JAC Advance Access published online on February 14, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn051
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Mutations in penicillin-binding proteins 1, 2 and 3 are responsible for amoxicillin resistance in Helicobacter pylori
1 Department of Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan 2 Endoscopy Center, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan
* Corresponding author. Tel: +81-426-76-5615; Fax: +81-426-76-5647; E-mail: noguchin{at}ps.toyaku.ac.jp
Received 11 November 2007; returned 21 January 2008; revised 13 December 2007; accepted 22 January 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Objectives: To elucidate the relationship between the mutations of penicillin-binding protein (PBP)1, PBP2 and PBP3 and amoxicillin resistance in Helicobacter pylori.
Methods: The mutations detected only in clinical amoxicillin-resistant strains were determined by comparison of the deduced amino acid sequences of PBP1(HP0597), PBP2(HP1556) and PBP3(HP1565) encoded by the pbp1, ftsI and pbp2 genes, respectively, in 13 clinical H. pylori strains and three ATCC strains. The contribution of the mutations in PBPs was analysed by the natural transformation of the amoxicillin-susceptible strain ATCC 700392 with various combinations of the pbp1, ftsI and pbp2 genes from the amoxicillin-resistant strain TH743 (MIC of amoxicillin: 8 mg/L).
Results: We initially identified six, four and two mutations of PBP1, PBP2 and PBP3, respectively, which were detected only in amoxicillin-resistant strains. By the natural transformation of an amoxicillin-susceptible strain ATCC 700392, we found that mutations in PBP1 and PBP3 conferred higher resistance to amoxicillin than mutations in PBP1 and PBP2, or mutations only in PBP1. Furthermore, mutations in PBP1, PBP2 and PBP3 conferred a 256-fold higher amoxicillin resistance when compared with ATCC 700392.
Conclusions: Multiple mutations in PBP2 and PBP3, in addition to mutations in PBP1, confer higher amoxicillin resistance in H. pylori.
Key Words: mechanisms of resistance , β-lactams , nucleotide sequencing , resistance genes , resistance genetics
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Helicobacter pylori is a pathogenic bacterium that causes chronic gastritis and peptic ulcers. Amoxicillin is one of the principal antimicrobial agents used in H. pylori infections. Amoxicillin-resistant H. pylori have been increasing recently and has become a major cause of eradication failure in certain areas.1
We previously analysed clinical amoxicillin-resistant H. pylori isolates in Japan and showed that multiple substitutions in the transpeptidase region of penicillin-binding protein 1 (PBP1) are necessary for the expression of amoxicillin resistance in H. pylori.2 Gerrits et al.3 also recently reported that multiple mutational changes in PBP1 are the predominant cause of amoxicillin resistance in H. pylori. The PBPs are peptidoglycan biosynthetic enzymes that have a transpeptidase in the C-terminal region. As β-lactams bind to the penicillin-binding motifs (SXXK, SXN and KTG) in the transpeptidase region, alterations in or around the motif possibly confer resistance due to reduced affinity to β-lactams. PBP1(HP0597), PBP2(HP1556) and PBP3(HP1565), which are encoded by the pbp1, ftsI and pbp2 genes, respectively, are found to be high molecular PBPs in H. pylori;4,5 however, there are few reports showing the relationship of mutations in PBP2 and PBP3 when compared with amoxicillin resistance. In the present study, we evaluated naturally transformed mutations in the pbp1, ftsI and pbp2 genes from a clinical amoxicillin-resistant strain to determine whether those mutations cause amoxicillin resistance.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Bacterial strains
Thirteen clinical H. pylori strains were isolated between 1995 and 2002 at the Tokyo Medical University Hospital in Japan. H. pylori ATCC 700392, ATCC 700824 and ATCC 43504 were also used in this study.
