Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%-and by 23% in combination with spoligotyping-among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold-by threefold in combination with spoligotyping-under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.The genotyping of Mycobacterium tuberculosis isolates contributes to tuberculosis (TB) control by, e.g., indicating possible epidemiological links between TB patients, detecting (un)suspected outbreaks and laboratory cross-contamination, and distinguishing exogenous reinfection from endogenous reactivation in relapse cases. For these purposes, IS6110 restriction fragment length polymorphism (RFLP) typing (48) has been used as the gold standard method for more than a decade. However, this method is labor-intensive, requires weeks for culturing the isolates and subsequent DNA purification, and suffers from problems of interpretability and portability of the complex banding patterns. In addition, it provides insufficient discrimination among isolates with low (Ͻ6) IS6110 copy numbers, a problem that is only partly overcome by using PCR-based spoligotyping as a secondary method (6).Genotyping based on variable numbers of tandem repeats (VNTRs) of different classes of interspersed genetic elements named mycobacterial interspersed repetitive units (MIRUs) (12,25,32,36,40,43,44) is increasingly used to solve these problems. This method relies on PCR amplification of multiple loci using primers specific for the flanking regions of each repeat locus and on the determination of the sizes of the amplicons...

Summary Methicillin-resistant Staphylococcus aureus (MRSA) is endemic in hospitals worldwide and a significant cause of morbidity and mortality. Healthcare-associated MRSA infections occur in individuals with predisposing risk factors for disease, such as surgery or presence of an indwelling medical device. By contrast, community-associated MRSA (CA-MRSA) infections often occur in otherwise healthy individuals who lack such risk factors. In addition, CA-MRSA infections are epidemic in some countries. These observations suggest that CA-MRSA strains are more virulent and transmissible than traditional hospital-associated MRSA strains. Relatively limited treatment options for CA-MRSA infections compound the problem of enhanced virulence and transmission. Although progress has been made toward understanding emergence of CA-MRSA, virulence, and treatment of infections, our knowledge in these areas remains incomplete. Here were review the most current knowledge in these areas and provide perspective on future outlook for prophylaxis and/or new therapies for CA-MRSA infections.

The production of most toxins and other exoproteins in Staphylococcus aureus is controlled globally by a complex polycistronic regulatory locus, agr. Secretory proteins are up‐regulated by agr whereas surface proteins are down‐regulated. agr contains two divergent promoters, one of which directs the synthesis of a 514 nucleotide (nt) transcript, RNAIII. In this report, we show that the cloned RNAIII determinant restores both positive and negative regulatory functions of agr to an agr‐null strain and that the RNA itself, rather than any protein, is the effector molecule. RNAIII acts primarily on the initiation of transcription and, secondarily in some cases, at the level of translation. In these cases, translation and transcription are regulated independently. RNAIII probably regulates translation directly by interacting with target gene transcripts and transcription indirectly by means of intermediary protein factors.

One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of Ϸ6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.One-third of the world's population is infected with Mycobacterium tuberculosis, and 3 million human deaths annually are attributed to the organism (1, 2). Although there is a very large global pool of infected individuals and considerable chromosomal heterogeneity based on restriction fragment length polymorphism (RFLP) patterns generated by probing with mobile insertion elements (3, 4), studies of drug resistance and pathogenesis have raised the possibility that synonymous (silent) nucleotide substitutions in structural genes may be limited (5). To investigate this apparent discrepancy from the perspective of molecular population genetics, we sequenced two megabases in 26 structural genes or loci in strains of M. tuberculosis and the three closely related members of the M. tuberculosis complex (M. africanum, M. bovis, and M. microti) collected worldwide. MATERIALS AND METHODSBacterial Isolates. The study is based on a sample of 842 M. tuberculosis complex isolates recovered from diverse geographic localities. The organisms include M. tuberculosis (n ϭ 715), M. bovis (n ϭ 109), and M. africanum and M. microti (n ϭ 9 each). M. tuberculosis isolates were recovered from diseased patients in the United States (five states), Latin America (Mexico, Honduras, Ecuador, Peru, Venezuela, Brazil, and Chile), Europe (Portugal, Spain, The Netherlands, Belgium, Germany, Switzerland, Italy, former Yugoslavia, and Romania), Africa (Kenya, Rwanda, Guinea, Algeria, Som...

