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A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility

Thegerström, John LU ; Matuschek, Erika; Su, Yu Ching; Riesbeck, Kristian LU and Resman, Fredrik LU (2018) In BMC Microbiology 18(1).
Abstract

Background: Identification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to β-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93-57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined. Results: Site-directed mutagenesis of a β-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion... (More)

Background: Identification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to β-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93-57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined. Results: Site-directed mutagenesis of a β-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion screening and broth microdilution determination of MICs for β-lactam agents were done with the NTHi3655-PBP3Y528H mutant and were compared with the NTHi3655 wild-type as well as the original clinical isolate NTHi93-57485. Introduction of the Y528H substitution in NTHi3655 resulted in positive screening for β-lactam resistance. MICs for aminopenicillins were increased in the mutant compared to the wild-type. However, the mutant remained susceptible to aminopenicillins according to EUCAST clinical breakpoints (assuming intravenous treatment) and the introduction of Y528H alone did not increase the resistance to the same level as NTHi93-57485. None of the isolates had frame shift insertions in the acrR gene regulating the AcrAB efflux pump. Conclusions: In parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility.

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Contribution to journal
publication status
published
subject
keywords
Ampicillin, ftsI, Haemophilus influenzae, PBP3, penicillin binding proteins, site directed mutagenesis, β-lactam resistance
in
BMC Microbiology
volume
18
issue
1
publisher
BioMed Central
external identifiers
  • scopus:85047907193
ISSN
1471-2180
DOI
10.1186/s12866-018-1196-6
language
English
LU publication?
yes
id
34c7cd1d-9d9d-4e9b-86e8-d324d2718021
date added to LUP
2018-06-12 14:03:42
date last changed
2018-06-13 03:00:02
@article{34c7cd1d-9d9d-4e9b-86e8-d324d2718021,
  abstract     = {<p>Background: Identification and characterization of non-typeable Haemophilus influenzae (NTHi) with reduced susceptibility to β-lactam antibiotics due to mutations in penicillin binding protein 3 (PBP3) is a clinical challenge. We analyzed a blood isolate, NTHi93-57485, that was categorized as aminopenicillin resistant but lacked key amino acid substitutions in PBP3 that have previously been associated with reduced aminopenicillin susceptibility. The significance of an alternative amino acid substitution (Y528H) in this isolate was examined. Results: Site-directed mutagenesis of a β-lactam susceptible H. influenzae (NTHi3655) was performed to introduce the Y528H substitution into wild-type ftsI (encoding for PBP3). Disc diffusion screening and broth microdilution determination of MICs for β-lactam agents were done with the NTHi3655-PBP3<sup>Y528H</sup> mutant and were compared with the NTHi3655 wild-type as well as the original clinical isolate NTHi93-57485. Introduction of the Y528H substitution in NTHi3655 resulted in positive screening for β-lactam resistance. MICs for aminopenicillins were increased in the mutant compared to the wild-type. However, the mutant remained susceptible to aminopenicillins according to EUCAST clinical breakpoints (assuming intravenous treatment) and the introduction of Y528H alone did not increase the resistance to the same level as NTHi93-57485. None of the isolates had frame shift insertions in the acrR gene regulating the AcrAB efflux pump. Conclusions: In parallel to the previously well-described PBP3-substitutions R517H and N526K, we demonstrate that Y528H confers reduced aminopenicillin susceptibility.</p>},
  articleno    = {48},
  author       = {Thegerström, John and Matuschek, Erika and Su, Yu Ching and Riesbeck, Kristian and Resman, Fredrik},
  issn         = {1471-2180},
  keyword      = {Ampicillin,ftsI,Haemophilus influenzae,PBP3,penicillin binding proteins,site directed mutagenesis,β-lactam resistance},
  language     = {eng},
  month        = {05},
  number       = {1},
  publisher    = {BioMed Central},
  series       = {BMC Microbiology},
  title        = {A novel PBP3 substitution in Haemophilus influenzae confers reduced aminopenicillin susceptibility},
  url          = {http://dx.doi.org/10.1186/s12866-018-1196-6},
  volume       = {18},
  year         = {2018},
}