Detection of Shiga toxin-producing Escherichia coli in meat marketed in Casablanca (Morocco).

The contamination of meat and meat products with Shiga toxin-producing O157:H7 and non-O157 Escherichia coli (STEC), obtained from markets in Casablanca, Morocco, was investigated. A total of 460 meat and meat products were sampled between March 2004 and July 2006 analysed and 176 strains of E. coli were isolated from these samples. The presence of the stx1, stx2, eae and ehxA genes, recognized as major virulence factors of STEC, was tested in E. coli isolates by polymerase chain reaction (PCR). STEC was detected in 4 (0.9%) samples. The result of serotyping by molecular method showed that two of these STEC isolates corresponded to the serotype O157:H7. The others Shiga toxin-producing E. coli non-O157 corresponded to O6:H21 and O76:H19. The presence of O157:H7 and non-O157 STEC in meat and meat products marketed in Casablanca, Morocco, emphasizes the importance of implementing the Hazard Analysis and Critical Control Point (HACCP) system, as well as the need for implementing, evaluating, and validating antimicrobial interventions to reduce the presence of potential pathogenic microorganisms.

A new multiplex PCR for differential identification of Shigella flexneri and Shigella sonnei and detection of Shigella virulence determinants.

Most of the multiplex PCR (mPCR) used to identify Shigella do not discriminate between Shigella species or serotypes. We designed a mPCR to differentiate between S. flexneri and S. sonnei strains based on the detection of markers associated with the she pathogenicity island described in Shigella. In addition, specific primers were included to detect the Shigella virulence determinants ShET-1 and ShET-2 enterotoxin genes. The analysis of 304 Shigella strains from Chile and 79 Shigella strains from other geographic locations indicated that the mPCR described here detected all Shigella species and specifically differentiated S. flexneri and S. sonnei. The technique was sensitive, reproducible, specific and simple to perform, providing a new tool with the potential to be employed for epidemiological and diagnostic purposes.

Molecular typing of Escherichia coli strains isolated from food in Casablanca (Morocco).

Serotyping of O- and H- antigens is regarded as the gold standard in classification of E. coli for taxonomic and epidemiological purposes similar to the Kaufmann-White scheme for Salmonella enterica. Molecular methods to replace or to support the serotyping were recently applied. Using the molecular polymorphism of the somatic (O- antigen) gene rfb cluster and flagella (H- antigen) gene fliC, 74 E. coli strains carrying the virulent genes isolated from food were characterised by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The results showed that 49 (66%) of the isolates revealed a reproducible and clear cut classification, with a very good correlation to the collection of reference strains, were found.

[Molecular epidemiology of tuberculosis in Guadeloupe from 1994 to 2000]. / Epidémiologie moléculaire de la tuberculose en Guadeloupe de 1994 à 2000.

In Guadeloupe, the incidence of tuberculosis decreased between 1994 and 2000. The rate of resistance to at least one antibiotic remained constant at 11%, whereas the rate of multiple-drug resistance increased from 0.9 to 2.4% in 2000. The proportion of patients of foreign origin (mainly from Haiti and the Dominican Republic) increased whereas the number of French patients decreased. These results show that the epidemiology of tuberculosis in Guadeloupe is similar to industrialized countries as older people, foreigners from countries where TB is endemic, and HIV+ patients are at a higher risk to declare tuberculosis disease. Molecular typing realized by spoligotyping showed the importance of previous successive colonizations and migrations as characterized by the presence of major phylogenetic families originating essentially from Northern Europe (Haarlem), Latin America and Mediterranean (LAM) and from Anglo-Saxon countries (X). The sub-typing of clustered strains by IS6110-RFLP and by a PCR method based on the variable number of tandem DNA repeats (VNTR), highlighted 29 clusters, corresponding to 44.8% of clustered strains, and allowed to estimate the rate of recent transmission at 32.2%. The epidemiologic data associated with fingerprinting results underlined the importance of reactivation cases among older people, a significant number of imported TB cases without evident links, and casual contacts.

[Molecular characterization and biodiversity of Mycobacterium tuberculosis in the Antilles-Guiana region and comparative analysis in a metropolitan region, Aquitaine]. / Caractérisation moléculaire et biodiversité de Mycobacterium tuberculosis dans la région Antilles-Guyane et analyse comparative à une région métropolitaine, l'Aquitaine.