Mycobacterium tuberculosis, which causes tuberculosis, is the greatest single infectious cause of mortality worldwide, killing roughly two million people annually. Estimates indicate that one-third of the world population is infected with latent M. tuberculosis. The synergy between tuberculosis and the AIDS epidemic, and the surge of multidrug-resistant clinical isolates of M. tuberculosis have reaffirmed tuberculosis as a primary public health threat. However, new antitubercular drugs with new mechanisms of action have not been developed in over thirty years. Here we report a series of compounds containing a nitroimidazopyran nucleus that possess antitubercular activity. After activation by a mechanism dependent on M. tuberculosis F420 cofactor, nitroimidazopyrans inhibited the synthesis of protein and cell wall lipid. In contrast to current antitubercular drugs, nitroimidazopyrans exhibited bactericidal activity against both replicating and static M. tuberculosis. Lead compound PA-824 showed potent bactericidal activity against multidrugresistant M. tuberculosis and promising oral activity in animal infection models. We conclude that nitroimidazopyrans offer the practical qualities of a small molecule with the potential for the treatment of tuberculosis.

Background: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.

Methicillin-resistant Staphylococcus aureus (MRSA) strains have become prevalent in health care facilities and in the community worldwide (3, 4). MRSA strains produce penicillin binding protein 2Ј or 2a, which is poorly acylated by ␤-lactam antibiotics (5,22,25). The mecA gene, encoding PBP2a, is carried on a peculiar type of mobile genetic element inserted into the staphylococcal chromosome, designated staphylococcal cassette chromosome mec (SCCmec) elements (12,14,24).SCCmec elements typically share four characteristics: first, they carry the mec gene complex (mec) consisting of the methicillin resistance determinant mecA and its regulatory genes and insertion sequences; second, they carry the ccr gene complex (ccr) consisting of ccr genes that are responsible for the mobility of the element and its surrounding sequences; third, they have characteristic directly repeated nucleotide sequences and inverted complementary sequences at both ends; and last, they integrate into the 3Ј end of an open reading frame (ORF), orfX.Despite these similarities, the structures of SCCmec elements are rather divergent. Allotypic differences that are used for SCCmec type definitions have been identified in both ccr and mec. Five types of ccr and four classes of mec have been reported. ccr types 1 to 4 carry the ccrA and ccrB genes, which share approximately 80% identity with each other, and the type 5 ccr carries the ccrC gene (10,11,17,19). Four classes of the mec gene complexes have been identified among methicillinresistant staphylococcal strains of various species: class A mec, consisting of IS431mec-mecA-mecR1-mecI; class B mec, consisting of IS431mec-mecA-⌬mecR1-IS1272; class C mec, consisting of IS431mec-mecA-⌬mecR1-IS431; and class D mec, consisting of IS431mec-mecA-⌬mecR1 with no insertion sequences downstream of ⌬mecR1 identified by PCR as of yet (13). In S. aureus strains, mec classes A, B, and C have been identified. Insertion sequences have sometimes been found to be integrated in or around the class A mec. A class A mec carrying IS431 downstream of mecI was found in Staphylococcus haemolyticus (13). Recently, Shore et al. identified MRSA strains carrying class A mec with an insertion of IS1182 in and around the mecI gene and designated them classes A3 and A4 (23).The SCCmec element type has been defined by the combination of ccr type and mec class. In MRSA strains, six types of SCCmec elements, that is, six combinations of ccr and mec, have been reported (Table 1). These six SCCmec elements have been further classified by differences in regions other than ccr and mec, which are designated junkyard (J) regions. The J regions comprise three parts: J1 (the region between ccr and the right-flanking chromosomal region), J2 (the region between mec and ccr), and J3 (the region between orfX and mec). The J regions are not always specific to each SCCmec type, but certain J regions are commonly shared among certain types of SCCmec elements. Of the three regions, we regard J1 as being the most fundamental, because we presume t...

Fifty million new infections with Mycobacterium tuberculosis occur annually, claiming 2-3 million lives from tuberculosis worldwide. Despite the apparent lack of significant genetic heterogeneity between strains of M. tuberculosis, there is mounting evidence that considerable heterogeneity exists in molecules important in disease pathogenesis. These differences may manifest in the ability of some isolates to modify the host cellular immune response, thereby contributing to the observed diversity of clinical outcomes. Here we describe the identification and functional relevance of a highly biologically active lipid species-a polyketide synthase-derived phenolic glycolipid (PGL) produced by a subset of M. tuberculosis isolates belonging to the W-Beijing family that show 'hyperlethality' in murine disease models. Disruption of PGL synthesis results in loss of this hypervirulent phenotype without significantly affecting bacterial load during disease. Loss of PGL was found to correlate with an increase in the release of the pro-inflammatory cytokines tumour-necrosis factor-alpha and interleukins 6 and 12 in vitro. Furthermore, the overproduction of PGL by M. tuberculosis or the addition of purified PGL to monocyte-derived macrophages was found to inhibit the release of these pro-inflammatory mediators in a dose-dependent manner.