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.

Characterization of human invasive isolates of Listeria monocytogenes in Sweden 1986-2007.

Since 1986, 68% of the Listeria monocytogenes isolates from human cases of invasive listeriosis in Sweden are available for retrospective studies. The aim of the present study was to characterize 601 human invasive isolates of L. monocytogenes in Sweden from 1986 to 2007 by using serotyping and pulsed-field gel electrophoresis. Since 1996, serovar 4b was permanently reduced to the second or third most common serovar in human cases in Sweden. During the latter period, 2000-2007, only 13% belonged to serovar 4b and 71% to 1/2a. The dendrogram, based on pulsovars, reveals two clusters with different serovars. Cluster 1 exhibits serovars 4b and 1/2b, whereas cluster 2 consists of serovar 1/2a. Serovar 1/2a seems to be more heterogeneous than serovar 4b.

Phenotypic and molecular characterization of Listeria monocytogenes strains isolated from a marine environment in Morocco.

Microbiological analysis of 1025 marine samples, including 345 from seawater, 337 from shellfish, and 343 from sediments collected between January 2000 and December 2002 from 18 shellfish sites on the Atlantic coast of mid-west of Morocco (Agadir region), yielded 143 strains of Listeria (Listeria monocytogenes: 38; L. innocua: 109; L. ivanovii: 1). The overall incidence of Listeria sp. in the coastal environment was 5.3%. Thirteen L. monocytogenes strains were isolated from seawater, 7 from sediment, and 12 from shellfish. The 38 strains of L. monocytogenes were phenotypically characterized. All belonged to two chemotypes according to appareillage et procédé d'identification (API) Listeria classification: 8 strains were type 2510, alpha-mannosidase-negative and hemolytic; and 30 strains were type 6510, alpha-mannosidase-positive, of which 8 strains were nonhemolytic. All the L. monocytogenes strains belonged to the 1/2 serogroup, with serovar 1/2b clearly prevalent (78.9%), although some nonhemolytic strains were serovar 1/2a. This collection of L. monocytogenes strains included 6 different pulsotypes as assessed by DNA macrorestriction with the restriction enzymes AscI and ApaI.

Pathogenomics of Listeria spp.

This review provides an overview of recent progress in the exploration of genomic, transcriptomic, and proteomic data in Listeria spp. to understand genome evolution and diversity, as well as physiological aspects of metabolism utilized by the bacteria when growing in diverse and varied environments.

Identification and molecular characterization of Listeria monocytogenes isolated in raw milk in the region of Algiers (Algeria).

Listeria monocytogenes was isolated from raw milk, whey and curdled milk produced and collected in the region of Algiers and Blida between September 2003 and July 2004. Four out of 153 (2.61%) farm milk samples and 6 out of 80 (7.50%) tankers' samples tested positive for L. monocytogenes. All samples of whey and curdled milk (n=12) tested negative for L. monocytogenes, but 2 of 22 (9%) samples of whey were contaminated by L. innocua. L. monocytogenes isolates were grouped by a multiplex PCR assay; all isolates belonged to the PCR-group IVb, which corresponds to serovars 4b, 4d and 4e. L. monocytogenes isolates were characterized by Pulsed-Field Gel Electrophoresis (PFGE). The combination of AscI and ApaI macrorestriction patterns yielded five different pulsovars (I to V). The results indicate that raw milk, and raw milk products are potential sources of the L. monocytogenes and represent a potential risk for consumers.

Genetic diversity of Listeria monocytogenes recovered from infected persons and pork, seafood and dairy products on retail sale in France during 2000 and 2001.