Tuberculosis is a highly contagious infectious disease in recrudescence whose epidemiologic monitoring is reinforced by molecular biology. In this context, we were particularly interested in the cases of tuberculosis of French West Indies and French Guiana (FWI-FG). This study covered a period of two years (1997 and 1998) and focused on the demographical and epidemiological characteristics of the cases diagnosed by an analysis of their genotypes. Our results were confronted with a French metropolitan area (Aquitaine) with similar demographic background. Moreover, Aquitaine area has privileged links with FWI-FG region and also has a similar network for monitoring tuberculosis as ours. So we used a PCR method called spoligotyping as a first line method to optimize the alternative IS6110-RFLP method which remains cumbersome. A total of 105 strains of FWI-FG and 172 strains of Aquitaine were typed by spoligotyping and by the standard IS6110-RFLP method. The results of the first grouping by spoligotyping were analyzed in comparison with IS6110-RFLP. The results obtained showed a rate of recent transmission of tuberculosis being 34.3% in FWI-FG and 10.5% in Aquitaine. These observations underlined a high degree of polymorphism in the Aquitaine region as compared to the FWI-FG region. Thanks to the various profiles obtained by spoligotyping, we could study their distribution in the three areas and highlight common types like type 53, 50 and 42 and types found locally like the types 33 and 14 found respectively in Aquitaine and FWI as well as endemic types like type 76 found only in FG. These results are discussed in the context of the evolution of clinical isolates of tubercle bacilli with time.

[Epidemiological study of tuberculosis in the area of Angers, France, as studied by 3 PCR-based fingerprinting methods]. / Epidémiologie moléculaire de la tuberculose dans l'agglomération angevine étudiée par une stratégie d'association de 3 méthodes de génotypage par PCR.

The new genotyping methods efficiently complement classical epidemiological investigation in order to attempt a global approach to TB control. In the present work, we have studied the genomic diversity of Mycobacterium tuberculosis isolated during the year 1998 within the district of Angers, France (260,000 inhabitants distributed in 29 districts), in order to identify recent transmission events and any related risk factors. The methods used included "spacer oligonucleotide typing" or spoligotyping, "variable number of DNA tandem repeats" or VNTR, and "double repetitive element PCR" or DRE-PCR. The resulting spoligotyping and VNTR results were also feeded to international databases and compared with >10,000 isolates for spoligotyping and 500 isolates for VNTR, representative of about 60 countries. The results obtained underlined that most of the TB cases in our setting probably reflected reactivation cases, as clustered cases indicative of potential events of recent transmission were rare. Furthermore, interrogation of international databases showed that most of the isolates from the Angers region belonged to major conserved families of TB isolates representative of Europe, with only rare cases of Asian origin, or those previously reported in specific epidemies reported from elsewhere.

Mycobacterium tuberculosis phylogeny reconstruction based on combined numerical analysis with IS1081, IS6110, VNTR, and DR-based spoligotyping suggests the existence of two new phylogeographical clades.

This paper deals with phylogenetic relationships among a set of 90 clinical strains representative of the worldwide diversity of the Mycobacterium tuberculosis complex (Kremer et al. 1999) using eight independent genetic markers: IS6110, IS1081, the direct repeat (DR) locus, and five variable number of tandem DNA repeat loci (VNTR). In a preliminary experiment, phylogenetic trees based on single markers were constructed that led to the detection of some similarities between the VNTR-based and the spoligotyping-based phylogenetic trees. In the second step, a more global phenetic approach based on pairwise comparison of strains within each typing system was used, followed by calculations of mean genetic distances based on all the eight loci and the use of the neighbor-joining algorithm for tree reconstruction. This analysis confirmed our preliminary observations and suggested the existence of at least two new phylogeographical clades of M. tuberculosis, one defined as the "East African-Indian family" (EA-I), which may find its origin on the African or Asian continents, and the other as the "Latin American and Mediterranean" (LA-M) family. The existence of these two families was also validated by an independent phylogenetic analysis of spoligotyping on a larger set of shared types (n = 252) and further corroborated by VNTR and katG-gyrA results. The potential origin of these families of bacilli is discussed based on cattle domestication and human migration history. In conclusion, the information contained in insertion sequence and repetitive DNAs may serve as a model for the phylogenetic reconstruction of the M. tuberculosis complex.

