Because the 16S rRNA gene evolves so slowly, phylogenetic information based on this molecule may not always be sufficient to distinguish closely related species or to resolve their evolution- ary relationships. In addition, when several copies of the 16S rRNA gene are present, sequence heterogeneity results in ambigu- ities in the sequence chromatograms derived from direct sequenc- ing of the PCR products (44). Nucleotide sequences of housekeep- ing protein-coding genes evolve more rapidly than 16S rRNA and may represent useful alternatives or complements to the 16S rRNA gene (45–47). Otherwise, housekeeping genes have been considered good monitoring tools for bacterial identification and strain typing. The gyrB gene-based mPCR presented in this study may be valuable in clinical microbiology laboratories because it provides a fast and cost-effective means of analyzing large num- bers of samples and allows the detection and discrimination of S. pneumoniae, S. oralis, and S. mitis from oral streptococci. Our protocol proved to be more accurate and sensitive than traditional culture methods.
Grouping of microorganisms in the B. cereus group accord- ing to gyrB gene sequences. Recently, a set of parallel 16S rRNA and partial (more then 60% of the whole gene length) gyrB sequences for B. cereus group isolates was placed in Gen- Bank. We classified these microorganisms (Table 4) according to subgroup-specific signatures found for 16S rRNA sequences (Fig. 1 and Table 2). Additional information about the 23S rRNA obtained from whole-genome sequences of B. cereus ATCC 10987 made it possible to identify this organism as belonging to the Cereus A subgroup (Fig. 2; Table 3) and, therefore, to differentiate it from bacteria of the Anthracis FIG. 3. Genetic relationship among B. cereus group strains. 16S rRNA (A) and 23S rRNA (B) phylogenetic trees obtained by minimum evolution method demonstrate the division of the B. cereus group into subgroups. Subgroup names are marked with bold on the right side of the brackets. Bootstrap volumes are reported on the branches. Numerals indicated in the quadrant parentheses denote the bootstrap volumes for each subgroup. During calculation of consensus parsimonious trees 7615 and 219 most-parsimonious trees were obtained for 16S rRNA and 23S rRNA, respectively. The percentages of most-parsimonious trees that support each subgroup in consensus parsimonious trees are presented in round parentheses. Bars indicate the scales of genetic distances.
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Phylogenetic analysis of about 200 strains of Salmonella, Shigella, and Escherichia coli was carried out using the nucleotide sequence of the gene for DNA gyrase B (gyrB), which was determined by directly sequencing PCR fragments. The results establish a new phylogenetic tree for the classification of Salmonella, Shigella, and Escherichia coli in which Salmonella forms a cluster separate from but closely related to Shigella and E. coli. In comparison with 16S rRNA analysis, the gyrB sequences indicated a greater evolutionary divergence for the bacteria. Thus, in screening for the presence of bacteria, the gyrB gene might be a useful tool for differentiating between closely related species of bacteria such as Shigella spp. and E. coli. At present, 16S rRNA sequence analysis is an accurate and rapid method for identifying most unknown bacteria to the genus level because the highly conserved 16S rRNA region is easy to amplify; however, analysis of the more variable gyrB sequence region can identify unknown bacteria to the species level. In summary, we have shown that gyrB sequence analysis is a useful alternative to 16S rRNA analysis for constructing the phylogenetic relationships of bacteria, in particular for the classification of closely related bacterial species.
