Identificação de <i>Achromobacter xylosoxidans</i> isoladas na fibrose cística: comparação entre as técnicas de <i>Multilocus Sequence Typing</i>, PCR para o gene <i>bla</i>_OXA-114 e sequenciamento do gene 16S rRNA

Abstract

<i>Achromobacter xylosoxidans</i> has been highlighted as an important pathogen in lung infections of patients with cystic fibrosis (CF). Phenotypic and genotypic similarity among species of the genus hampers identification and the real frequency is possibly underestimated. The proper identification is required to understand epidemiology and clinical impacts of different species of <i>Achromobacter</i>. This study compared the results obtained of identification of species of the genus by 16S rRNA gene, by Multilocus Sequence Typing (MLST) and by PCR for <i>bla</i>_OXA-114-like gene. Thirty <i>Achromobacter</i> spp. samples were obtained from 17 patients with CF treated in two reference centers Rio de Janeiro city and belonging to 18 clones previously established by PFGE were evaluated. The 16S rRNA gene sequencing identified all samples as <i>A. xylosoxidans</i>. The sequencing of the amplification products of <i>bla</i>_OXA-114-like gene revealed compatibility of 17 samples with some gene variation and were identified as <i>A. xylosoxidans</i>. Eleven samples were consistent with the <i>bla</i>_OXA-258 gene, and were classified as <i>A. ruhlandii</i>. Two samples didn t obtain amplification. MLST analysis showed 11 samples matching four STs (2, 13, 35, 36) provided by MLST <i>Achromobacter</i> site. In 15 samples were characterized ten new alleles, grouped in eight new STs (198, 199, 200, 201, 202, 203, 204 and 205). Two new combinations of alleles were observed in 2 samples featuring 2 new STs (206 and 207). Two samples didn t have their STs defined. The 30 samples corresponded to 14 STs and four species of <i>Achromobacter</i> spp. Seventeen samples were identified as <i>A. xylosoxidans</i>, nine as <i>A. ruhlandii</i>, onde as <i>A.dolens</i> and one as <i>A. insuavis</i>. The three techniques were consistent only to identify 17 samples as <i>A. xylosoxidans</i> (56.6%). The identification of nine samples of <i>A. ruhlandii</i> were concordant in MLST, PCR and sequencing for OXA-114 techniques. A. dolens and <i>A. insolitus</i> were identified only by MLST. The comparison between PFGE and MLST showed that results of the two techniques aren t entirely consistent. PFGE technique characterized 18 clones while MLST identified 14 STs. Among different samples, PFGE different clones were correspondent to the same ST and some PFGE / MLST clones were unique per patient. Results of this study show that 16S rRNA sequencing was a good tool for gender characterization and PCR gene <i>bla</i>_OXA-114 was useful for identification of two most common species in the FC (<i>A. xylosoxidans</i> and <i>A.ruhlandii</i>). However, MLST had the best performance featuring all described species. Additional studies are required to elucidate the infection dynamics and contribution of the different species of the genus <i>Achromobacter</i> in FC.