Molecular mechanisms of Bartonella henselae resistance to azithromycin, pradofloxacin and enrofloxacin.

Sir, Bartonella henselae are fastidious, facultative intracellular bacilli that can cause bacteraemia, endocarditis, cat-scratch disease in immunocompetent patients, and bacillary angiomatosis and peliosis hepatitis in immunocompromised patients. There are a limited number of studies that have addressed the antibiotic treatment of cats infected with B. henselae. Azithromycin, a macrolide compound, has seemingly become the drug of choice to treat cats and dogs for B. henselae infection. However, relapses after antibiotic withdrawal have been reported. Azithromycin, which is derived from erythromycin, binds to the 50S subunit of the bacterial ribosome and, thus, inhibits the translation of mRNA. Fluoroquinolone antibiotics exert their antibacterial effects by inhibiting certain bacterial topoisomerase enzymes. Pradofloxacin, a third-generation fluoroquinolone, is being exclusively developed for use in veterinary medicine. Enrofloxacin is a broad-spectrum antimicrobial agent with bactericidal activity against Gram-negative and Grampositive bacteria, mycobacteria and rickettsia. As for B. henselae, potential mechanisms of resistance to azithromycin, pradofloxacin and enrofloxacin are not known. Therefore, the objective of this study was to select in vitro azithromycin-resistant, pradofloxacin-resistant and enrofloxacin-resistant mutants to determine the molecular mechanism of resistance. Six B. henselae isolates (BhH1, Mina Mia, Stray7, Bh94FO73, BhFO1946 and Bh95FO101) were used in this study. Four of these isolates (Mina Mia, Stray7, Bh94FO73 and BhFO1946) were derived from cats from the USA. BhH1 is the ATCC type strain obtained by blood culture from a febrile, HIV-infected patient in Houston, Texas. Isolate Bh95FO101 was from a sick pet cat from Israel. Pradofloxacin (5 mg) and enrofloxacin (5 mg) discs were purchased from AB Biodisk (Solna, Sweden) and supplied by Bayer HealthCare, Germany. Azithromycin (15 mg) discs were purchased from VWR International, USA. The selection of antibiotic-resistant mutants was performed by serial passages of each B. henselae isolate on blood agar plates containing an antibiotic disc. Bartonella strains were considered resistant when the inhibition zone was ,6 mm. The B. henselae azithromycin-susceptible strains and the azithromycin-resistant mutants were screened by PCR and sequencing using primers for 23S rRNA, and L4 and L22 ribosomal proteins. Strains resistant and susceptible to pradofloxacin and enrofloxacin were screened by PCR and sequencing using primers for gyrA, gyrB, parC and parE. Six B. henselae azithromycin-resistant mutants were obtained after the second in vitro passage (Table 1). Compared with the parental strain, each B. henselae azithromycin-resistant mutant had a homogenous single nucleotide substitution at position 2058 (A2058G, Escherichia coli numbering) in the 23S rRNA gene. Mutations at A2058 for certain macrolides confer the highest levels of resistance. Many independent lines of evidence indicate that adenosine 2058 is the key nucleotide involved in macrolide interaction with the bacterial ribosome. An A2058 to G transition was the first rRNA mutation shown to confer erythromycin resistance and is presently the most frequent substitution found in clinical isolates. Our in vitro results might explain relapses or treatment failures observed in vivo when using azithromycin as the sole antibiotic for treatment of Bartonella-related infections. We did not find any change in the L4 and L22 ribosomal proteins for the B. henselae azithromycinresistant mutants. All B. henselae isolates became resistant to pradofloxacin and enrofloxacin after differing numbers of subculture passages; however, in contrast to azithromycin, at least five passages