Since FQs are often prescribed as broad-spectrum antibiotics for the treatment of undiagnosed respiratory infections, and because TB patients are not treated nor- matively, FQ-resistant TB has become more prevalent . With the occurrence of extensively drug-resistant TB in recent years, concerns about FQ-resistant TB have been raised [7-9]. Reports show that the majority (approximately 50%~90%) of FQ-resistant MTB isolates carry mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene [2,10-15], and that a small number have mutations in the gyrB gene [16,17]. Much research has focused on the mutations in gyrA/ gyrB of MTB to determine the drug susceptibility to FQs [15,18]. A correlation between quinolone suscept- ibility patterns and nucleotide sequences in the A and B subunits of DNA gyrase in 14 mycobacterial species has been described . There are, however, no data on the correlation between quinolone susceptibility patterns
The assay allowed for the identification of 35 mycobacterial species, which is larger than that previously reported (18–21), and was able to differentiate between M. tuberculosis and M. bovis. The final list of Mycobacterium species detected by the assay includes 35 species, 27 of which were found in the clinical isolates of two regions of Russia: M. tuberculosis complex (MTC) (non-M. bovis), M. bovis, M. intracellulare, M. avium subspecies, M. mantenii, M. intracellulare/M. colombiense, M. kansasii, M. gordonae, M. xenopi, M. kumamotonense, M. senuense, M. lentiflavum, M. interjectum, M. intermedium, M. mageritense, M. neoaurum, M. fortuitum, M. septicum/M. peregrinum, M. houstonense, M. iranicum, M. muco- genicum, M. duvalii, M. flavescens, M. phlei, M. smegmatis, M. chelonae, and M. abscessus. The capability to detect 8 more species (M. marinum, M. avium subsp. paratuberculosis, M. gastri, M. scrofulaceum, M. szulgai, M. malmoense, M. asiaticum, and M. simiae) was tested with synthetic DNA fragments based on Gen- Bank sequences of the gyrB gene.
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DNA-DNA hybridization and sequence analysis are the most widely accepted methods for identifying species, includ- ing Acinetobacter (3). DNA-DNA hybridization is considered the gold standard but is labor-intensive and impractical in most clinical laboratories (15). Sequence analysis of housekeeping genes, including the 16S rRNA gene (13), 16S-23S rRNA in- tergenic spacer (ITS) (6), recA gene (15), rpoB and flanking spacers (16), and gyrB gene (27), also allows identification of Acinetobacter species. Among these genes, the 16S rRNA gene is most commonly used (14). It has been suggested that an unknown isolate can be assigned to a species when its 16S rRNA sequence has a similarity of ⱖ 99% with the reference sequence of a well-defined species and when it has at least 0.5% sequence difference to the second nearest species (4, 14). In the present study, we used genotypic methods to identify Acinetobacter baylyi in six bacteremic patients. Previously, this species was only known to live in the soil. The identification procedures, antimicrobial susceptibility, natural transformability of the isolates and clinical features of the patients are described.
Materials and methods: A total of 150 non-duplicate clinical strains of M. catarrhalis were obtained from individuals with signs of upper respiratory tract infection. Bacterial identification was corroborated on the basis of phenotypic and biochemical characteristics as well as with the use of molecular tests. The antimicrobial susceptibility of M. catarrhalis isolates was determined using the disk diffusion method and Etest. Mutations in the gyrase (gyrA and gyrB) and topoi- somerase (parC and parE) genes were determined by polymerase chain reaction and sequencing. Results: It was observed that 16.7% of the studied isolates were drug resistant. Resistance to tetracycline was detected for 12% of the strains. Resistance to nalidixic acid, moxifloxacin, and levofloxacin was exhibited by 2.7% of the strains; 1.3% of the strains were resistant to trimethoprim/sulfamethoxazole and 0.7% to erythromycin. Minimum inhibitory concentra- tion values of the four strains demonstrating fluoroquinolone resistance were: 6–12 mg/L for nalidixic acid, 1–1.5 mg/L for levofloxacin, 1 mg/L for moxifloxacin, and 0.25–0.5 mg/L for ciprofloxacin. The research resulted in the detection of mutations in 4 strains, in gyrase gyrA and gyrB genes. In gyrA gene, there occurred mutation G412C as well as four silent transition mutations. Within gyrB gene, there occurred mutation, substitution A1481G, as well as two identical silent mutations.
Among the FQ-resistant strains, mutations in gyrA codons 90, 91, and 94 were observed in 86% cases (36 of 43). Four isolates (9%) contained substitutions within codons 70 and 88 (the gyrA gene), and two isolates had mutations in the gyrB gene only (D500H, and N538D). One more strain bearing R485H substitution in gyrB gene was resistant to OFX and sensitive to LVX and MFX; however, the role of this mutation in FQ resistance was not confirmed by a gyrase assay [2,3]. Five strains with double mutations in gyrA were detected. They could be either true double mutant, either the result of heteropeaks due to the presence of different clones . However, the spoligotyping revealed the unambiguous pattern for every strain. The sensitivity and specificity of the method relating to FQ resistance were 98 and 100%, respectively.
