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Antimicrobial Agents and Chemotherapy, May 2007, p. 1589-1595, Vol. 51, No. 5
0066-4804/07/$08.00+0     doi:10.1128/AAC.01545-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Horizontal Gene Transfer of ftsI, Encoding Penicillin-Binding Protein 3, in Haemophilus influenzae{triangledown}

Sho Takahata,* Takashi Ida, Nami Senju, Yumiko Sanbongi, Aiko Miyata, Kazunori Maebashi, and Shigeru Hoshiko

Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan

Received 12 December 2006/ Returned for modification 20 January 2007/ Accepted 15 February 2007


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ABSTRACT
 
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 β-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 β-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 x 10–7 to 1.5 x 10–6. 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.


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INTRODUCTION
 
Haemophilus influenzae is naturally competent for DNA uptake from the surrounding environment. Recognition and efficient uptake of species-specific DNA molecules is mediated by the presence of uptake signal sequences (USSs) in donor DNA. The consensus sequence of the USS in H. influenzae was identified as 5'-AAGTGCGGT-3' (6). A recent study has revealed that USSs exist in the genomes of other species of the family Pasteurellaceae, suggesting that this element is utilized for the uptake and exchange of DNA between H. influenzae and related species (2). A total of 1,471 copies of the USS were found in the H. influenzae Rd strain by searching its complete genome (15). However, horizontal gene transfer in H. influenzae has been reported for a small number of genes. For instance, the genetic transfer of ompP2, a gene encoding major outer membrane protein P2, was reported among clinical isolates of H. influenzae (7, 16). Intergeneric lateral transfer of the tryptophanase gene cluster between Escherichia coli and H. influenzae was also identified (11).

Penicillin-binding protein 3 (PBP 3), which is encoded by the ftsI gene, is a transpeptidase needed for cross-linking of the septal cell wall peptidoglycan during cell division. The essentiality of this enzyme in H. influenzae was demonstrated by targeted gene disruption (18). A remarkable increase of β-lactamase-nonproducing ampicillin-resistant (BLNAR) H. influenzae strains has been reported in Japan (14). These resistant strains have been shown to possess various mutations in the ftsI gene, leading to amino acid substitutions responsible for β-lactam resistance, such as substitutions at positions 526 (Asn->Lys), 517 (Arg->His), 377 (Met->Ile), 385 (Ser->Thr), and 389 (Leu->Phe) in the PBP 3 protein (12, 19). Due to their rapid increase and nationwide spread, it is difficult to consider that BLNAR strains emerged independently by accumulating multiple mutations in the ftsI gene. Moreover, totally identical ftsI sequences from different BLNAR isolates have frequently been observed (14). These observations drove us to hypothesize that the genetic transfer of the ftsI gene occurs in H. influenzae strains. In this study, evidence for the genetic transfer of the ftsI gene in H. influenzae was obtained by carefully analyzing the nucleotide sequence of the ftsI genes from various clinical isolates and also by in vitro gene transfer experiments.


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MATERIALS AND METHODS
 
Bacterial strains. The strains used in this study are listed in Table 1. H. influenzae Rd (ATCC 51907) and type strains of Haemophilus species (H. haemolyticus ATCC 33390, H. parainfluenzae ATCC 33392, H. aphrophilus ATCC 33389, H. paraphrophilus ATCC 29241, 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 RdRIF, was obtained by selecting a clone grown on chocolate agar containing 25 µg/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 β-NAD+ (V factor) and hemin (X factor) for growth, by the nucleotide sequence of the 16S rRNA gene (4), and also by hemolytic activity assessment.


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  		TABLE 1. Strains used in this study

Media, antimicrobial susceptibility testing, and antibacterial agents. Chocolate II agar plates (Becton Dickinson, Sparks, MD) and brain heart infusion broth supplemented with 2% defibrinated, lysed horse blood plus 15 µg/ml β-NAD+ (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 were used in this study: rifampin (Sigma Chemical Co., St. Louis, MO), cefdinir (Kemprotec, Ltd., Middlesbrough, United Kingdom), and fosfomycin (Meiji Seika Kaisha, Ltd., Tokyo, Japan).

