Isolates of the gastric pathogen Helicobacter pylori harvested from different individuals are highly polymorphic. Strain variation also has been observed within a single host. To more fully ascertain the extent of H. pylori genetic diversity within the ecological niche of its natural host, we harvested additional isolates of the sequenced H. pylori strain J99 from its human source patient after a 6-year interval. Randomly amplified polymorphic DNA PCR and DNA sequencing of four unlinked loci indicated that these isolates were closely related to the original strain. In contrast, microarray analysis revealed differences in genetic content among all of the isolates that were not detected by randomly amplified polymorphic DNA PCR or sequence analysis. Several ORFs from loci scattered throughout the chromosome in the archival strain did not hybridize with DNA from the recent strains, including multiple ORFs within the J99 plasticity zone. In addition, DNA from the recent isolates hybridized with probes for ORFs specific for the other fully sequenced H. pylori strain 26695, including a putative traG homolog. Among the additional J99 isolates, patterns of genetic diversity were distinct both when compared with each other and to the original prototype isolate. These results indicate that within an apparently homogeneous population, as determined by macroscale comparison and nucleotide sequence analysis, remarkable genetic differences exist among single-colony isolates of H. pylori. Direct evidence that H. pylori has the capacity to lose and possibly acquire exogenous DNA is consistent with a model of continuous microevolution within its cognate host.

The prevalence of recently described mutation V176F, located in the beginning of the rpoB gene and associated with rifampin resistance and the wild-type cluster I sequence, was determined by analyzing the distribution of rpoB mutations among 80 rifampin (RIF)-resistant Mycobacterium tuberculosis strains isolated in Germany during 1997. The most frequent rpoB mutations were changes in codon 456 (52 isolates, 65%), followed by changes in codon 441 (13 isolates, 16%) and codon 451 (11 isolates, 14%). The V176F mutation was detected in one isolate of the study population and in 5 of 18 RIF-resistant strains with no cluster I mutation from six previously published studies. In three isolates, a mixture of resistant and susceptible subpopulations (heteroresistance) prohibited the detection of rpoB mutations in the initial analysis; however, in these isolates, cluster I mutations could be verified after a passage on RIF-containing medium. IS6110 DNA fingerprinting of 76 strains revealed eight clusters comprising 27 strains with identical restriction fragment length polymorphism patterns that mainly also show identical rpoB mutations and identical or similar drug resistance patterns. In conclusion, our results indicate that the V176F mutation should be included in molecular tests for prediction of RIF resistance in M. tuberculosis. We further demonstrated that heteroresistance caused by a mixture of mycobacterial subpopulations with different susceptibilities to RIF may influence the sensitivity of molecular tests for detection of resistance.

NCT01138891 (http://www.clinicaltrials.gov).

A clinical isolate of Helicobacter pylori that developed resistance to rifabutin during therapy carried an rpoB gene that retained a wild-type cluster region sequence but had acquired a novel codon 149 (V149F) mutation. In transformation experiments, the mutation was shown to confer high-level rifabutin resistance. The equivalent mutation (V176F) was present in several resistant isolates of Mycobacterium tuberculosis.Rifabutin and other derivatives of rifampin are inhibitory against Helicobacter pylori at very low concentrations in vitro (1, 4). Triple therapy including rifabutin, amoxicillin, and a proton pump inhibitor has been effective in the eradication of H. pylori after failure of other therapies and in spite of resistance to other antibiotics (10). Resistance to rifampin and rifabutin is caused by amino acid exchanges in the ␤ subunit of the DNA-directed RNA polymerase (RpoB). Mutations at codons 146, 507 to 533, 563 to 572, and 687 of the rpoB gene in Escherichia coli (11) or at codons 507 to 533 (cluster region) in Mycobacterium tuberculosis (8, 9) have been shown to induce resistance.All resistant mutants of H. pylori ATCC 43504 selected in vitro in a previous investigation (4) showed mutations in the cluster region encompassing codons 525 to 545 and codon 586.Here we describe a clinical isolate of H. pylori that developed resistance during therapy. The patient was treated with lanzoprazole and rifabutin, but amoxicillin was discontinued due to intolerance. Isolates of H. pylori before (DR62a) and after (DR62n) treatment were available for evaluation. Culture, storage, E-test, agar dilution assay, PCR, and sequencing of the cluster region were performed as described recently (4). Table 1 shows the primers for the amplification and evaluation of large and small segments of the respective rpoB regions.Sequencing was performed in both directions (Perkin-Elmer Applied Biosystems).DR62n was resistant to rifampin (E-test, MIC of Ͼ256 g/ ml) and rifabutin but, in contrast to previous findings (4), showed no difference from the published wild-type sequence (14) in the cluster region. The MICs of rifabutin for H. pylori DR62a and DR62n were 0.002 and 8 g/ml, respectively (Table 2). Both strains yielded homologous patterns when typed by arbitrarily primed PCR (2; unpublished work). Amplification and sequencing of DR62n rpoB (rifabutin resistant) from bp 387 to bp 916 revealed a codon exchange, GTC3TTC (V149F). DR62a (rifabutin susceptible) showed wild-type sequence in both the V149 region and the cluster region.In order to confirm that the amino acid mutation V149F is responsible for high-level resistance, large fragments of the rpoB gene (bp 54 to 916 harboring codon 149 and bp 1271 to 2106 harboring the cluster region) from DR62n and DR62a were amplified and sequenced. The amino acid mutation V149F was the only difference between DR62n and DR62a or the published wild-type sequence (14). All fragments were transformed into a competent, rifabutin-susceptible strain, H. pylori 2802A. Transformation of H. pylori ...

Background Capsular fibrosis is a severe complication after breast implantation with an uncertain etiology. Microbial colonization of the prosthesis is hypothesized as a possible reason for the low-grade infection and subsequent capsular fibrosis. Current diagnostic tests consist of intraoperative swabs and tissue biopsies. Sonication of removed implants may improve the diagnosis of implant infection by detachment of biofilms from the implant surface. Methods Breast implants removed from patients with Baker grades 3 and 4 capsular contracture were analyzed by sonication, and the resulting sonication fluid was quantitatively cultured. Results This study investigated 22 breast implants (6 implants with Baker 3 and 16 implants with Baker 4 capsular fibrosis) from 13 patients. The mean age of the patients was 49 years (range, 31-76 years). The mean implant indwelling time was 10.4 years (range, 3 months to 30 years). Of the 22 implants, 12 were used for breast reconstruction and 10 for aesthetic procedures. The implants were located subglandularly (n = 12), submuscularly (n = 6), and subcutaneously (n = 4). Coagulasenegative staphylococci, Propionibacterium acnes, or both were detected in the sonication fluid cultures of nine implants (41%), eight of which grew significant numbers of microorganisms ([100 colonies/ml of sonication fluid). Conclusions Sonication detected bacteria in 41% of removed breast implants. The identified bacteria belonged to normal skin flora. Further investigation is needed to determine any causal relation between biofilms and capsular fibrosis.

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