In general, the most frequent clinical isolate among Acineto- bacter spp. has been reported to be A. baumannii (2). Current molecular methods have revealed 27 valid species within the Acin- etobacter genus (22). In the present study, we used molecular tech- niques to investigate the frequency of Acinetobacter isolates caus- ing bacteremia at a tertiary hospital in Japan. As with reports from other countries, this study showed that the clinical isolates of Acin- etobacter spp. from blood cultures included a high percentage of non-A. baumannii species. The present study also revealed a high isolation rate for A. soli, although there have been few reports from elsewhere in the world about this microbe. A. soli was first re- ported by Kim et al. in 2008 (4). In the present study, we identified A. soli by sequence analysis of the rpoB gene (zones 1 and 2), the 16S rRNA gene, and the gyrB gene. According to the results of our genetic analyses, 13 isolates showed an extremely high similarity to A. soli (GenBank accession no. HQ148175, EU290155, and JQ411222), which strongly suggests that they were A. soli strains. The 13 A. soli strains isolated from blood cultures in the present study were divided into 8 clusters according to PFGE (Fig. 1). Strains 10397 and 11584 (cluster A) were isolated from patients in the intensive care unit (ICU) but were isolated at different times, and there was no interaction between the two patients, so com- mon hospital equipment and/or staff need to be considered when examining the potential relationship between the two cluster A strains isolated from the ICU.
When the phylogenetic trees based on 16S and gyrB were com- pared, the strains classified as N. paucivorans, N. veterana, and N. ignorata remained together in both representations. However, strains belonging to N. beijingensis orN. wallacei grouped together in the 16S-based tree by species and separately in the gyrB-based tree. To a lesser extent, this happens in strains proceeding from N. elegans, N. flavorosea, N. higoensis, N. rhamnosiphila, and N. testa- cea. This shows the greater discriminating capacity of gyrB (Fig. 2). Changes in GyrB showed that ⬎ 70% of the SNPs were silent, above all in the strains of N. arthritidis and N. veterana. Nonsyn- onym substitution gathered predominantly between positions 778 and 787 with respect to the N. farcinica IFM10152 gyrB gene partial sequence (GenBank accession no. NC_006361), which cor- responds to positions 260 to 264 of the N. farcinica IFM10152 GyrB protein sequence (YP_116212).
Background: Indonesia have many different geographic areas which could be various on the variant strains of Mycobacterium tuberculosis. The gyrB gene codes GyrB protein as sub unit compound of Gyrase enzyme that functioning in multiplication of bacteria. Detection of gyrB gene could be a marker of active multiplication of viable bacteria in the specimen from patients; and some of the DNA sequence regions were conserved and specific in the strain of Mycobacterium tuberculosis that would be a marker for identification. This research ai ms to analyze the sequence of gyrB gene of Mycobacterium tuberculosis clinical isolates from sputum of pulmonary TB patients in Indonesia, and determine the specific region. Method: Mycobacterium tuberculosis clinical isolates have been collected from sputum of the patients with pulmonary TB that live in some area in Indonesia. Isolation and identification of Mycobacterium tuberculosis clinical isolates using standard culture method; sequence analysis using PCR-direct sequencing of the part bases region of gyrB. Results: this study revealed that nucleotide sequence on a fragment 764 bases of gyrB gene Mycobacterium tuberculosis strains among clinical isolates almost identically to a wild type strain Mycobacterium tuberculosis H37Rv and subspecies member of Mycobacterium tuberculosis complex (MTBC), with a little difference of SNPs; there are many difference nucleotide sequence with MOTT and Gram positive or negative bacteria, except Corynebacterium diphtheriae identically with MTBC. Conclusion: the gyrB sequence in Mycobacterium tuberculosis strains among these clinical isolates from sputum of pulmonary TB patients in Indonesia have the conserved specific DNA region that almost identically with wild type strain H37Rv and MTBC.