Determining the sequence of the ftsI genes from type strains of Haemophilus species. Reported nucleotide sequences of the ftsI genes from species of the family Pasteurellaceae (H. influenzae Rd, H. ducreyi 35000HP, H. somnus 2336, Mannheimia succiniciproducens MBEL55E, Pasteurella multocida Pm70, and Actinobacillus pleuropneumoniae 4074 [GenBank accession nos. NC 00097, NC 002940, NZ AACJ01000008, NC 006300, NC 002663, and NZ AACK01000005, respectively]) were aligned in order to search for conserved regions. Degenerate oligonucleotide primers specific for the conserved regions upstream and downstream of the coding region of the ftsI gene were constructed. One of two upstream forward primers, primer PH1 (5'-TTGGTAAAGCNATTMWGCC-3') and primer PH2 (5'-GWAGTGCVRTATTACG-3'), was used for PCR amplification, along with a downstream reverse primer, primer PH3 (5'-TGATAATCNARATGATCRCG-3'). Primers PH2 and PH3 were used to amplify the full-length ftsI genes of H. haemolyticus, H. parainfluenzae, H. aphrophilus, and H. paraphrophilus. Primers PH1 and PH3 were used for H. segnis. The following two primers were constructed for specific amplification of the ftsI genes from H. parahaemolyticus and H. paraphrohaemolyticus: 5'-GTGAGGCAGAAATAGAGG-3' and 5'-AAAGGTTCTGCCATCCAC-3'. PCR amplifications were performed as follows: 30 s of denaturation at 98°C and 30 cycles of denaturation at 95°C for 30 s, annealing at 45 to 55°C for 30 s, and extension at 72°C for 3 min. The cycling reaction was performed with a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA). Sequencing was carried out with a BigDye Terminator (version 1.1) cycle sequencing kit and a 3730 DNA analyzer (Applied Biosystems). The sequences were confirmed by sequencing at least two independent PCR products for each strain.

Nucleotide sequencing of the ftsI genes from clinical isolates. The full-length ftsI genes were amplified by PCR with the following primers: 5'-CTCGTTATCCGTTACAGCAG-3' and 5'-GCCAAACCGTGTGATGAAAC-3'. PCR amplifications were performed as follows: 2 min of denaturation at 94°C and 30 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 3 min. The cycling reaction and DNA sequencing were performed as described above.

PFGE. The preparation of the plugs containing bacteria and pulsed-field gel electrophoresis (PFGE) were performed as described previously (13), with slight modifications. Briefly, cells grown in sBHI broth were collected and suspended in 10 mM EDTA buffer (pH 8.0). The suspension was mixed with an equivalent volume of 2% low-melting-point agarose to produce agarose plugs. After solidification, the plugs were incubated in 100 mM EDTA buffer containing 50 µg lysozyme per ml at 37°C for 1 h. Then the plugs were treated with 250 mM EDTA buffer containing 10 mg proteinase K per ml and 1% N-lauroylsarcosine overnight at 50°C. After the plugs were washed with extensive volumes of 10 mM Tris-HCl (pH 8.0)-1 mM EDTA buffer, the DNA was digested with SmaI. The resulting DNA fragments were subjected to PFGE in 0.5x TBE (0.045 M Tris-borate, 1 mM EDTA) buffer with a CHEF Mapper electrophoresis system (Bio-Rad Laboratories, Hercules, CA). The DNA fragments were visualized with ethidium bromide.

In vitro genetic transfer. Cells grown on chocolate II agar were suspended in sBHI broth at a density of about 108 CFU/ml. After preincubation at 37°C for 1 h, H. influenzae RdRIF and one of the BLNAR strains (strain MSC06647, MSC06651, or MSC06663) were mixed at a ratio of 1:1. The effect of DNase I on transfer was assessed by adding 100 U of the enzyme (Takara Bio Inc., Otsu, Japan). The cell suspensions were incubated at 37°C for 2 h and plated on chocolate agar containing 16 µg/ml of rifampin and 0.5 µg/ml of cefdinir. Nucleotide sequencing of the ftsI gene from doubly resistant mutants was performed as indicated above.

Nucleotide sequence accession numbers. The complete DNA sequences of the ftsI genes from type strains of Haemophilus species and from clinical isolates of H. influenzae and H. haemolyticus, as determined in the present study, appear in the DDBJ/EMBL/GenBank nucleotide sequence databases under the accession nos. AB267855 to AB267861 (for the type strains) and AB267863 to AB267867 (for the clinical isolates).


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RESULTS
 
Determination of ftsI gene sequences from Haemophilus species. Analysis of the nucleotide sequences of the ftsI genes from 621 H. influenzae isolates revealed that at least 10 strains harbored mosaic structures of the ftsI gene which contained a sequence divergent from that of the Rd strain. The nucleotide sequences of these regions showed no similarity with those of the reported ftsI genes. To identify the origin of the mosaic sequence, the complete sequence of the ftsI genes of Haemophilus species, whose natural host is only humans, was determined. Figure 1 shows the deduced amino acid sequences of PBP 3 for type strains of seven Haemophilus species. The PBP 3 sequences of H. haemolyticus and H. parainfluenzae showed high degrees of homology with that of H. influenzae. The identities of the amino acid sequences were 92.5% and 83.1%, respectively. The ftsI genes of H. parahaemolyticus and H. paraphrohaemolyticus were quite different from that of H. influenzae and much closer to those of H. ducreyi and Actinobacillus pleuropneumoniae.


