Molecular characterization of Mycobacterium tuberculosis isolates from Izmir, Turkey.

In recent years, molecular typing methods have been used in epidemiologic studies of Mycobacterium tuberculosis isolates in various areas of the world. However, there have been few data on this issue in Turkey. We describe the molecular characterization of 56 Mycobacterium tuberculosis isolates recovered from individual patients in Izmir and the surrounding area by three different molecular methods. Isolated M. tuberculosis strains were characterized by IS6110 RFLP, spoligotyping and major genetic group designation. In total, 51 RFLP and 35 spoligopatterns were identified. Fourteen (25%) isolates were indicated as low copy number. Based on three genotypic characterization methods together, five clusters with two isolates each were identified. Most of the isolates (98.2%) were assigned as genetic groups 2 or 3. Only one isolate was identified as Beijing family strain (principal genetic group 1). The shared international clades were found to be Beijing-family, var T1 (ST 37), LAM (Latin-American-Mediterranean) 7 (ST 41), LAM 9 (ST 42), Haarlem 1 (ST 47), Haarlem 3 (ST 50) and T1 (ST 53). In this study, IS6110 RFLP, spoligotyping and major genetic group designation were found to be useful methods for molecular epidemiologic studies.

Molecular characterization of nontypeable group B streptococcus.

Traditionally, the capsular polysaccharide (CPS) antigen has been used to distinguish between the nine known serotypes of group B streptococcus (GBS) by classical antibody-antigen reactions. In this study, we used PCR for all CPSs and selected protein antigens, multilocus sequencing typing (MLST), and pulsed-field gel electrophoresis (PFGE) to molecularly characterize 92 clinical isolates identified as nontypeable (NT) by CPS-specific antibody-antigen reactivity. The PCR and MLST were performed on blinded, randomly numbered isolates. All isolates contained the cfb gene coding for CAMP factor. While most (56.5%) contained a single CPS-specific gene, 40 isolates contained either two or three CPS-specific genes. Type V CPS-specific gene was present in 66% of the isolates, and all serotypes except types IV, VII, and VIII were represented. Most (44.5%) of the isolates contained a single protein antigen gene (bca, bac, rib, alp1, or alp3), and the remaining isolates had multiple protein antigen genes. Of the 61 isolates that had the V CPS-specific gene, 48 (78.6%) had the alp3 gene. PFGE analysis classified the isolates into 21 profile groups, while MLST analysis divided the isolates into 16 sequence types. Forty-two (69%) of 61 isolates with the V CPS-specific gene were in PFGE profile group 4; 41 of these 42 were sequence type 1 by MLST. These data shed new light on the antigenic complexity of NT GBS isolates, information that can be valuable in the formulation of an effective GBS vaccine.

Identification of novel cps locus polymorphisms in nontypable group B Streptococcus.

Group B Streptococcus (GBS) is an important pathogen responsible for a variety of diseases in newborns and the elderly. A clinical GBS isolate is considered nontypable (NT) when serological methods fail to identify it as one of nine known GBS serotypes. Eight clinical isolates (designated A1-A4, B1-B4) showed PFGE profiles similar to that of a GBS serotype V strain expressing R1, R4 surface proteins. These unique isolates were further characterized by immunologic and genetic methods. Rabbit sera to isolates A1 and A2 reacted weakly with concentrated HCl extracts of A1-A4 isolates, but not with those of B1-B4 isolates. In addition, a type V capsular polysaccharide (CPS) inhibition ELISA revealed that cell wall extracts from isolates A1-A4, but not from B1-B4, expressed low but measurable amounts of type V CPS. Molecular serotyping with PCR analysis showed that all eight isolates contained a type V-specific CPS gene (cpsO) and harboured the gene encoding the surface protein Alp3. Multilocus sequence typing identified isolate A1 as belonging to a new sequence type (ST) designated ST-173, whereas the other seven isolates keyed to ST-1. Sequencing of the 18 genes (17 736 bp) in the cps locus showed that each NT isolate harboured one to three unique polymorphisms, and also identified an IS1381 element in cpsE of the B4 isolate. Collectively, genetic and immunologic analyses revealed that these NT isolates expressing R1, R4 proteins have a genetic profile consistent with that of type V, an emergent, antigenically diverse and increasingly prevalent GBS serotype.

Comparative sequencing of the serine-aspartate repeat-encoding region of the clumping factor B gene (clfB) for resolution within clonal groups of Staphylococcus aureus.

