were under control in 21 plants. In the remaining plant, un-effective pasteurization or cross-contamination during production processes could be the cause of the contamination. Consequently, it is imperative that producers operate under the total respect of the Good Manufacturing Practices and following the principles of the Hazard Analysis Critical Control Point plans, in order to contain contamination throughout the whole processing.

The evolution and epidemiology of Listeria monocytogenes in Europe and the United States.

Listeria monocytogenes is an opportunistic food-borne pathogen responsible for listeriosis, a disease associated with high mortality rates. L. monocytogenes causes invasive syndromes and case-fatality can be as high as 30%, in specific high-risk population groups such as the elderly, immuno-compromised individuals, fetuses and newborns. Acquisition of the disease is mainly due to consumption of contaminated (predominantly ready-to-eat) food. We aimed to provide a state-of-the-art collection of different likely evolutionary models, based on recombination and positive selection, and the phylogenetic relationship between lineages of L. monocytogenes and between them and other Listeria species. We described the most recent findings in comparative pan-genomics, considering the core and accessory genome in relation to virulence and adaptation to different environments. Finally, this review illustrates L. monocytogenes epidemiology and transmission in humans, foods and animals, the surveillance systems of the European Union and United States and the application of molecular techniques as a core tool in epidemiological investigation.

Microbial ecology of Gorgonzola rinds and occurrence of different biotypes of Listeria monocytogenes.

In this study we investigated the microbiota of Gorgonzola rinds and maturing shelf swabs collected in 5 different maturing cellars in the Northwest part of Italy, in association with the detection and characterization of Listeria monocytogenes. Culture-dependent and -independent methods were performed in order to profile the main microbial populations present on the rinds and in the maturing shelves and species-specific PCR and Pulsed Field Gel Electrophoresis (PFGE) were used to identify and type L. monocytogenes isolates. The microflora was predominated by lactic acid bacteria and coagulase negative cocci, while enterococci and yeasts were very variable between the samples. Arthrobacter sp., Carnobacterium sp., Staphylococcus sp. and Brevibacterium linens, as bacteria, and Debaryomyces hansenii, as yeast, were detected by Denaturing Gradient Gel Electrophoresis (DGGE). Cluster analysis of the DGGE profiles clearly highlighted a cellar-specific microflora. L. monocytogenes was isolated in 11.1% of the rinds and 29.4% of the swabs and the molecular characterization of the isolates suggests a route of contamination from the maturing shelves to the rinds. No correlation was found between DGGE profiles and presence or absence of L. monocytogenes.

Comparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy.

Listeria monocytogenes ranks among the most frequent causes of death due to foodborne illness (20-30% case fatality rate). Discriminative subtyping methods are important to detect the relatedness of isolates and verify epidemiologic associations. AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. In this study, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance. A total of 103 unrelated L. monocytogenes strains isolated from different environmental and food sources were analyzed. Strains were isolated from samples obtained from food-production plants, supermarkets and small food markets in Piedmont, Italy. All methods clustered L. monocytogenes strains into two genetic lineages, Lineage I and II. The three methods were compared using the 82 isolates which were typeable with all techniques. The calculated pair-wise Pearsons correlation coefficients (r) showed close agreement between all three methods. Our findings suggest that the AFLP II method can be successfully used to subtype L. monocytogenes strains isolated from foods and food processing facilities.

A five year surveillance report on PFGE types of Listeria monocytogenes isolated in Italy from food and food related environments