MICs of amoxicillin, benzylpenicillin, ceftriaxone and ceftazidime were determined using the agar dilution method according to the CLSI's instructions.6 Strains were considered to be resistant, low susceptible and susceptible to amoxicillin when the MIC was 
0.5, 0.063–0.25 and 
0.031 mg/L, respectively.
The genome DNA of H. pylori was isolated, as described previously.2 To amplify the pbp1, ftsI and pbp2 genes, PCR was performed with Ex Taq polymerase (Takara, Kyoto, Japan) using the primers pbp1F (5'-TGCGAACACCCTTTTAAAT-3') and pbp1R (5'-GCGACAATAAGAGTGGCA-3'), ftsIF (5'-TTATTGAGTCCTTTCTCTGAGC-3') and ftsIR (5'-TTGCTCTAATTTTACATTATTTTGA-3'), and pbp2F (5'-GAAACACTTGCTCACTAACCC-3') and pbp2R (5'-CAGAGTGAAAAGACCAGGAAAT-3'), respectively. Each fragment was purified and sequenced using the method described previously.2
The amoxicillin-susceptible strain, ATCC 700392, was transformed using the method described by Ge and Taylor.7 PCR products were obtained using amplification of the pbp1, ftsI and pbp2 genes from the amoxicillin-resistant strain, TH743, under the same conditions mentioned earlier. Transformants were selected on Brain Heart Infusion (Oxoid, Basingstoke, UK) agar plates containing 5% horse blood and 0.25, 1 or 2 mg/L benzylpenicillin, or 1 mg/L ceftriaxone. No spontaneous mutants were found on the plates used for the selection.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Susceptibilities of clinical H. pylori isolates to β-lactams
Among the 13 clinical isolates, TH743 and TS1289 were resistant to amoxicillin, whereas TS281 and TS1112 had low susceptibilities to amoxicillin. The remaining nine clinical isolates, ATCC 43504, ATCC 700392 and ATCC 700824 were susceptible to amoxicillin (Table 1). The amoxicillin-resistant strain TH743 also revealed high MICs of benzylpenicillin, ceftazidime and ceftriaxone (16, 64 and 32 mg/L, respectively).
|
Mutations of PBPs of amoxicillin-resistant H. pylori strains
The mutations of PBP1, PBP2 and PBP3 that are detected only in amoxicillin-resistant strains are shown in Table 1. In addition to the previously reported six mutations (A369T, V374L, S414R, L423F, N562Y and T593A) in PBP1,2 other mutations were detected in TH743; four mutations (A296V, S494H, A541M and E572G) in PBP2 and two mutations (A499V and E536K) in PBP3. The mutations S414R and N562Y in PBP1 and S494H in PBP2 were common in the amoxicillin-resistant strains TH743 and TS1289. The mutations N562Y in PBP1 and S494H in PBP2 were not detected in strains TH1112 and TS281, categorized as low susceptible to amoxicillin, but mutation A369T in PBP1 was detected in both TS1112 and TS281.
Susceptibilities of amoxicillin-resistant transformants to β-lactams
To elucidate the relationship between the mutations in PBPs and amoxicillin resistance, we determined the susceptibilities of transformants obtained by the natural transformation of amoxicillin-susceptible strain ATCC 700392 with the pbp1, ftsI and pbp2 genes of amoxicillin-resistant strain TH743 (Table 2). The TF1a and TF1b obtained by the natural transformation with the pbp1 gene of TH743 revealed an 8- to 16-fold higher resistance to amoxicillin, benzylpenicillin and ceftriaxone than the recipient strain. The TF2 obtained by the natural transformation with the ftsI gene of TH743 revealed almost equal susceptibility to amoxicillin and benzylpenicillin, but had a 4- to 8-fold higher resistance to ceftriaxone and ceftazidime when compared with the recipient strain. No transformant was obtained by the natural transformation of ATCC 700392 with the pbp2 gene of TH743 by three repetitions of the experiments (<8.6 x 10–7 transformants/cfu/µg of DNA). TF3a and TF3b were obtained by the natural transformation of TF1a and TF1b, respectively, with the ftsI gene of TH743; TF4a and TF4b were obtained by the natural transformation of TF1a and TF1b, respectively, with the pbp2 gene of TH743. Compared with the susceptibilities of TF1a and TF1b, TF3a and TF3b were revealed to be almost equally resistant to amoxicillin and benzylpenicillin, and TF4a and TF4b were 4–8- and 2-fold more resistant to amoxicillin and benzylpenicillin, respectively. These transformants revealed almost equal susceptibility to ceftazidime and ceftriaxone when compared with the recipient strain. TF5 obtained by the transformation of TF3a with the pbp1, ftsI and pbp2 genes of TH743 demonstrated 16-, 8-, 2- and 2-fold higher resistance to amoxicillin, benzylpenicillin, ceftazidime and ceftriaxone, respectively, than the recipient strain TF3a. That is, TF5 was 256-fold more resistant to amoxicillin than the original recipient strain, ATCC 700392.