Molecular techniques such as spa typing and multilocus sequence typing use DNA sequence data for differentiating Staphylococcus aureus isolates. Although spa typing is capable of detecting both genetic micro- and macrovariation, it has less discriminatory power than the more labor-intensive pulsed-field gel electrophoresis (PFGE) and costly genomic DNA microarray analyses. This limitation hinders strain interrogation for newly emerging clones and outbreak investigations in hospital or community settings where robust clones are endemic. To overcome this constraint, we developed a typing system using DNA sequence analysis of the serine-aspartate (SD) repeat-encoding region within the gene encoding the keratin- and fibrinogen-binding clumping factor B (clfB typing) and tested whether it is capable of discriminating within clonal groups. We analyzed 116 S. aureus strains, and the repeat region was present in all isolates, varying in sequence and in length from 420 to 804 bp. In a sample of 36 well-characterized genetically diverse isolates, clfB typing subdivided identical spa and PFGE clusters which had been discriminated by whole-genome DNA microarray mapping. The combination of spa typing and clfB typing resulted in a discriminatory power (99.5%) substantially higher than that of spa typing alone and closely approached that of the whole-genome microarray (100.0%). clfB typing also successfully resolved genetic differences among isolates differentiated by PFGE that had been collected over short periods of time from single hospitals and that belonged to the most prevalent S. aureus clone in the United States. clfB typing demonstrated in vivo, in vitro, and interpatient transmission stability yet revealed that this locus may be recombinogenic in a primarily clonal population structure. Taken together, these data show that the SD repeat-encoding region of clfB is a highly stable marker of microvariation, that in conjunction with spa typing it may serve as a DNA sequence-based alternative to PFGE for investigating genetically similar strains, and that it is useful for analyzing collections of isolates in both long-term population-based and local epidemiologic studies.

Detection of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis using denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with rifampin (RIF) resistance in the rpoB gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and is comparable to DNA sequencing in detecting DNA alterations. Specific mutations are often recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five DGGE primer sets that scanned for DNA alterations across 775 bp of rpoB were developed. These primer sets were used to scan rpoB for DNA alterations in 296 M. tuberculosis patient isolates from the United States-Mexico border states of Texas and Tamaulipas. The most useful primer set scanned for mutations in the rifampin resistance-determining region (RRDR) and detected mutations in 95% of the RIF-resistant isolates compared to 2% of RIF-susceptible isolates. Thirty-four different alterations were observed within the RRDR by DGGE. In addition, isolates harboring mixtures of DNA within rpoB were readily detected by DGGE. A second PCR primer set was used to detect the V146A mutation in 5 to 7% of RIF-resistant isolates. A third primer set was used to detect mutations in 3% of RIF-resistant isolates, some of which also harbored mutations in the RRDR. Only 1 of 153 RIF-resistant isolates did not have a detectable rpoB mutation as determined by DGGE and DNA sequencing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with drug resistance in M. tuberculosis.

Detection by denaturing gradient gel electrophoresis of pncA mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis isolates from the United States-Mexico border region.

Denaturing gradient gel electrophoresis (DGGE) was used to probe for mutations associated with pyrazinamide (PZA) resistance in the pncA gene of Mycobacterium tuberculosis. DGGE scans for mutations across large regions of DNA and rivals sequencing in its ability to detect DNA alterations. Specific mutations can often be recognized by their characteristic denaturation pattern, which serves as a molecular fingerprint. Five PCR target fragments were designed to scan for DNA alterations across 600 bp of pncA in 181 M. tuberculosis isolates from patients residing in the U.S-Mexico border states of Texas and Tamaulipas, respectively. A region of pncA was observed with a high GC content and a melting temperature approaching 90 degrees C that was initially refractory to denaturation, and a DGGE target fragment was specifically designed to detect mutations in this region. DGGE detected pncA mutations in 82 of 83 PZA-resistant isolates. By contrast, only 1 of 98 PZA-susceptible isolates harbored a detectable DNA alteration. The pncA gene was sequenced from 41 isolates, and 32 DNA alterations in 32 PZA-resistant isolates were identified, including 11 new mutations. DGGE also detected nine isolates whose susceptibility to PZA appeared to be incorrect, and DNA sequencing confirmed these apparent errors in drug susceptibility testing. These results demonstrate the power and usefulness of DGGE in detecting mutations associated with PZA resistance in M. tuberculosis.

Molecular characteristics of nosocomial and Native American community-associated methicillin-resistant Staphylococcus aureus clones from rural Wisconsin.

