Occurrence and persistence of Listeria spp. in the environment of ewe and cow's milk cheese dairies in Portugal unveiled by an integrated analysis of identification, typing and spatial-temporal mapping along production cycle.
ABSTRACT Eight dairies, located in two distant geographic regions of Portugal, were screened along the production cycle in order to evaluate the presence and distribution of Listeria spp. in their environment. Three dairies in each region were positive for the presence of listeriae and 213 isolates were obtained. Based on an integrated analysis of RAPD fingerprints with three primers, molecular identification and genomic typing of isolates was performed followed by spatial and temporal mapping on dairy plants. The occurrence of Listeria species by region was noticeable different. Listeria monocytogenes prevailed in South Portugal dairies and L. innocua presented the highest occurrence in Azores, whereas L. seeligeri and L. ivanovii were detected in distinct regions. Dairies were at risk of contamination, from more than one source, whatever the stage in the production cycle and the surface materials used. For the three prevalent species, most of the genomic types were dairy and sampling time specific. Nonetheless, more than one type could be found in each dairy at a particular site and, in a few cases, even for different species. Some dairies also shared types, mainly for L. innocua and usually at the same stage of the production cycle. For L. monocytogenes, PCR serotyping was applied and 52% of genomic types were serotype 4b. An equal frequency of genomic types (24%) was found for serotypes 1/2b or 3b and 1/2a or 3a. The global pattern of types within a dairy is not constant, suggesting cycles of elimination and recontamination along the production cycle.
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ABSTRACT: While increasing data on bacterial evolution in controlled environments are available, our understanding of bacterial genome evolution in natural environments is limited. We thus performed full genome analyses on four Listeria monocytogenes, including human and food isolates from both a 1988 case of sporadic listeriosis and a 2000 listeriosis outbreak, which had been linked to contaminated food from a single processing facility. All four isolates had been shown to have identical subtypes, suggesting that a specific L. monocytogenes strain persisted in this processing plant over at least 12 years. While a genome sequence for the 1988 food isolate has been reported, we sequenced the genomes of the 1988 human isolate as well as a human and a food isolate from the 2000 outbreak to allow for comparative genome analyses. The two L. monocytogenes isolates from 1988 and the two isolates from 2000 had highly similar genome backbone sequences with very few single nucleotide (nt) polymorphisms (1 - 8 SNPs/isolate; confirmed by re-sequencing). While no genome rearrangements were identified in the backbone genome of the four isolates, a 42 kb prophage inserted in the chromosomal comK gene showed evidence for major genome rearrangements. The human-food isolate pair from each 1988 and 2000 had identical prophage sequence; however, there were significant differences in the prophage sequences between the 1988 and 2000 isolates. Diversification of this prophage appears to have been caused by multiple homologous recombination events or possibly prophage replacement. In addition, only the 2000 human isolate contained a plasmid, suggesting plasmid loss or acquisition events. Surprisingly, besides the polymorphisms found in the comK prophage, a single SNP in the tRNA Thr-4 prophage represents the only SNP that differentiates the 1988 isolates from the 2000 isolates. Our data support the hypothesis that the 2000 human listeriosis outbreak was caused by a L. monocytogenes strain that persisted in a food processing facility over 12 years and show that genome sequencing is a valuable and feasible tool for retrospective epidemiological analyses. Short-term evolution of L. monocytogenes in non-controlled environments appears to involve limited diversification beyond plasmid gain or loss and prophage diversification, highlighting the importance of phages in bacterial evolution.BMC Genomics 12/2008; 9:539. · 4.07 Impact Factor
Article: Listeria monocytogenes biofilm-associated protein (BapL) may contribute to surface attachment of L. monocytogenes but is absent from many field isolates.[show abstract] [hide abstract]
ABSTRACT: Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.Applied and environmental microbiology 06/2008; 74(17):5451-6. · 3.69 Impact Factor