|
Mutations of PBPs in amoxicillin-resistant transformants
The mutations of PBP1, PBP2 and PBP3 detected in the transformants are shown in Table 2. Among the six mutations in PBP1 of TH743, four mutations were introduced in TF1a and TF1b, whereas the remaining two mutations (A369T and V374L) were not detected in TF1b. All four mutations in PBP2 and a mutation of PBP3 were introduced in all transformants. No unexpected mutation in the pbp1, ftsI and pbp2 genes was detected in transformants obtained in this study.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
The correlation between the mutations of PBP2 and PBP3 and amoxicillin resistance has not been elucidated, although it has been reported that the mutations of PBP1 confer amoxicillin resistance in H. pylori.2,3,8–10 Here, we show for the first time the mutations in PBP2 and PBP3 that confer heightened amoxicillin resistance in H. pylori.
In addition to the mutations in PBP1 previously reported,2 we identified the mutations in PBP2 and PBP3 that were detected only in amoxicillin-resistant or low-susceptibility strains. To demonstrate the contribution of the mutations in PBP1, PBP2 and PBP3 to amoxicillin resistance, the amoxicillin-susceptible strain ATCC 700392 was transformed with various combinations of the pbp1, ftsI and pbp2 genes from the amoxicillin-resistant strain TH743. Although transformants with mutations of PBP1 revealed low susceptibilities to amoxicillin, TF2-possessing PBP2 mutations was susceptible to amoxicillin. These data suggest that the mutation of PBP2 was not critical for the first step in a change in the susceptibility to amoxicillin in H. pylori. However, the level of resistance to ceftriaxone and ceftazidime was increased by the mutations of PBP2. DeLoney and Schiller5 reported that the primary function of PBP with molecular masses of 63 kDa (PBP63) in H. pylori is in septum formation and that ceftriaxone preferentially binds to PBP63. Using an estimate of the molecular mass and function, PBP63 corresponded to PBP2 encoded by the ftsI gene.4 These reports support our data, showing that the mutations in PBP2 confer resistance to ceftriaxone and ceftazidime in H. pylori.
DeLoney and Schiller5 also report amoxicillin bound to PBP60 (corresponding to PBP3 encoded by the pbp2 gene) very strongly compared with other PBPs. Although no transformants possessing PBP3 mutations were obtained in this study, we did obtain TF4a- and TF4b-possessing mutations in PBP1 and PBP3, which revealed higher resistance to amoxicillin than TF1a and TF1b. In addition, the levels of resistance to amoxicillin in TF4a and TF4b were higher than in TF3a and TF3b. These data indicate that mutations in PBP3 are more significant in increasing the level of amoxicillin resistance than those in PBP2. Furthermore, transformants possessing mutations in PBP1, PBP2 and PBP3 revealed a 256-fold higher resistance to amoxicillin than the recipient strain ATCC 700392. This suggests that multiple mutations in PBP1, PBP2 and PBP3 contribute to a greater increase in the level of amoxicillin resistance. As four mutations in PBP2 and one mutation in PBP3 were introduced in all transformants analysed in this study, these mutations possibly confer β-lactam resistance. Further investigation is necessary to determine the relationship between individual mutations and amoxicillin resistance.