Growth of the food-borne human pathogen Listeria monocytogenes to large numbers in ready-to-eat food products greatly increases the risk of disease for susceptible consumers. A better knowledge of the population structure of L. monocytogenes present in retailed food could allow better prevention strategies to be developed. We present the analysis of 450 L. monocytogenes isolates, 179 responsible for sporadic human cases of listeriosis and 271 isolated from foods collected from retailers. All isolates were investigated by multiplex PCR (food isolates), allowing serovar predictions, or serotyped (human isolates), and DNA macrorestriction patterns were determined. Isolates from different sources were significantly differently distributed into PCR groups. PCR group IIa, corresponding to serovars 1/2a and 3a, was predominant in food isolates (58%; OR=3.19; P<1 x 10(-7)). A larger proportion of human isolates belonged to PCR group IVb, corresponding to serovars 4b, 4d and 4e (44%; OR=5.69; P<1 x 10(-7)). DNA macrorestriction pattern analysis of PCR group IIa isolates showed that isolates from pork products had a very low diversity (ID=0.905) whereas isolates from humans were more diverse (ID=0.976). Furthermore, 78% of the pork product isolates belonging to PCR group IIa exhibited only two AscI profiles, a(1) and a(2), which were very similar (94%). DNA array analysis of representative isolates showed that isolates with a(1) and a(2) profiles constitute a homogeneous population, whereas isolates exhibiting non a(1)-a(2) profiles are more diverse. Six of the isolates with a(1) and a(2) profiles were selected and investigated for their gene content using a DNA array. With respect to 295 strains present in our data collection, a specific pattern of the presence and absence of 15 genes was identified. Five are predicted to encode internalins and cell surface proteins, and eight of the genes were missing in this group. They code for cell surface proteins, transcriptional regulators, an acylase, a sugar phosphorylase and proteins of unknown functions. The ability of strains to multiply in different niches may be determined by the presence or absence of these genes.

Emoção e experiência corporal na trajetória da conversão: um estudo de caso / Emotion and corporal experience in the trajectory of conversion: a case study

O texto tem por objetivo discutir a articulação existente entre doença, corpo e emoções nas narrativas de cura religiosa. Partimos de casos concretos de mulheres convertidas ao neopentecostalismo que contam ter encontrado nos ritos e rituais religiosos uma alternativa para lidar com sofrimentos e aflições pessoais e/ou familiares. As histórias de conversão relatam uma vida caracterizada por um sentimento de desvalorização de si e a conversão ocorre durante um episódio de crise ou de uma experiência extraordinária que quebra o curso normal da trajetória de vida. As convertidas são quase unânimes em afirmar que a conversão foi resultado da busca de uma solução para uma doença; no entanto, a doença relatada pelas fiéis não é senão um quadro genérico no interior do qual são organizadas evidências que visam deslocar a atenção daqueles que as escutam para o caráter eficaz dos rituais religiosos. A motivação inicial da conversão não estava assim ligada unicamente à doença, mas a um conjunto de situações problemáticas.(AU)

This article discusses the relationship that exists between illness, emotions, and the body in narratives concerning religious cures. The point of departure are concrete cases of women who have converted to neo-Pentecostalism and who relate how they discovered an alternative to dealing with their afflictions and suffering through religious rites and rituals. Their stories of conversion revolve around lives characterized by loss of self-esteem, and their subsequent conversions took place during moments of crisis or as a result of extraordinary events that interrupted the normal course of their lives. The converted women are almost unanimous in affirming that their conversions came about as the result of a search for the cure for an illness. However, the illness they refer to is a generic view beneath which lies evidence that shifts the listeners' attention to the efficient nature of the religious rituals. Thus, their initial motivation in seeking for conversion was not related solely to their illnesses, but to a prior series of problem situations that went beyond the actual physical ailments in themselves. Inspired by an anthropology of emotions, this text aims to analyze how religious conversion helped these women to better face their personal and family conflicts, creating a self-awareness that helped them to reemerge from the depths of their despair.(AU)

Emoção e experiência corporal na trajetória da conversão: um estudo de caso / Emotion and corporal experience in the trajectory of conversion: a case study

O texto tem por objetivo discutir a articulação existente entre doença, corpo e emoções nas narrativas de cura religiosa. Partimos de casos concretos de mulheres convertidas ao neopentecostalismo que contam ter encontrado nos ritos e rituais religiosos uma alternativa para lidar com sofrimentos e aflições pessoais e/ou familiares. As histórias de conversão relatam uma vida caracterizada por um sentimento de desvalorização de si e a conversão ocorre durante um episódio de crise ou de uma experiência extraordinária que quebra o curso normal da trajetória de vida. As convertidas são quase unânimes em afirmar que a conversão foi resultado da busca de uma solução para uma doença; no entanto, a doença relatada pelas fiéis não é senão um quadro genérico no interior do qual são organizadas evidências que visam deslocar a atenção daqueles que as escutam para o caráter eficaz dos rituais religiosos. A motivação inicial da conversão não estava assim ligada unicamente à doença, mas a um conjunto de situações problemáticas.

Use of DNA arrays for the analysis of outbreak-related strains of Listeria monocytogenes.