Spoligotype database of Mycobacterium tuberculosis: biogeographic distribution of shared types and epidemiologic and phylogenetic perspectives.

We give an update on the worldwide spoligotype database, which now contains 3,319 spoligotype patterns of Mycobacterium tuberculosis in 47 countries, with 259 shared types, i.e., identical spoligotypes shared by two or more patient isolates. The 259 shared types contained a total of 2,779 (84%) of all the isolates. Seven major genetic groups represented 37% of all clustered isolates. Two types (119 and 137) were found almost exclusively in the USA and accounted for 9% of clustered isolates. The remaining 1,517 isolates were scattered into 252 different spoligotypes. This database constitutes a tool for pattern comparison of M. tuberculosis clinical isolates for global epidemiologic studies and phylogenetic purposes.

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France.

We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100% (16/16) from France and 89% (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2%), 526, 513 and 533 (18.7% each). In Brazilian strains the most common mutations were in codons 531 (72.2%), 526 (11.1%) and 513 (5.5%). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients.

Use of spoligotyping to study the evolution of the direct repeat locus by IS6110 transposition in Mycobacterium tuberculosis.

Based on the variability of 43 spacers within the direct repeat (DR) locus of Mycobacterium tuberculosis complex organisms, spoligotyping is a rapid method that aids in the study of the epidemiology of tuberculosis. It was recently hypothesized that despite its presence in the DR locus, spacer 31 could not be amplified in M. tuberculosis clinical isolates belonging to spoligotype 50 due to the insertion of an extra copy of IS6110 between spacers 31 and 32 that could lead to an asymmetrical split of the primer targets (I. Filliol, C. Sola, and N. Rastogi, J. Clin. Microbiol. 38:1231--1234, 2000). In the present investigation, previous observations were extended to 25 clinical isolates of type 50 showing that the primer set IS6-DRb that selectively amplified the left and central DR regions was indeed able to demonstrate the presence of spacer 31. IS6110-restriction fragment length polymorphism (RFLP) and DR-RFLP showed that type 50 isolates were characterized by the presence of two copies of IS6110 associated with the DR locus and an additional double IS6110 band of 1.4 kb. The primer set IS3-IS6 was then used to selectively amplify a 750-bp inter-IS6110 fragment within the DR locus. The sequencing of the central DR region corroborated our previous findings and showed that the absence of spacer 31 among the type 50 isolates was due to the asymmetric insertion of an extra copy of IS6110 between spacers 31 and 32, leading to an unequal split of the DRa-DRb target into two portions, of 6 and 30 bp, respectively. These results show that the DR locus constitutes an ideal IS6110 preferential locus (ipl), permitting the insertion of two or more copies of IS6110, and provide new clues for epidemiological and phylogenetic interpretation of changes in IS6110-RFLP and spoligotyping profiles.

Mutations in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis strains isolated in Brazil and France

We evaluated the mutations in a 193bp of the rpoB gene by automated sequencing of rifampicin (RMP)-resistant and susceptible Mycobacterium tuberculosis strains isolated from Brazil (25 strains) and France (37 strains). In RMP-resistant strains, mutations were identified in 100 percent (16/16) from France and 89 percent (16/18) from Brazil. No mutation was detected in the 28 RMP-susceptible strains. Among RMP-resistant or RMP-susceptible strains deletion was observed. A double point mutation which had not been reported before was detected in one strain from France. Among French resistant strains mutations were found in codons 531 (31.2 percent), 526, 513 and 533 (18.7 percent each). In Brazilian strains the most common mutations were in codons 531 (72.2 percent), 526 (11.1 percent) and 513 (5.5 percent). The heterogeneity found in French strains may be related to the fact that most of those strains were from African or Asian patients

Genetic diversity of Mycobacterium africanum clinical isolates based on IS6110-restriction fragment length polymorphism analysis, spoligotyping, and variable number of tandem DNA repeats.

