Wolbachia pipientis growth kinetics and susceptibilities to 13 antibiotics determined by immunofluorescence staining and real-time PCR

Abstract

Wolbachia spp. are strict intracellular bacteria that infect a wide range of arthropods and filarial nematodes. Filarial nematodes are important causes of human diseases. There is increasing evidence that Wolbachia spp. influence important functions in the biology of the hosts, specifically, infertility. Preliminary experiments with humans and animals have suggested that antibiotics with activity against Wolbachia may help to treat filariasis. In this study, we determined using a real-time quantitative PCR assay the growth kinetics of a strain of Wolbachia pipientis from a mosquito grown in Aa23 cells. The doubling time was estimated to be 14 h. We then determined the susceptibilities of this strain to 13 antibiotics by two methods: an immunofluorescent-antibody test and a real-time quantitative PCR assay. Both techniques gave similar results. Doxycycline and rifampin were the most effective compounds, with MICs of 0.125 and 0.06 to 0.125 micro g/ml, respectively. Fluoroquinolones were less effective, with MICs of 2 to 4 micro g/ml for ciprofloxacin, 2 micro g/ml for ofloxacin, and 1 micro g/ml for levofloxacin. beta-Lactams (penicillin G, amoxicillin, ceftriaxone) were not effective at concentrations up to 128 micro g/ml. The MIC of erythromycin was >32 micro g/ml, whereas that of telithromycin was 8 micro g/ml. Other antibiotic compounds were bacteriostatic only at high concentrations, including gentamicin, co-trimoxazole, and thiamphenicol. The real-time PCR assay was a convenient and reliable technique for determination of the antibiotic susceptibilities of WOLBACHIA: It may help in the future to simplify antibiotic susceptibility testing of strict intracellular pathogens.

FIG. 1.

Growth kinetics of W. pipientis in culture as determined by quantitative PCR assay.

FIG. 2.

Light Cycler PCR melting curve obtained with standard concentrations of W. pipientis showing the specificity of the PCR product as a single peak.

FIG.3.

Phylogenetic tree based on 16S rRNA gene sequences showing the association of the intracellular bacteria of the Rickettsia spp., Ehrlichia spp., Anaplasma spp., Neorickettsia spp., W. pipientis, and several Wolbachia endosymbionts from nematods and correlation between doxycycline, fluoroquinolone, and erythromycin susceptibilities. R, resistant; S, susceptible. For doxycycline susceptibility was considered an MIC ≤4 μg/ml, for fluoroquinolones susceptibility was considered an MIC ≤2 μg/ml and resistance was considered an MIC >2 μg/ml, for erythromycin susceptibility was considered an MIC ≤1 μg/ml and resistance was considered an MIC >1 μg/ml, for rifampin susceptibility was considered an MIC ≤1 μg/ml and resistance was considered an MIC >1 μg/ml, and for chloramphenicol susceptibility was considered an MIC ≤8 μg/ml and resistance was considered an MIC >8 μg/ml. The numbers at the nodes represent the bootstrap confidence value after 100 replicates. Coxiella burnetii was defined as the outgroup. The sequences of the following species were used in the construction of the tree (GenBank accession numbers are given in parentheses): C. burnetii (D89799), R. typhi (L36221), R. prowazekii (M21789), R. massiliae (L36106), R. rickettsii (L36217), N. risticii (AF037211), N. sennetsu (M73225), N. helminthoeca (U12457), W. pipientis (X61768), A. marginale (AF311303), A. platys (AF287153), A. phagocytophila (M73224), Cowdria ruminantium (U03777), E. ewingii (U96436), E. canis (AF373613), E. muris (U15527), E. chaffeensis (U60476), Wuchereria bancroftii endosymbiont (AF093510), B. malayi endosymbiont (AF051145), Onchocerca volvulus endosymbiont (AF069069), L. sigmodontis endosymbiont (AF069068), and Dirofilaria immitis endosymbiont (Z49261).