Christine Jacquet

Differentiation of the Major Listeria monocytogenes Serovars by Multiplex PCR

ABSTRACT A new multiplex PCR assay was developed to separate the four major Listeria monocytogenes serovars isolated from food and patients (1/2a, 1/2b, 1/2c, and 4b) into distinct groups. The PCR test, which constitutes a rapid and practical alternative to laborious classical serotyping, was successfully evaluated with 222 Listeria strains.

New Aspects Regarding Evolution and Virulence of Listeria monocytogenes Revealed by Comparative Genomics and DNA Arrays

ABSTRACT Listeria monocytogenes is a food-borne bacterial pathogen that causes a wide spectrum of diseases, such as meningitis, septicemia, abortion, and gastroenteritis, in humans and animals. Among the 13 L. monocytogenes serovars described, invasive disease is mostly associated with serovar 4b strains. To investigate the genetic diversity of L. monocytogenes strains with different virulence potentials, we partially sequenced an epidemic serovar 4b strain and compared it with the complete sequence of the nonepidemic L. monocytogenes EGDe serovar 1/2a strain. We identified an unexpected genetic divergence between the two strains, as about 8% of the sequences were serovar 4b specific. These sequences included seven genes coding for surface proteins, two of which belong to the internalin family, and three genes coding for transcriptional regulators, all of which might be important in different steps of the infectious process. Based on the sequence information, we then characterized the gene content of 113 Listeria strains by using a newly designed Listeria array containing the “flexible” part of the sequenced Listeria genomes. Hybridization results showed that all of the previously identified virulence factors of L. monocytogenes were present in the 93 L. monocytogenes strains tested. However, distinct patterns of the presence or absence of other genes were identified among the different L. monocytogenes serovars and Listeria species. These results allow new insights into the evolution of L. monocytogenes, suggesting that early divergence of the ancestral L. monocytogenes serovar 1/2c strains from the serovar 1/2b strains led to two major phylogenetic lineages, one of them including the serogroup 4 strains, which branched off the serovar 1/2b ancestral lineage, leading (mostly by gene loss) to the species Listeria innocua. The identification of 30 L. monocytogenes-specific and several serovar-specific marker genes, such as three L. monocytogenes serovar 4b-specific surface protein-coding genes, should prove powerful for the rapid tracing of listeriosis outbreaks, but it also represents a fundamental basis for the functional study of virulence differences between L. monocytogenes strains.

A Molecular Marker for Evaluating the Pathogenic Potential of Foodborne Listeria monocytogenes

BACKGROUND Internalin mediates entry of Listeria monocytogenes into some human cultured cell lines and crossing of the intestinal barrier in transgenic mice expressing its receptor, human E-cadherin, in enterocytes. The relevance of these findings for humans is challenged by the observation that some L. monocytogenes isolates express a truncated nonfunctional form of internalin. METHODS We investigated expression of internalin by use of immunoblot assay in 300 clinical strains obtained in France in a single year and a representative set of 150 strains obtained from food products during the same period. RESULTS Clinical strains expressed full-length internalin far more frequently (288/300 strains [96%]) than did strains recovered from food products (98/150 strains [65%]; odds ratio, 12.73; 95% confidence interval, 6.27-26.34; P<1 x 10(-7)). All 61 strains (100%) from pregnancy-related cases, 55 (98%) of 56 strains from patients with central nervous system infections, and 151 (93%) of 162 strains from patients with bacteremia expressed full-length internalin. All 110 strains belonging to serovar 4b, the most frequently implicated serovar in human listeriosis, expressed full-length internalin. CONCLUSIONS This study demonstrates the critical role of internalin in the pathogenesis of human listeriosis. It provides a molecular explanation for the predominance of serovar 4b among clinical strains and supports the usefulness of studying the expression of internalin as a marker of virulence in humans.

Listeriosis Outbreak Associated with the Consumption of Rillettes in France in 1993

An outbreak of listeriosis involving 38 patients occurred in France between 18 June and 5 October 1993. The epidemic clone was characterized by serovar 4b, phagovar 2671:108:312, and DNA macrorestriction patterns 12 and 13. Thirty-one case-patients were materno-neonatal patients and 7 patients were nonpregnant adults. Preliminary analysis of a case-control study implicated a pork product, rillettes, of a particular brand (odds ratio, 18; 95% confidence interval, 2.2-208) as the vehicle of infection. Rillettes is a ready-to-eat food prepared with ham meat cooked with grease. The implicated lots of rillettes were recalled in mid-August, and the French authorities issued a warning to the general public. Microbiologic analysis of unopened plastic cans of rillettes confirmed the results of the case-control study 3 weeks after the recall. Final analysis showed that the rillettes was the major vehicle of the outbreak but suggested that other brand A meat products could also have been involved.

Expression of ActA, Ami, InlB, and Listeriolysin O in Listeria monocytogenes of Human and Food Origin

ABSTRACT Expression of proteins involved in the adhesion of Listeria monocytogenes to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of L. monocytogenes strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90; P = 1 × 10−4). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89; P = 1 × 10−2) and central nervous system infections were associated with group ActA2 strains (OR = 3.04; P = 1 × 10−3) and group ActA3 strains (OR = 3.91; P = 1 × 10−3).