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Methods: Sand flies were collected from two locations (peri-urban and jungle biotype) in the Department of Sucre (Caribbean coast of Colombia). A total of 752 Lutzomyia evansi intestines were dissected. In this study, 125 bacterial strains were isolated from different culture media (LB Agar, MacConkey Agar). Different methods were used for bacterial identification, including ribosomal intergenic spacer analysis (RISA) and analysis of the 16S rRNA and gyrB gene sequences. The genetic profiles of the bacterial populations were generated and temporal temperature gradient gel electrophoresis (TTGE) was used to compare them with total gut DNA. We also used PCR and DNA sequence analysis to determine the presence of Wolbachia endosymbiont bacteria and Leishmania parasites. Results: The culture-dependent technique showed that the dominant intestinal bacteria isolated belong to Acinetobacter, Enterobacter, Pseudomonas, Ochrobactrum, Shinella and Paenibacillus in the larval stage; Lysobacter, Microbacterium, Streptomyces, Bacillus and Rummeliibacillus in the pupal stage; and Staphylococcus, Streptomyces, Brevibacterium, Acinetobacter, Enterobacter and Pantoea in the adult stage. Statistical analysis revealed significant differences between the fingerprint patterns of the PCR-TTGE bands in bacterial communities from immature and adult stages. Additionally, differences were found in bacterial community structure in fed females, unfed females, males and larvae. The intestinal bacteria detected by PCR-TTGE were Enterobacter cloacae and Bacillus thuringiensis, which were present in different life stages of Lu. evansi, and Burkholderia cenocepacia and Bacillus gibsonii, which were detected only in the larval stage. Wolbachia and Leishmania were not detected in gut samples of Lutzomyia evansi.
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Conclusion. In this study, we have attempted to characterize the genetic mechanism of resistance in the XDR isolates, wherein we saw good concordance with the phenotypic resis- tance in the cases of isoniazid, rifampin, fluoroquinolones, and second-line aminoglycosides. As XDR M. tuberculosis is emerging as a highly threatening pathogen in a pervasive as- sociation with the AIDS epidemic, rapid tests to promptly identify resistance to first- and second-line antitubercular agents are urgently required. It has been observed that point mutations in rpoB, inhA, katG, gyrA, gyrB, and rrs can accu- rately predict resistance to these drugs. The RLBH assay is proposed to be an adequate tool for direct analysis of XDR M. tuberculosis from clinical specimens, thus fulfilling many attri- butes of an ideal TB diagnostic test, as proposed by the WHO for developing countries.
We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influen- zae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.
Table 5 demonstrates the proportion of fluoroquinolone-re- sistant and -susceptible M. tuberculosis isolates with polymor- phisms according to region of gyrA and gyrB. As expected, fluoro- quinolone-resistant isolates were more likely to have mutations in the QRDR of gyrA (68% versus 6% in susceptible isolates). How- ever, resistant isolates were not more likely to have mutations in the QRDR of gyrB (4% versus 6% in susceptible isolates). Inter- estingly, fluoroquinolone-susceptible isolates were significantly more likely to have polymorphisms in gyrA at codons 1 to 73 than fluoroquinolone-resistant isolates (20% versus 0%, respectively). Of the 10 isolates with polymorphisms in this region, each had a polymorphism at a different codon. The global test suggested that at least one region had a significant difference in mutation rate, thus assuring that the observed difference was not due to inflation of type I error by multiple comparisons. There were no other regions that had significantly more polymorphisms in fluoroquin- olone-resistant versus -susceptible isolates.
Viridans group streptococci (VGS) are a heterogeneous group of medically important bacteria that cannot be accurately assigned to a particular species using conventional phenotypic methods. Although multilocus sequence analysis (MLSA) is considered the gold standard for VGS species-level identification, MLSA is not yet feasible in the clinical setting. Conversely, molecular meth- ods, such as sodA and 16S rRNA gene sequencing, are clinically practical but not sufficiently accurate for VGS species-level iden- tification. Here, we present data regarding the use of an ⬃400-nucleotide internal fragment of the gene encoding DNA gyrase subunit B (GyrB) for VGS species-level identification. MLSA, internal gyrB, sodA, full-length, and 5= 16S gene sequences were used to characterize 102 unique VGS blood isolates collected from 2011 to 2012. When using the MLSA species assignment as a reference, full-length and 5= partial 16S gene and sodA sequence analyses failed to correctly assign all strains to a species. Precise species determination was particularly problematic for Streptococcus mitis and Streptococcus oralis isolates. However, the inter- nal gyrB fragment allowed for accurate species designations for all 102 strains. We validated these findings using 54 VGS strains for which MLSA, 16S gene, sodA, and gyrB data are available at the NCBI, showing that gyrB is superior to 16S gene and sodA sequence analyses for VGS species identification. We also observed that specific polymorphisms in the 133-amino acid sequence of the internal GyrB fragment can be used to identify invasive VGS species. Thus, the GyrB amino acid sequence may offer a more practical and accurate method for classifying invasive VGS strains to the species level.