Figure 1
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  		FIG. 1. Deduced amino acid sequences of PBP 3 from seven Haemophilus species. The complete PBP 3 sequences for H. haemolyticus ATCC 33390, H. parainfluenzae ATCC 33392, H. aphrophilus ATCC 33389, H. paraphrophilus ATCC 29241, H. segnis ATCC 33393, H. parahaemolyticus ATCC 10014, and H. paraphrohaemolyticus ATCC 29237 were aligned with that of H. influenzae Rd. Three conserved motifs in the active site are boxed, and an asterisk shows the position of the catalytic serine residue. Dots, identical amino acids.

Recombination of the ftsI gene between H. influenzae and H. haemolyticus. Comparison of the mosaic ftsI genes with the corresponding genes of Haemophilus species (H. parainfluenzae, H. haemolyticus, H. aphrophilus, H. paraphrophilus, H. segnis, H. parahaemolyticus, and H. paraphrohaemolyticus) indicated that homologous recombination between H. haemolyticus and H. influenzae had occurred in some isolates (Fig. 2). While the overall divergence of the nucleotide sequences between H. haemolyticus ATCC 33390 and H. influenzae Rd was 12.7% (233 bp of 1,833 bp), both genes possessed two USSs at the same position. One of the H. influenzae isolates (isolate MSC07169) possessed a sequence at the 3'-terminal region (positions 1647 to 1833) almost identical to that of H. haemolyticus ATCC 33390. Another H. influenzae isolate (isolate MSC07771) was also found to have an H. haemolyticus-derived sequence at positions 402 to 831. Furthermore, an H. haemolyticus isolate (isolate MSC07286) harboring a partial ftsI gene of H. influenzae at the 3'-terminal region (positions 897 to 1833) was also identified among the culture collection. These data provide direct evidence for the homologous recombination of the ftsI gene between H. influenzae and H. haemolyticus.


Figure 2
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  		FIG. 2. Schematic representation of the ftsI genes of H. influenzae and H. haemolyticus strains. The nucleotide sequence divergences between regions of the ftsI genes compared with the sequences of the corresponding regions in the ftsI gene of H. influenzae Rd are shown as percentages. Solid bars, nucleotides that differ from the corresponding nucleotides in H. influenzae strains. The sites and orientations of the USSs are shown as arrows under the H. influenzae Rd and H. haemolyticus ATCC 33390 sequences.

Evidence for intraspecies genetic transfer of the ftsI gene. To investigate the genetic transfer of the ftsI gene between H. influenzae strains, particularly among ampicillin-resistant variants, BLNAR and β-lactamase-positive ampicillin-clavulanic acid-resistant (BLPACR) strains possessing ftsI genes with identical nucleotide sequences were selected for genotyping by PFGE (Table 1, strains 1 to 10). Four strains isolated from different hospitals (strains MSC02070, MSC01869, MSC01400, and MSC01855) possessed identical nucleotide sequences for ftsI (sequence 1). Another ftsI sequence (sequence 2) was found in three strains isolated from three separate hospitals (strains MSC07237, MSC02104, and MSC02149). Strains MSC02022, MSC01363, and MSC01432 were isolated at the same hospital and harbored ftsI genes with identical sequences (sequence 3). As shown in Fig. 3A, strains isolated from different hospitals harboring identical ftsI genes exhibited distinctive PFGE patterns (lanes 1 to 4 and lanes 5 to 7), indicating that these isolates are phylogenetically independent, despite the identity of their ftsI genes. On the other hand, three BLNAR strains harboring identical ftsI genes from the same hospital showed the same PFGE pattern (lanes 8 to 10), suggesting their clonal spread.


Figure 3
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  		FIG. 3. PFGE patterns of BLNAR and BLPACR isolates, and strains used for in vitro gene transfer. (A) Lane numbers correspond to the strain numbers in Table 1. Identical ftsI genes were identified among strains 1 to 4 (ftsI sequence 1), 5 to 7 (ftsI sequence 2), and 8 to 10 (ftsI sequence 3). (B) Lane numbers 11 to 14 correspond to the strain numbers in Table 1. Lanes 15, 16, and 17, PFGE patterns of rifampin- and cefdinir-resistant mutants from MSC06647 (RdRIF/MSC06647), MSC06651 (RdRIF/MSC06651), and MSC06663 (RdRIF/MSC06663), respectively. Lanes M, molecular size marker (bacteriophage lambda ladder DNA).