Listeria monocytogenes can cause severe invasive disease in humans and has been isolated from a variety of foods. This study aimed to investigate type diversity and distribution across sources by subtyping via PFGE a set of 300 L. monocytogenes isolates collected in Italy from foods over a five year period (from 2003 to 2007). The most frequent serotypes were 1/2a (45%), 1/2c (22%), and 4b/4e (16%); 5% of the isolates were untypeable by conventional serotyping. Significant associations were observed between serotype 1/2a with dairy (O.R.=13.9) and 1/2c with meat (O.R.=33.3). All isolates were typeable, generating 164 combined PFGE profiles. Of these, 121 were unique, being displayed by only one isolate. The other 43 profiles grouped the remaining isolates and were shared between two (N=22), three (N=10), four (N=3) and five isolates (N=4). The remaining 4 profiles were shared between 7, 14, 17 and 46 isolates, respectively. Some profiles (N=7) were retrieved in samples collected in different years, indicating persistency in foods and processing plants. This research may pose the ground for designing a broad typing database which could ease the understanding of L. monocytogenes diversity and could be used for facilitating epidemiological investigations for the identification of listeriosis outbreaks. Data show how large subtype databases may facilitate the identification of common and source-specific types. More comprehensive databases may be needed to fully understand L. monocytogenes diversity and to provide useful data to be considered in epidemiological investigations.

Analysis of additional virulence genes and virulence gene regions in Listeria monocytogenes confirms the epidemiologic relevance of multi-virulence-locus sequence typing.

Previous molecular subtyping studies have defined four epidemic clones (ECs) of Listeria monocytogenes (ECI, ECII, ECIII, and ECIV). Partial sequences of eight virulence genes were previously shown to be identical within individual ECs of L. monocytogenes. The present study was conducted to determine if the sequences of other virulence genes and virulence gene regions are also conserved within these ECs. Six additional virulence genes--bsh, hly, inlJ, IplA1, pgdA, and srtA--and three additional virulence gene regions of actA, inlA, and inlB were selected based on their role in L. monocytogenes virulence, and intragenic regions of each gene were sequenced. Sequencing was performed on a diverse set of 44 to 48 L. monocytogenes strains. Results demonstrated that the sequenced regions of the nine virulence genes were identical within each of the ECs, and 257 new single nucleotide polymorphism (SNPs) were identified. ECIII (lineage II) was easily distinguishable from the other ECs, as 238 SNPs were observed in ECIII due to its significant evolutionary divergence from lineage I. With regard to the other ECs, there were 5 SNPs that represented an informative set, since these SNPs were able to differentiate specific ECs from all other unrelated strains used in this study. This study confirms our previous finding that virulence gene sequences are highly conserved within individual ECs and contain stable SNPs that can be used to very accurately differentiate ECs of L. monocytogenes from each other and from other diverse strains.

Detection of virulence-associated genes and epidemic clone markers in Listeria monocytogenes isolates from PDO Gorgonzola cheese.

Fifty-three Listeria monocytogenes isolates obtained from Gorgonzola cheese and previously characterized with biochemical typing, serotyping and pulsed field gel electrophoresis (PFGE), were analyzed in this study. Seven virulence-associated genes were selected (actA, inlC, inlJ, plcA, prfA, hlyA and iap) and their presence was investigated using PCR. All virulence-associated genes were detected in 51 isolates. One isolate did not show amplification of the inlC gene and one other isolate, previously mis-identified as L. monocytogenes probably due to atypical phenotypes, resulted negative by PCR for all virulence genes and was identified as Listeria innocua by 16S rRNA gene sequencing analysis. A multiplex PCR assay was used to evaluate the presence of markers specific for epidemic clones (ECs) of L. monocytogenes. The marker specific for the recently identified epidemic clone V (ECV) was detected in 38 of 43 (88%) of serotype 1/2a isolates. These findings suggest that Gorgonzola cheese can represent a significant source of L. monocytogenes and potential health risk for listeriosis as almost all isolates (94%) could be potentially virulent and that 38 (~72%) were presumptive positive ECV. PCR screening for both virulence-associated genes and EC markers may be useful for rapidly evaluating the epidemic potential and public health risk posed by L. monocytogenes in PDO Gorgonzola cheese and other dairy products.

Novel Multiplex Single Nucleotide Polymorphism-Based Method for Identifying Epidemic Clones of Listeria monocytogenes

ABSTRACT A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of Listeria monocytogenes and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of L. monocytogenes.

Ruminant Rhombencephalitis-Associated Listeria monocytogenes Strains Constitute a Genetically Homogeneous Group Related to Human Outbreak Strains

ABSTRACT Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cows milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.