A collection of 105 clinical isolates originally identified as Mycobacterium africanum were characterized using both phenotypic and genotyping methods. The phenotypic methods included routine determination of cultural properties and biochemical tests used to discriminate among the members of the M. tuberculosis complex, whereas genotypic characterization was based on IS6110-restriction fragment length polymorphism (IS6110-RFLP) analysis, IS1081-RFLP analysis, direct repeat-based spacer oligonucleotide typing (spoligotyping), variable number of tandem DNA repeats (VNTR), and the polymorphism of the oxyR, pncA, and mtp40 loci. The results obtained showed that a majority of M. africanum isolates were characterized by a specific spoligotyping pattern that was intermediate between those of M. tuberculosis and M. bovis, which do not hybridize with spacers 33 to 36 and spacers 39 to 43, respectively. A tentative M. africanum-specific spoligotyping signature appeared to be absence of spacers 8, 9, and 39. Based on spoligotyping, as well as the polymorphism of oxyR and pncA, a total of 24 isolates were excluded from the final study (19 were identified as M. tuberculosis, 2 were identified as M. canetti, and 3 were identified as M. bovis). The remaining 81 M. africanum isolates were efficiently subtyped in three distinct subtypes (A1 to A3) by IS6110-RFLP analysis and spoligotyping. The A1 and A2 subgroups were relatively more homogeneous upon spoligotyping than A3. Further analysis of the three subtypes by VNTR corroborated the highly homogeneous nature of the A2 subtype but showed significant variations for subtypes A1 and A3. A phylogenetic tree based on a selection of isolates representing the three subtypes using VNTR and spoligotyping alone or in combination confirmed the subtypes described as well as the heterogeneity of subtype A3.

Molecular typing of Mycobacterium tuberculosis based on variable number of tandem DNA repeats used alone and in association with spoligotyping.

Fingerprinting based on variable numbers of tandem DNA repeats (VNTR), a recently described methodology, was evaluated for molecular typing of Mycobacterium tuberculosis in an insular setting. In this study, VNTR fingerprinting was used alone or as a second-line test in association with spoligotyping, double-repetitive-element PCR (DRE-PCR), and IS6110 restriction fragment length polymorphism (RFLP) analysis, and the discriminatory power for each method or the combination of methods was compared by calculating the Hunter-Gaston discriminative index (HGI). The results obtained showed that in 6 out of 12 (50%) cases, VNTR-defined clusters were further subdivided by spoligotyping, compared to 7 out of 18 (39%) cases where spoligotyping-defined clusters were further subdivided by VNTR. When used alone, VNTR was the least discriminatory method (HGI = 0.863). Although VNTR was significantly more discriminatory when used in association with spoligotyping (HGI = 0.982), the combination of spoligotyping and DRE-PCR (HGI = 0.992) was still the most efficient among rapid, PCR-based methodologies, giving results comparable to IS6110 RFLP analysis. Nonetheless, VNTR typing may provide additional phylogenetical information that may be helpful to trace the molecular evolution of tubercle bacilli.

Detection of a previously unamplified spacer within the DR locus of Mycobacterium tuberculosis: epidemiological implications.

Spoligotyping, a method based on the variability of distribution of the 43 inter-direct repeat (DR) spacers of Mycobacterium tuberculosis and Mycobacterium bovis BCG, is useful to study the molecular epidemiology of bovine and human tuberculosis. Recently, a major family of M. tuberculosis clinical isolates named the Haarlem family, which did not contain spacers 31 and 33 to 36, was reported in a multicenter study. Independently, a data bank containing all the published spoligotypes showed that the two most prevalent spoligotypes in the world differed only by the presence or absence of spacer 31. A careful analysis of the DR locus sequence led us to hypothesize that spacer 31 may not have been amplified in some isolates with the primer sets DRa and DRb currently used for spoligotyping. Consequently, a modified spoligotyping method based on different combinations of the 36-bp DR and IS6110 primers was devised that was able to discriminate between the left and the right parts of the DR locus and demonstrated the presence of the previously unamplified spacer 31 for some of the clinical isolates. By analogy, we suggest that a single-spacer difference in some epidemiologically linked cases of tuberculosis may simply arise due to the insertion of an extra copy of IS6110 within the DR locus, leading to its asymmetrical disruption and subsequent lack of the DRa or DRb targets. The influence of the IS6110 preferential insertion sites within the DR locus on spoligotyping results should be further investigated.