mutations in grlA (8, 25). Indeed, data from various studies have shown that most S. aureus isolates having decreased sus- ceptibility to fluoroquinolones harbor mutations at codons 80 (Ser-80 to Phe or Tyr) and/or 84 (Glu-84 to Lys or Val or Gly) of grlA (23, 26, 27, 30). Single mutations in grlA appear to be sufficient to reach MICs of ciprofloxacin that exceed the lab- oratory breakpoints for susceptibility (8). On the other hand, for new fluoroquinolones, single grlA mutations are associated with a reduced susceptibility to these compounds but are not sufficient for clinical resistance. Additional mutations in gyrA and, less commonly, in grlB and gyrB, encoding the B subunits of the DNA topoisomerase IV and DNA gyrase, respectively, will usually generate full resistance to new fluoroquinolones and an increased level of ciprofloxacin resistance (8). In addi- tion to mutations in these loci, altered expression of norA, a gene encoding a multidrug efflux pump, can also confer low- level resistance to fluoroquinolones (8, 25). It has been shown that S. aureus isolates with existing first-step grlA mutations are more likely to acquire subsequent mutations that result in clinical resistance to the new fluoroquinolones (5). Therefore, the development of novel methods that can rapidly identify mutations in the S. aureus grlA gene would provide a useful tool for the appropriate use of new fluoroquinolones in clinical settings.
was identified by gyrB RFLP analysis; however, the spoligotype profiles were not always readable. To exclude PCR amplification problems, M. microti DNA was quantified by the Artus M. tuber- culosis PCR kit (Qiagen). All of the SB000 samples showed an amount of target DNA comparable to that in the SB0118 samples (data not shown). We could assign spoligotypes to 118 samples, with 84 showing spoligotype SB0118, while 34 did not show any spacers. The frequencies of the two spoligotypes in tissue samples (SB0118, 84/118 [71%]; SB000, 34/118 [29%]) are consistent with those found among the isolates (SB0118, 19/23 [83%]; SB000, 4/23 [17%]). By georeferencing the spoligotype profiles obtained from tissue samples, we noticed a geographical clustering of geno- types (Fig. 1). The profile SB000 is predominant in the area near Garda Lake. SB000 was occasionally present also in BG and CO in 2007, but it was not detected in the following years. Conversely, SB0118 prevailed in CO, VA, and BG, and only occasionally ap- pears in BS.
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and challenging group as the Nocardia. Although DDH has long been held to be the gold standard for prokaryotic species demarcation, MLSA may soon supplant DDH to become the primary measure of bacterial diversity and species identifica- tion (34). While the results of MLSA correlate well with DDH taxonomic classifications (5, 28, 30, 31), it is technically easier to perform and allows the rapid concurrent comparison of multiple strains. Compared to single-locus sequence-based identification schemes (e.g., 16S rRNA), it yields a fuller un- derstanding of the genome by sampling multiple alternate re- gions of the DNA. The use of multiple loci buffers against stochastic genetic variation and horizontal gene transfer with homologous recombination (12, 17, 35). MLSA is less cumber- some than biochemical testing, yet it demonstrates greater discriminatory power (12, 35).
We report on the correlation between MICs of MXF and mutations in the gyrA gene in a selection of clinical isolates in KZN, South Africa. Fluoroquinolone resistance in MTB is most frequently attributed to mutations occurring in the QRDR of the gyrA gene. The QRDR of the gyrA gene consists of a short region, coding for amino acids 74–113. In our study, we sequenced the QRDR of both the gyrA and gyrB genes, as well as flanking regions. We found the C269T mutation within the QRDR of the gyrA gene, which corre- sponds with the amino acid change A90V, correlated with the high MICs seen in the XDR MTB isolates that we studied. The A90V mutation in gyrA has been described as one of the most frequent mutations associated with fluoroquinolone resistance. 17 In our study, based on WHO-recommended