We evaluated the discriminative power and usefulness of the DNA array technology as compared to DNA macrorestriction pattern analysis for monitoring epidemiologically related clusters of Listeria monocytogenes strains that differ slightly in DNA macrorestriction patterns. We show that this approach allows clarifying the genetic basis of the pattern variations. In the reported outbreak, the differences were due to phage excision, showing the power of this technique in epidemiological studies.

Pulsed-field gel electrophoresis of Listeria monocytogenes strains: the PulseNet Europe Feasibility Study.

A feasibility study on standardized subtyping of Listeria monocytogenes by pulsed-field gel electrophoresis (PFGE) was performed in 2003 in Europe. The aim of the project was to identify veterinary, food, and public health reference laboratories that were willing to participate in the molecular surveillance of Listeria infections in Europe, as is done with PulseNet USA, and to test if the participants could generate results that were comparable with each other. A panel of strains and the methodology were sent to European laboratories, and images of PFGE gels were transferred electronically, then analyzed visually and with image analysis software. Only nine out of 43 gels were not fully satisfactory at the visual evaluation step stage. Computerized analysis showed that only three out of 33 gels gave dendrograms totally different from the reference dendrograms. Thus, the objectives of the study were met: most human and food European laboratories concerned with typing L. monocytogenes participated, images were transmitted electronically, and the overall quality of the gels was good.

Genotypes of Listeria monocytogenes strains isolated from 2000 to 2002 in Poland.

Pulsed field gel electrophoresis (PFGE), multiplex PCR and multilocus sequence typing (MLST) methods were used for genotyping study of seventy-three L. monocytogenes isolates collected in Poland between 2000 and 2002 from human, food, environment and a diseased goat. The multiplex PCR, which is an alternative method to classical serotyping, divided the isolates into four PCR groups, IIa (42.5%), IIb (27.4%), IIc (4.1%) and IVb (26%). The isolates displayed 33 distinct PFGE profiles. Twenty eight strains were further characterised by MLST based on sequence analyses of seven housekeeping genes. The combined sequence analyses revealed a total of 10 different allelic profiles from which 3 were not described earlier. It is intended that results obtained in this study will be the first data of a national database of L. monocytogenes genotypes in Poland.

Comparative characterization of Listeria monocytogenes isolated from Portuguese farmhouse ewe's cheese and from humans.

In order to investigate the possible relationships between Listeria monocytogenes strains isolated from farmhouse ewe's cheese and clinical strains collected, in partially overlapping dates, from the same geographical area in Portugal, a total of 109 isolates from seven ewe's cheese manufactures (n=94) and from humans (n=15) were characterized by serotyping, RAPD, PFGE and allelic analysis of the virulent actA gene. Serotyping indicated the presence of four different serovars: 1/2a, 1/2b, 1/2c and 4b. The 15 clinical isolates were either serovar 4b (86.7%) or serovar 1/2b (13.3%). Among the 94 isolates from cheese and related environments the serovars prevalence was 1/2a (1.1%), 1/2b (17.0%), 1/2c (12.8%) and, unexpectedly, 4b (69.1%). Based on results obtained with PFGE typing of the strains, 25 genotypes were identified, 10 from farmhouses and 15 from human cases. Isolates from serovars 1/2a and 1/2c were assigned to single genotypes, respectively. Within serovars 1/2b and 4b three and 20 genotypes were established, respectively. RAPD typing of the isolates rendered 18 types indicating the lack of accuracy of the primers used in strain differentiation within serovar 4b. The actA gene typing of the strains showed a prevalence of actA gene type I (90.4%) compared with the rest of the strains that were all actA gene type II (9.6%). In spite of the fact that all the farmhouses were completely independent, the distribution of L. monocytogenes genotypes, intra and inter cheese manufactures, was relatively homogeneous, suggesting the existence of resident strains. In contrast, among human isolates there was a great genetic diversity. There was no common genotype between L. monocytogenes implicated in the cases of listeriosis and these cheese-related isolates, suggesting the absence of a causal relationship.

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.

Cellular lipid fatty acid pattern heterogeneity between reference and recent food isolates of Listeria monocytogenes as a response to cold stress.