[Recent developments of spoligotyping as applied to the study of epidemiology, biodiversity and molecular phylogeny of the Mycobacterium tuberculosis complex]. / Développements récents du spoligotypage appliqués à l'étude de l'épidémiologie, de la biodiversité, et de la phylogénie moléculaire du complexe Mycobacterium tuberculosis.

Spoligotyping (for 'spacer-oligonucleotide-typing'), a rapid method for genotyping of Mycobacterium tuberculosis complex using the principle of reverse hybridization, is based on the structure of the direct repeat (DR) locus. The DR locus is made up of a variable number of 36 bp DR repeats that are separated by unique inter-DR sequences of 35 to 41 bp. Fast and highly discriminatory, spoligotyping is an useful alternative to the IS6110-RFLP reference method for molecular typing of M. tuberculosis, in particular for isolates possessing five or few copies of IS6110. In this paper, we review the state of the art of spoligotyping through its main current applications. After a brief introduction to the principle of the technique and its description, we successively review recently published results concerning the molecular epidemiology of tuberculosis in humans and cattle, and discuss the main genotyping strategies currently in use to fingerprint the M. tuberculosis complex organisms. We also describe the recent applications of spoligotyping to study ancient DNA and report on recent developments of this technique to study the biodiversity of the M. tuberculosis complex, its contribution towards improved taxonomy and phylogenetics of the M. tuberculosis complex. Last but not least, potential applications of spoligotyping to study DNA recombination mechanisms are also discussed.

Tuberculosis in the Caribbean: using spacer oligonucleotide typing to understand strain origin and transmission.

We used direct repeat (DR)-based spacer oligonucleotide typing (spoligotyping) (in association with double-repetitive element polymerase chain reaction, IS6110-restriction fragment length polymorphism [RFLP], and sometimes DR-RFLP and polymorphic GC-rich sequence-RFLP) to detect epidemiologic links and transmission patterns of Mycobacterium tuberculosis on Martinique, Guadeloupe, and French Guiana. In more than a third of the 218 strains we typed from this region, clusters and isolates shared genetic identity, which suggests epidemiologic links. However, because of limited epidemiologic information, only 14.2% of the strains could be directly linked. When spoligotyping patterns shared by two or more isolates were pooled with 392 spoligotypes from other parts of the world, new matches were detected, which suggests imported transmission. Persisting foci of endemic disease and increased active transmission due to high population flux and HIV-coinfection may be linked to the recent reemergence of tuberculosis in the Caribbean. We also found that several distinct families of spoligotypes are overrepresented in this region.

[Genotypic diversity of Mycobacterium tuberculosis in the Guiana-Antilles region]. / Diversité génotypique de Mycobacterium tuberculosis dans la région Antilles-Guyane.

This investigation dealt with 226 strains (1 isolate/patient) of Mycobacterium tuberculosis isolated in the French West Indies and French Guiana over a three-year period (1994-1996). The genotypic diversity of the isolates was investigated using various molecular markers; essentially two PCR-based rapid methods, namely spoligotyping and double-repetitive-element (DRE)-PCR, as well as three restriction fragment length polymorphism (RFLP)-based methods, namely IS6110-RFLP, DR-RFLP and PGRS-RFLP. Out of 226 isolates investigated, a total of 166 isolates were distributed in 31 spoligotype-defined clusters containing 2-31 strains, which corresponded to a rate of 73% of primary clustering. After secondary typing with DRE-PCR, IS6110-RFLP, DR-RFLP and/or PGRS-RFLP, molecular clonality was established for 73 isolates organised in 25 clusters (32% of clustered isolates). Considering one reactivation case per cluster, the rate of recent transmission was estimated to a minimal rate of 21%, however the available epidemiologic information led to the positive conclusion for only 14% of cases. The data obtained demonstrated the presence of common genotypes of M. tuberculosis among the three overseas French territories, i.e. Guadeloupe, Martinique and French Guiana. The results obtained during this retrospective study clearly indicate the importance of future prospective epidemiological investigations around the clustered cases of tuberculosis, so as to detect the persisting foci of endemic disease and characterize the chain of transmission as well as the subpopulations which are at an increased risk of contracting and/or propagating the disease. Last but not least, the present study also deals with a first phylogenetic approach of M. tuberculosis based on a comparison of the spoligotyping results obtained locally with those reported elsewhere in the world.