Horizontal Gene Transfer of ftsI , Encoding Penicillin-Binding Protein 3, in Haemophilus inﬂuenzae (cid:1)

Horizontal gene transfer has been identiﬁed in only a small number of genes in Haemophilus inﬂuenzae , an organism which is naturally competent for transformation. This report provides evidence for the genetic transfer of the ftsI gene, which encodes penicillin-binding protein 3, in H. inﬂuenzae . Mosaic structures of the ftsI gene were found in several clinical isolates of H. inﬂuenzae . To identify the origin of the mosaic sequence, complete sequences of the corresponding gene from seven type strains of Haemophilus species were determined. Comparison of these sequences with mosaic regions identiﬁed a homologous recombination of the ftsI gene between H. inﬂuenzae and Haemophilus haemolyticus . Subsequently, ampicillin-resistant H. inﬂuenzae strains harboring identical ftsI sequences were genotyped by pulsed-ﬁeld gel electrophoresis (PFGE). Divergent PFGE patterns among (cid:1) -lactamase-nonproducing ampicillin-resistant (BLNAR) strains from different hospitals indicated the potential for the genetic transfer of the mutated ftsI gene between these isolates. Moreover, transfer of the ftsI gene from BLNAR strains to (cid:1) -lactamase-nonproducing ampicillin-susceptible (BLNAS) H. inﬂuenzae strains was evaluated in vitro. Coincubation of a BLNAS strain (a rifampin-resistant mutant of strain Rd) and BLNAR strains resulted in the emergence of rifampin- and cefdinir-resistant clones at frequencies of 5.1 (cid:2) 10 (cid:3) 7 to 1.5 (cid:2) 10 (cid:3) 6 . Characterization of these doubly resistant mutants by DNA sequencing of the ftsI gene, susceptibility testing, and genotyping by PFGE revealed that the ftsI genes of BLNAR strains had transferred to BLNAS strains during coincubation. In conclusion, horizontal transfer of the ftsI gene in H. inﬂuenzae can occur in an intraspecies and an interspecies manner. H. segnis ATCC 33393, H. parahaemolyticus ATCC 10014, and H. paraphrohaemolyticus ATCC 29237) were obtained from the American Type Culture Collection. A spontaneous rifampin-resistant derivative of H. inﬂuenzae Rd, designated H. inﬂuenzae Rd RIF , was obtained by selecting a clone grown on chocolate agar containing 25 (cid:4) g/ml of rifampin. Complete sequencing of the rpoB gene in this mutant revealed a point mutation, G436T, that resulted in the replacement of Val-146 to Phe-146 in the RpoB protein. Other H. inﬂuenzae and H. haemolyticus strains were clinical isolates collected in Japan between 1997 and 2003 (12, 14). The isolates were identiﬁed through their requirements for (cid:3) -NAD (cid:1) (V factor) and hemin (X factor) for growth, by the nucleotide sequence of the 16S rRNA gene (4), and also by hemolytic activity assessment. Media, antimicrobial susceptibility testing, and antibacterial agents. Choco- late II agar plates MD) and brain heart infusion broth supplemented with 2% deﬁbrinated, lysed horse blood plus 15 (cid:4) g/ml (cid:3) -NAD (cid:1) (sBHI broth) were routinely used for bacterial growth. MICs were determined by the broth dilution method according to the guidelines of the Clinical and Laboratory Standards Institute (3). The following antibiotics study: fosfomycin the sequence the ftsI genes from type strains of nucleotide sequences of the ftsI genes aligned in order to search for conserved regions. Degenerate oligonucleotide primers speciﬁc for the conserved regions upstream and down- stream of the coding region of the ftsI of two forward primers, a downstream reverse primer, primer PH3