Listeria monocytogenes isolated from vegetable salads sold at supermarkets in Santiago, Chile: Prevalence and strain characterization

Between 2000 and 2005, 717 samples of three types of salads were analysed for Listeria monocytogenes in Santiago, Chile in order to provide information to Chilean health authorities on the presence of the pathogen in vegetable salad samples and to ascertain the risk of these products for consumers. L. monocytogenes isolates were found in 88 out of 347 (25.4%) samples of frozen vegetable salads and in 22 out of 216 (10.2%) freshly supermarkets prepared, cooked or raw ready-to-eat vegetable salads; no Listeria was isolated from 154 samples of raw minimally processed salads industrially prepared. Enumeration of L. monocytogenes was done by plate count for 20 positive frozen samples, randomly chosen. Most of them (90%) had < 10 cfu/g. MPN technique was performed for 34 another positive samples; 12 had > or = 1100/g, five ranged between 240 and 93, eight between 23 and three and nine had < 3.0. No L. monocytogenes was recovered after cooking 12 contaminated frozen samples. Isolation of strains was done using three selective agars. Sixty-two L. monocytogenes were isolated from lithium chloride phenylethanol moxalactam agar, 95 from Listeria selective agar Oxford formulation, and 103 from polymixin acriflavine lithium chloride ceftazidime aesculin mannitol agar. Fifty isolates (45.5%) belong to PCR group IIb (including strains serovar 1/2b), 41 (37.3%) to PCR group IVb (including strains serovar 4b), 17 (15.5%) to PCR group IIa (including strains serovar 1/2a), and 2 (1.8%) to PCR group IIc. With the use of DNA macrorestriction patterns analysis, 17 different clusters were detected among 71 isolates, with P10, the most frequent with 25 isolates (35.2%) of PCR group IIb.

Role of FliF and FliI of Listeria monocytogenes in Flagellar Assembly and Pathogenicity

ABSTRACT Flagellar structures have been shown to participate in virulence in a variety of intestinal pathogens. Here, we have identified two potential flagellar genes of Listeria monocytogenes: lmo0713, encoding a protein similar to the flagellar basal body component FliF, and lmo0716, encoding a protein similar to FliI, the cognate ATPase energizing the flagellar export apparatus. Expression of fliF and fliI appears to be downregulated at 37°C, like that of flaA, encoding flagellin. By constructing two chromosomal deletion mutants, we show that inactivation of either fliF or fliI (i) abolishes bacterial motility and flagella production, (ii) impairs adhesion and entry into nonphagocytic epithelial cells, and (iii) also reduces uptake by bone marrow-derived macrophages. However, the ΔfliF and ΔfliI mutations have only a minor impact on bacterial virulence in the mouse model, indicating that the flagellar secretion apparatus itself is not essential for survival in this animal model. Finally, among 100 human clinical isolates of L. monocytogenes tested, we found 20 strains still motile at 37°C. Notably, all these strains adhered less efficiently than strain EGD-e to Caco-2 cells at 37°C but showed no defect of intracellular multiplication. These data suggest that expression of the flagella at 37°C might hinder optimal adhesion to epithelial cells but has no impact on intracytosolic survival of L. monocytogenes.

Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b: toward an international standard.

The performance of a multiplex PCR assay that separates the four major serovars of the pathogenic Listeria monocytogenes into four distinct PCR groups was evaluated through a multicenter typing study. Identical panels of 90 Listeria isolates were distributed to five participating laboratories that were blind to the nature of the isolates. Isolates were characterized using the previously standardized protocol. Overall concordance was 96.6 to 100%, sufficient for the assay to be used as an alternative to serotyping and confidently applied in laboratories involved in L. monocytogenes typing.

Assignment of Listeria grayi and Listeria murrayi to a single species, Listeria grayi, with a revised description of Listeria grayi

The genomic relatedness between Listeria grayi and Listeria murrayi was reevaluated by using DNA-DNA hybridization, multilocus enzyme electrophoresis, and rRNA restriction fragment length polymorphism techniques. The high levels of similarity observed between the strains of these two species confirmed the data published since 1973 and indicated that they should be considered members of a single species. On grounds of priority, the species should be named L. grayi.

Listeria ivanovii subsp. londoniensis subsp. nov.

An analysis of 23 Listeria ivanovii strains in which we used multilocus enzyme electrophoresis at 18 enzyme loci showed that this bacterial species could be divided into two main genomic groups. The results of DNA-DNA hybridizations and rRNA gene restriction patterns confirmed this finding. The DNA homology data suggested that the two genomic groups represent two subspecies, L. ivanovii subsp. ivanovii and L. ivanovii subsp. londoniensis subsp. nov. The two subspecies can be distinguished biochemically on the basis of the ability to ferment ribose and N-acetyl-β-d-mannosamine. The type strain of L. ivanovii subsp. londoniensis is strain CLIP 12229 (=CIP 103466).