Cells of four reference strains (Scott A, LO 28, CNL 895807 and ATCC 19115) and of five recent food isolates (A00M011, A00M018, A00M087, A00M092 and A00M123) of Listeria monocytogenes were grown until late exponential phase in Brain Heart Broth at two different temperatures (37 degrees C and 4 degrees C). Our results show that significant differences exist between the cellular lipid fatty acid profile of reference and recent food isolates. Like the reference strains, and in keeping with previous reports on the cellular lipid fatty acid profile of L. monocytogenes, the recent food isolates were characterised by the presence of ai15:0, i15:0 and ai17:0. In addition, the fatty acid ai13:0 was observed in all of the recent food isolates grown at 4 degrees C, whereas only two reference strains, Scott A and LO 28, showed ai13:0 in their cellular lipid fatty acid profile at 4 degrees C. When grown at 4 degrees C, the recent food isolates showed a mean aiC15/aiC17 ratio of 66, while reference strains were characterised by significantly lower ratios, ranging between 4.3 (ATCC 19115) and 28.9 (Scott A). These results showed that all of the recent food isolates, Scott A and LO28 strains use chain length and anteiso-branching (ai15:0) as their major response to cold temperature adaptation. However, the cold adaptation response of reference strains CNL 895807 and ATCC 19115 appears to be different.

Role of FliF and FliI of Listeria monocytogenes in flagellar assembly and pathogenicity.

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 degrees 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 DeltafliF and DeltafliI 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 degrees C. Notably, all these strains adhered less efficiently than strain EGD-e to Caco-2 cells at 37 degrees C but showed no defect of intracellular multiplication. These data suggest that expression of the flagella at 37 degrees C might hinder optimal adhesion to epithelial cells but has no impact on intracytosolic survival of L. monocytogenes.

Differentiation of the major Listeria monocytogenes serovars by multiplex PCR.

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.

Sequence and binding activity of the autolysin-adhesin Ami from epidemic Listeria monocytogenes 4b.

Ami is an autolytic amidase from Listeria monocytogenes that is targeted to the bacterial surface via its C-terminal cell wall anchoring (CWA) domain. We recently showed that the CWA domain from Ami of L. monocytogenes EGD (serovar 1/2a) (Ami 1/2a) mediated bacterial binding to mammalian cells. Here we studied the sequence and binding properties of Ami from CHUT 82337 (serovar 4b) (Ami 4b). The Ami 4b polypeptide is predicted to be 770 amino acids long (compared with the 917 amino acids of Ami 1/2a from EGD). Ami 1/2a and Ami 4b are almost identical in the N-terminal enzymatic domain (approximately 98% amino acid identity), but the sequence is poorly conserved in the C-terminal CWA domain, with only approximately 54% amino acid identity and eight GW modules in Ami 1/2a compared with six GW modules in Ami 4b. The purified Ami 4b CWA domain efficiently bound serovar 4b bacterial cells and only poorly bound serovar 1/2a bacterial cells. The Ami 4b CWA domain was also significantly less able to bind Hep-G2 human hepatocytic cells than the Ami 1/2a CWA domain. We sequenced the ami regions encoding CWA domains of reference strains belonging to the 12 L. monocytogenes serovars. The phylogenic tree constructed from the sequences yielded a binary division into group I (serovars 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, and 7) and group II (serovars 4a, 4b, 4c, 4d, and 4e). This is the first direct evidence of divergence between serovars 1/2a and 4b in a gene involved in the adhesion of L. monocytogenes to mammalian cells, as well as the first demonstration of allelic polymorphism correlated with the somatic antigen in this species.

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.

New aspects regarding evolution and virulence of Listeria monocytogenes revealed by comparative genomics and DNA arrays.

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.

Efflux pump Lde is associated with fluoroquinolone resistance in Listeria monocytogenes.

Five Listeria monocytogenes isolates (CLIP 21369, CLIP 73298, CLIP 74811, CLIP 75679, and CLIP 79372) were found to be resistant to fluoroquinolones during the screening for antibiotic resistance of 488 L. monocytogenes isolates from human cases of listeriosis in France. On the basis of a fourfold or greater decrease in the ciprofloxacin MIC in the presence of reserpine, fluoroquinolone resistance was attributed to active efflux of the drugs. The lde gene (Listeria drug efflux; formerly lmo2741) encodes a 12-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of secondary transporters that displayed 44% identity with PmrA from Streptococcus pneumoniae. Insertional inactivation of the lde gene in CLIP 21369 indicated that the corresponding protein was responsible for fluoroquinolone resistance and was involved in the level of susceptibility to dyes such as ethidium bromide and acridine orange.