In vitro transfer of ftsI gene from BLNAR to BLNAS. A spontaneous rifampin-resistant mutant of H. influenzae Rd, designated RdRIF (a β-lactamase-nonproducing ampicillin-susceptible [BLNAS] strain), and three BLNAR isolates (isolates MSC06647, MSC06651, and MSC06663) were used to evaluate the genetic transfer of the ftsI gene in vitro. The spontaneous rates of rifampin resistance in the BLNAR strains were 4.3 x 10–9 to 8.7 x 10–9. After the cultures of RdRIF and one of the BLNAR strains were mixed, mutants resistant to both rifampin and cefdinir were selected. Doubly resistant mutants were obtained after the coincubation of RdRIF with MSC06647, MSC06651, and MSC06663 at frequencies of 5.1 x 10–7, 1.2 x 10–6, and 1.5 x 10–6, respectively (Table 2). Treatment with DNase I at 100 U totally abolished the emergence of doubly resistant mutants (below the lower limit of detection). Nucleotide sequencing of the ftsI genes from doubly resistant mutants revealed that the mutants possessed sequences identical to that of each parent BLNAR strain (data not shown). As shown in Table 3, the susceptibilities of the mutant clones (clones RdRIF/MSC06647, RdRIF/MSC06651, and RdRIF/MSC06663) to rifampin and fosfomycin were equivalent to those of RdRIF. Moreover, as predicted from the ftsI sequence, the susceptibility of these mutants to cefdinir was reduced to a level similar to that of each of the parent BLNAR strains, suggesting that doubly resistant mutants were derivatives of the RdRIF strain that acquired the ftsI gene from each BLNAR strain. The genotypes of the doubly resistant mutants were confirmed by PFGE (Fig. 3B). Doubly resistant mutants (lanes 15 to 17) had the same pattern with RdRIF (lane 14) and had patterns that were divergent from those of the BLNAR strains (lanes 11 to 13).


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  		TABLE 2. Transfer of ftsI genes from BLNAR to RdRIF (BLNAS)


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  		TABLE 3. Susceptibilities of parent strains and doubly resistant derivatives


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DISCUSSION
 
The nucleotide sequences of the ftsI gene of BLNAR strains are quite different from that of the H. influenzae Rd strain. For instance, the sequences of ftsI from strains MSC02070 (sequence 1; GenBank accession no. AB267863), MSC07237 (sequence 2; GenBank accession no. AB257096), and MSC02022 (sequence 3; GenBank accession no. AB267864), the strains used in PFGE analysis, differed from that of Rd by 2.7% (49 bp of 1,833 bp), 3.0% (55 bp of 1,833 bp), and 5.6% (102 bp of 1,833 bp), respectively. The existence of genotypically divergent strains harboring identical ftsI genes (Fig. 3A) strongly suggests the occurrence of genetic transfer of the ftsI gene in H. influenzae. The existence of USSs in the middle of and just downstream of the ftsI gene supports the efficient uptake and recombination of this gene (Fig. 2), resulting in horizontal gene transfer among species.

Extensive sequence analysis of the ftsI genes from numerous clinical isolates of H. influenzae identified a mosaic structure of the ftsI gene in some strains. Complete sequences of the ftsI genes from Haemophilus species other than H. influenzae, such as H. parainfluenzae, H. haemolyticus, H. aphrophilus, H. paraphrophilus, and H. segnis, were determined and compared with that of the mosaic ftsI gene. Homologous recombination of the ftsI gene derived from H. haemolyticus was identified in some isolates (Fig. 2). Moreover, an H. haemolyticus strain harboring a partial ftsI gene from H. influenzae was also found. This is the first report describing the homologous recombination of the gene encoding a PBP in H. influenzae. It has been reported that other naturally competent gram-negative bacteria, Neisseria species (Neisseria gonorrhoeae and N. meningitidis), develop resistance to β-lactams by acquiring a mosaic-like structure of PBP 2, encoded by the penA gene. PBP 2 of Neisseria species is a homologue of PBP 3 in H. influenzae (36% amino acid sequence identity). It has been suggested that the horizontal genetic exchange of penA genes between commensal Neisseria species, such as N. cinerea, N. perflava, and N. flavescens, resulted in the mosaic structure of PBP 2 in N. gonorrhoeae and N. meningitidis (17). As H. haemolyticus is often recovered from the oropharyngeal area (10), it is possible that genetic exchange between colocalized H. influenzae and H. haemolyticus isolates or other Haemolyticus species takes place.