In central and northern Wisconsin methicillin-resistant Staphylococcus aureus (MRSA) was first detected in 1989. Over the next 10-year period, 581 MRSA isolates were collected, 17.2% of which came from patients who were treated at five Native American clinics. These isolates were typed by SmaI-macrorestricted pulsed-field gel electrophoresis (PFGE). The PFGE patterns clustered the isolates into six major clonal groups (MCGs), i.e., MCGs 1 to 6, and 19 minor clonal groups (mCGs). The 25 clonal groups were represented by 109 unique PFGE types. Sixty-five percent of the MCG-2 isolates were recovered from patients who were treated at Native American clinics. Ninety-four percent of the MCG-2 isolates harbored the staphylococcal cassette chromosome mec (SCCmec) IVa. These isolates also had PFGE profiles that were clonally related to the midwestern community-associated MRSA (CA-MRSA) strain, MW2. The representative isolates from MCG-2 had the multilocus sequence type allelic profile 1-1-1-1-1-1-1 and contained pvl genes. They were also susceptible to various antibiotics, a finding consistent with the CA-MRSA phenotype. SCCmec IV was also present in other mCGs. Unlike MCG-2, isolates from the remaining five MCGs harbored SCCmec II and were resistant to multiple antibiotics, suggesting their nosocomial origin. The 19 mCGs were represented by diverse SCCmec types and three putative new variants referred to as SCCmec Ib, IIa, and IIb.

Novel polymorphisms in mec genes and a new mec complex type in methicillin-resistant Staphylococcus aureus isolates obtained in rural Wisconsin.

We determined allelic polymorphisms in the mec complexes of 524 methicillin-resistant Staphylococcus aureus isolates by partial or complete sequencing of three mec genes, mecA, mecI, and mecR1. The isolates had been collected over a 10-year period from patients living in rural Wisconsin, where the use of antibiotics was expected to be lower than in the bigger cities. Of the 18 mutation types identified, 16 had not been described previously. The five most common mutations were a mutation 7 nucleotides (nt) upstream from the start site (G-->T) in the mecA promoter (34.7%), an E246G change encoded by mecA (2.2%), a cytosine insertion at codon 257 in mecA (13.2%), an N121K change encoded by mecI (7.8%), and a major mecI-mecR1 deletion designated as a class B1 mec complex deletion type (25.4%). There was a high degree of conservation of the amino acid sequence of MecR1. Strains with the same mutations had variable resistance to oxacillin, and the median MIC for isolates that harbored the 7-nt-upstream mutation was lower than that for strains harboring other mutations. Our data suggest that the mecA promoter mutation plays a more important role in determining methicillin resistance than mutations in mecI and mecA do. Eighty-five percent of the tested isolates (n = 148) with the mecA promoter mutation and the class B1 mec complex deletion belonged to the same major clonal group, identified as MCG-2, and harbored the type IV staphylococcal cassette chromosome mec DNA. There was also a wide range of oxacillin MICs for strains with wild-type mecA, mecI, and mecR1 sequences, suggesting that the genetic backgrounds of clinical strains are significant in determining susceptibility to methicillin.

Genetic polymorphism in Mycobacterium tuberculosis isolates from patients with chronic multidrug-resistant tuberculosis.

Multidrug-resistant tuberculosis (MDR-TB) is a major public health problem because treatment is complicated, cure rates are well below those for drug-susceptible tuberculosis (TB), and patients may remain infectious for months or years, despite receiving the best available therapy. To gain a better understanding of MDR-TB, we characterized serial isolates recovered from 13 human immunodeficiency virus-negative patients with MDR-TB, by use of IS6110 restriction fragment-length polymorphism analysis, spacer oligonucleotide genotyping (i.e., "spoligotyping"), and sequencing of rpoB, katG, mabA-inhA (including promoter), pncA, embB, rpsL, rrs, and gyrA. For all 13 patients, chronic MDR-TB was caused by a single strain of Mycobacterium tuberculosis; 8 (62%) of the 13 strains identified as the cause of MDR-TB belonged to the W-Beijing family. The sputum-derived isolates of 4 (31%) of the 13 patients had acquired additional drug-resistance mutations during the study. In these 4 patients, heterogeneous populations of bacilli with different resistance mutations, as well as mixtures of drug-susceptible and drug-resistant genotypes, were observed. This genetic heterogeneity may require treatment targeted at both drug-resistant and drug-susceptible phenotypes.

spa typing method for discriminating among Staphylococcus aureus isolates: implications for use of a single marker to detect genetic micro- and macrovariation.