The level of resistance to amoxicillin in those transformants that had mutations in PBP1, PBP2 and PBP3 was slightly lower than in the original clinical amoxicillin-resistant TH743 strain. It is possible that other factors, such as mutations in the porin proteins,10 function as an additional mechanism for high-level amoxicillin resistance in clinical H. pylori isolates.
The results of our study show that multiple mutations in PBP2 and PBP3, in addition to those in PBP1, confer amoxicillin resistance in H. pylori.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
This work was supported by the High-Tech Research Centre Project for Private Universities provided by the Ministry of Education, Culture, Sports, Science and Technology and by the Matching Fund Subsidy for Private Schools of Japan.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
We thank M. Tanuma, Y. Saigo, Y. Miyata and R. Tamura for their contributions to the study.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
1 . Dore MP, Piana A, Carta M, et al. Amoxycillin resistance is one reason for failure of amoxycillin–omeprazole treatment of Helicobacter pylori infection. Aliment Pharmacol Ther (1998) 12:635–9.[CrossRef][Web of Science][Medline]
2 . Rimbara E, Noguchi N, Kawai T, et al. Correlation between substitutions in penicillin-binding protein 1 and amoxicillin resistance in Helicobacter pylori. Microbiol Immunol (2007) 51:939–44.[Web of Science][Medline]
3 . Gerrits MM, Godoy AP, Kuipers EJ, et al. Multiple mutations in or adjacent to the conserved penicillin-binding protein motifs of the penicillin-binding protein 1A confer amoxicillin resistance to Helicobacter pylori. Helicobacter (2006) 11:181–7.[CrossRef][Web of Science][Medline]
4
.
Goffin C, Ghuysen JM. Biochemistry and comparative genomics of SxxK superfamily acyltransferases offer a clue to the mycobacterial paradox: presence of penicillin-susceptible target proteins versus lack of efficiency of penicillin as therapeutic agent. Microbiol Mol Biol Rev (2002) 66:702–38.
5
.
DeLoney CR, Schiller NL. Competition of various β-lactam antibiotics for the major penicillin-binding proteins of Helicobacter pylori : antibacterial activity and effects on bacterial morphology. Antimicrob Agents Chemother (1999) 43:2702–9.
6 . Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing—Seventeenth Informational Supplement: Approved Standard M100-S17 (2007) Villanova, PA, USA: CLSI.
7 . Ge Z, Taylor DE. H. pylori DNA transformation by natural competence and electroporation. In: Helicobacter pylori Protocols—Clayton CL, Mobley HLT, eds. (1997) New Jersey: Human Press Inc. 145–52.
8
.
Okamoto T, Yoshiyama H, Nakazawa T, et al. A change in PBP1 is involved in amoxicillin resistance of clinical isolates of Helicobacter pylori. J Antimicrob Chemother (2002) 50:849–56.
9
.
Kwon DH, Dore MP, Kim JJ, et al. High-level β-lactam resistance associated with acquired multidrug resistance in Helicobacter pylori. Antimicrob Agents Chemother (2003) 47:2169–78.
10
.
Co EM, Schiller NL. Resistance mechanisms in an in vitro-selected amoxicillin-resistant strain of Helicobacter pylori. Antimicrob Agents Chemother (2006) 50:4174–6.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  E. Rimbara, N. Noguchi, T. Kawai, and M. Sasatsu Novel Mutation in 23S rRNA That Confers Low-Level Resistance to Clarithromycin in Helicobacter pylori Antimicrob. Agents Chemother., September 1, 2008; 52(9): 3465 - 3466. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||