The molecular evolution of BLNAR strains has been considered to occur through the acquisition of point mutations in the ftsI gene by antibiotic pressure. However, our results raise the possibility of the development of resistance through homologous recombination from related species, which is a well-recognized mechanism known in N. gonorrhoeae (17) and Streptococcus pneumoniae (5, 9). As shown in Fig. 4, the nucleotide sequences of BLNAR strains are highly similar to that of H. haemolyticus around the region encoding the conserved SSN motif. Indeed, PBP 3 of H. haemolyticus, as well as those of BLNAR strains, possesses 377-Ile, which is identified as the amino acid residue associated with β-lactam resistance. Therefore, the ftsI gene of H. haemolyticus and/or related species might be the source of BLNAR. On the other hand, no sequence homology was observed in the region downstream of the KTG motif between BLNAR strains and H. haemolyticus or other Haemophilus species. These observations suggest that primary mutations of BLNAR, i.e., Asn526Lys and Arg517His, have emerged from the point mutation during antibiotic pressure.


Figure 4
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  		FIG. 4. Partial alignment of ftsI genes of H. influenzae Rd, four BLNAR strains (strains MSC06651, MSC06663, MSC02070, and MSC07237), and H. haemolyticus ATCC 33390. The nucleotide sequences from positions 1001 to 1200 and positions 1501 to 1600 are shown. Sequences coding for the conserved motifs, SSN and KTG, are boxed. Amino acid sequences associated with β-lactam resistance are shown above the corresponding nucleotide sequences. Dots, identical nucleotides.

A recent study of the bacterial flora in patients with acute upper respiratory tract infections and healthy subjects revealed that H. influenzae strains are frequently detected in the nasopharynges from both groups aged 6 years or younger (8). Therefore, the colocalization of or coinfection with multiple H. influenzae strains could happen in some children, giving the opportunity for H. influenzae strains to exchange DNA. In Japan, oral cephalosporins are extensively used for the treatment of upper respiratory tract infections in children. BLNAR strains harboring mutations in PBP 3 are resistant to most of the oral cephems but are not resistant to cefditoren. The use of those cephems with poor activities against BLNAR strains not only results in the failure of the treatment (i.e., the failure to eliminate BLNAR strains) but also leads to the selection of BLNAR strains mediated by genetic transfer of the mutated ftsI gene to BLNAS (i.e., the failure to prevent the emergence of BLNAR strains).

Direct evidence for horizontal gene transfer of the ftsI gene between H. influenzae strains was obtained from in vitro gene transfer experiments. Coincubation of BLNAS and BLNAR strains resulted in the transfer of the ftsI gene from BLNAR to a BLNAS strain and recombination (Table 2). Addition of DNA fragments of the ftsI gene derived from BLNAR strains by PCR amplification, at final concentrations of 1 to 10 ng/ml, to the culture of BLNAS (strain RdRIF) had equivalent effects on the emergence of rifampin- and cefdinir-resistant mutants as coincubation of BLNAR and BLNAS strains (data not shown). Treatment with 100 U of DNase I abolished the emergence of resistant mutants not only after the transformation of DNA fragments to RdRIF strain but also after the coincubation of BLNAS and BLNAR strains. Although cell-to-cell contact has previously been implicated in chromosomal gene transfer (1), the mechanism of in vitro gene transfer of the ftsI gene was found to involve classical transformation.

In conclusion, horizontal transfer of the ftsI gene in H. influenzae was identified for the first time. The transfer of mutated ftsI genes might be associated with the rapid increase in BLNAR strains in Japan. Additionally, in vitro gene transfer experiments will be useful in evaluating the evolution of BLNAR strains.


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ACKNOWLEDGMENTS
 
We thank Kimiko Ubukata (Kitasato University) for kindly providing clinical isolates of H. influenzae.


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FOOTNOTES
 
* Corresponding author. Mailing address: Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama 222-8567, Japan. Phone: 81-45-545-3139. Fax: 81-45-541-1768. E-mail: sho_takahata{at}meiji.co.jp Back

{triangledown} Published ahead of print on 26 February 2007. Back


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Antimicrobial Agents and Chemotherapy, May 2007, p. 1589-1595, Vol. 51, No. 5
0066-4804/07/$08.00+0     doi:10.1128/AAC.01545-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Tristram, S. G., Pitout, M. J., Forward, K., Campbell, S., Nichols, S., Davidson, R. J. (2008). Characterization of extended-spectrum {beta}-lactamase-producing isolates of Haemophilus parainfluenzae. J Antimicrob Chemother 61: 509-514 [Abstract] [Full Text]  

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