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.Listeria monocytogenes is a facultative intracellular pathogen that can cause serious illness in susceptible individuals. Persons with specific immunocompromising conditions, pregnant women, newborns, and the elderly are particularly at risk for listeriosis (9, 23). Although rare, listeriosis remains of great public health concern due to its high mortality rate (20 to 30%) (16). Ingestion of contaminated foods is considered to be the primary source of infection for both sporadic and epidemic human listeriosis cases (19). Because of the importance of L. monocytogenes strain characterizations for epidemiological investigations, a number of discriminatory subtyping methods have been described for this organism (2,4,5,18,20,24,25). Pulsed-field gel electrophoresis (PFGE) typing, which has provided the most sensitive strain discrimination up to now, has rapidly become the standard subtyping method to detect listeriosis outbreaks (4, 11). However, this method is labor-intensive and time-consuming and thus for practical purposes is often preceded by serotyping. Since all major outbreaks of the invasive form of listeriosis are due to serovar 4b strains, an infrequent serovar in foods compared to 1/2a strains (6, 9), the procedure adopted for outbreak investigations relies upon serovar characterization to provide valuable information for rapid screening of groups of strains. Indeed, the serovar information allows discrimination between isolates probably belonging to an outbreak and those that are not part of the outbreak and thus decreases the number of strains which need to be characterized by PFGE in order to improve discrimination beyond the serovar level. Moreover, serotyping is widely used for long-term microbiological surveillance of human listeriosis. For the food industry, where the presence of L. monocytogenes is a big concern, tracing contaminating strains within the food chain and the plant environment is of primary importance. Again serotyping is often used as a first-line typing method. Although 13 serovars are described for the species L. monocytogenes, at least 95% of the strains isolated from foods and patients are of serovars 1/2a, 1/2b, 1/2c, and 4b (12,21,22). Routine analysis of L. monocytogenes by serotyping with traditional agglutination methods is limited by cost, availability, and the need for technical expertise to perform the assay. Furthermore, the reproducibility of serotyping is not always satisfactory. Schonberg et al. (20) concluded in a multicenter study that a critical need exists for high-quality antisera. A new enzyme-linked immunosorbent assay serotyping format used in conjunction with a commercially available kit to make serotyping more efficient and more ...
SummaryHuman dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34 + hematopoietic progenitors in presence of GM-CSF+TNFet for 12 d. The present study demonstrates that cord blood CD34 + HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CDla and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CDS0, CD83, CD86, CD58, high HLA class II). CDla + precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14 + progenitors mature into CDla + DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIlla described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA + naive T cells. Interestingly, the CD14 + precursors, but not the CDla + precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T ceils.Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14+-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.
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.
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.
Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34 + hematopoietic progenitors in presence of GM-CSF+TNFet for 12 d. The present study demonstrates that cord blood CD34 + HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CDla and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CDS0, CD83, CD86, CD58, high HLA class II). CDla + precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14 + progenitors mature into CDla + DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIlla described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA + naive T cells. Interestingly, the CD14 + precursors, but not the CDla + precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T ceils.Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14+-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.Preliminary results were presented at
To define the prevalence of antibiotic resistance in Listeria species pathogenic for humans and animals, 1100 isolates (60 from cases of listeriosis and 1040 from food and environment) collected worldwide were screened. Of the 61 tetracycline- and minocycline-resistant strains (37 Listeria monocytogenes), 57 harbored tet(M); 4 non-L. monocytogenes isolates contained tet(S). One Listeria innocua isolate was also resistant to streptomycin and contained the tet(M) and aad6 genes. An L. monocytogenes isolate was trimethoprim-resistant, a characteristic not reported previously in Listeria species, because of the presence of a yet-uncharacterized gene. Three clinical isolates of L. monocytogenes were resistant to low levels of streptomycin. Since the tet(M), tet(S), and aad6 genes are common in enterococci and streptococci, these data suggest transfer from the latter to Listeria species. Uniform susceptibility to tetracycline, minocycline, trimethoprim, and streptomycin cannot be assumed any longer for Listeria species.
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.Listeria monocytogenes is widely distributed in the environment and can cause serious human infections, such as bacteremia and central nervous system infections, primarily in neonates and immunocompromised adults, and abortions (22). Food-borne transmission is recognized as the main route of acquisition of the infection during epidemic and sporadic listerioses (3, 18). Listeriosis differs from most food-borne diseases by its high fatality rate (20 to 30% of cases), despite the administration of appropriate antibiotics (3, 9). L. monocytogenes is generally susceptible to a wide range of antibiotics but is not susceptible to cephalosporins and fosfomycin (8). However, during the last few years, increasing numbers of strains resistant to one or more antibiotics have been reported (1,2,8,11,19,20).Although indications for the use of fluoroquinolones do not include listeriosis, they can, due to their increasing use for other pathologies such as respiratory tract infections, select resistant Listeria. In gram-positive bacteria, resistance usually results from mutational alterations in the so-called quinolone resistance-determining regions (QRDRs) of the intracellular targets of fluoroquinolones, the type II DNA topoisomerases gyrase and topoisomerase IV, or active export of the drugs via efflux pumps (12). We have detected ciprofloxacin-resistant L. monocytogenes clinical isolates and characterized the efflux pump involved in resistance.(An initial report of this work was presented at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy [S. Godreuil, M. Galimand, G. Gerbaud, and P. Courvalin, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. UL-11, 2001].) MATERIALS AND METHODSStrains, plasmids, and growth conditions. Screening of 488 L. monocytogenes isolates responsible for human listeriosis in France for resistance to fluoroquinolones was carried out on brain heart infusion agar (Difco Laboratories, Detroit, Mich.) containing 4 g of ciprofloxacin per ml. Strains that were found to be resistant and susceptible L. monocytogenes EGD were studied ( Table 1)....
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