Strain typing of microbial pathogens has two major aims: (i). to index genetic microvariation for use in outbreak investigations and (ii). to index genetic macrovariation for use in phylogenetic and population-based analyses. Until now, there has been no clear indication that one genetic marker can efficiently be used for both purposes. Previously, we had shown that DNA sequence analysis of the protein A gene variable repeat region (spa typing) provides a rapid and accurate method to discriminate Staphylococcus aureus outbreak isolates from those deemed epidemiologically unrelated. Here, using the hypothesis that the genetic macrovariation within a low-level recombinogenic species would accurately be characterized by a single-locus marker, we tested whether spa typing could congruently index the extensive genetic variation detected by a whole-genome DNA microarray in a collection of 36 isolates, which was recovered from 10 countries on four continents over a period of four decades, that is representative of the breadth of diversity within S. aureus. Using spa and coa typing, pulsed-field gel electrophoresis (PFGE), and microarray and multilocus enzyme electrophoresis (MLEE) data in molecular epidemiologic and evolutionary analyses, we determined that S. aureus likely has a primarily clonal population structure and that spa typing can singly index genetic variation with 88% direct concordance with the microarray and can correctly assign isolates to phylogenetic lineages. spa typing performed better than MLEE, PFGE, and coa typing in discriminatory power and in the degree of agreement with the microarray at various phylogenetic depths. This study showed that genetic analysis of the repeat region of protein A comprehensively characterizes both micro- and macrovariation in the primarily clonal population structure of S. aureus.

Genotypic analysis of multidrug-resistant Mycobacterium tuberculosis isolates from Monterrey, Mexico.

Thirty-seven multidrug-resistant and 13 pan-susceptible isolates of Mycobacterium tuberculosis were analysed for the diversity of genotypes associated with known drug-resistance mechanisms. The isolates were obtained from patients attending a university tuberculosis clinic in Monterrey, Mexico. A total of 25 IS6110-RFLP patterns were obtained from the multidrug-resistant tuberculosis (MDR-TB) isolates. Approximately 65% of the MDR-TB isolates were attributed to secondary resistance. Different drug-susceptibility patterns were seen with the clustered isolates. The percentage of isolates resistant to isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and streptomycin (STR) was 100, 97.3, 48.7 and 67.6, respectively. The most common resistance-associated polymorphisms for the four drugs were as follows: INH, Ser315Thr (67.6%) in katG; RIF, Ser450Leu (41.7%) in rpoB; EMB, Met306Ile/Val/Leu (66.7%) in embB; and STR, Lys43Arg (24%) in rpsL. Drug-resistance-associated mutations were similar to changes occurring in isolates from other areas of the world, but unique, previously unreported, mutations in katG (n=5), rpoB (n=1) and rrs (n=3) were also identified.

Mycobacterium tuberculosis growth at the cavity surface: a microenvironment with failed immunity.

Protective immunity against pulmonary tuberculosis (TB) is characterized by the formation in the lungs of granulomas consisting of macrophages and activated T cells producing tumor necrosis factor alpha and gamma interferon, both required for the activation of the phagocytes. In 90% of immunocompetent humans, this response controls the infection. To understand why immunity fails in the other 10%, we studied the lungs of six patients who underwent surgery for incurable TB. Histologic examination of different lung lesions revealed heterogeneous morphology and distribution of acid-fast bacilli; only at the surface of cavities, i.e., in granulomas with a patent connection to the airways, were there numerous bacilli. The mutation profile of the isolates suggested that a single founder strain of Mycobacterium tuberculosis may undergo genetic changes during treatment, leading to acquisition of additional drug resistance independently in discrete physical locales. Additional drug resistance was preferentially observed at the cavity surface. Cytokine gene expression revealed that failure to control the bacilli was not associated with a generalized suppression of cellular immunity, since cytokine mRNA was up regulated in all lesions tested. Rather, a selective absence of CD4(+) and CD8(+) T cells was noted at the luminal surface of the cavity, preventing direct T-cell-macrophage interactions at this site, probably allowing luminal phagocytes to remain permissive for bacillary growth. In contrast, in the perinecrotic zone of the granulomas, the two cell types colocalized and bacillary numbers were substantially lower, suggesting that in this microenvironment an efficient bacteriostatic or bactericidal phagocyte population was generated.

Single nucleotide polymorphisms in genes associated with isoniazid resistance in Mycobacterium tuberculosis.

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG, inhA, kasA, ndh, and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drug's mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS6110 profiles. Spoligotyping was done with isolates with five or fewer IS6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG, mabA, or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24, Rv1592c, Rv1772, Rv0340, and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.