BACKGROUND: Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. One of the mechanisms of resistance to β-lactams is the alteration of the transpeptidase region of penicillin binding protein 3 (PBP3) which is caused by mutations in the ftsI gene. It was shown that exposure to beta-lactams has a stimulating effect on increase of prevalence of H. influenzae strains with the non-enzymatic mechanism of resistance. OBJECTIVES: The aim of our study was to compare the mutational potential of ampicillin and cefuroxime in H. influenzae strains, determination of minimum inhibitory concentration and the evolution of mutations over time, focusing on amino acid substitutions in PBP3. METHODS: 30 days of serial passaging of strains in liquid broth containing increasing concentrations of ampicillin or cefuroxime was followed by whole-genome sequencing. RESULTS: On average, cefuroxime increased the minimum inhibitory concentration more than ampicillin. The minimum inhibitory concentration was increased by a maximum of 32 fold. Substitutions in the PBP3 started to appear after 15 days of passaging. In PBP3, cefuroxime caused different substitutions than ampicillin. CONCLUSIONS: Our experiment observed differences in mutation selection by ampicillin and cefuroxime. Selection pressure of antibiotics in vitro generated substitutions that do not occur in clinical strains in the Czech Republic.

• MeSH
• ampicilin * farmakologie   MeSH
• antibakteriální látky * farmakologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• cefuroxim * farmakologie   MeSH
• Haemophilus influenzae * genetika   účinky léků   MeSH
• hemofilové infekce mikrobiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti * MeSH
• molekulární evoluce MeSH
• mutace * MeSH
• proteiny vázající penicilin * genetika   metabolismus   MeSH
• sekvenování celého genomu MeSH
• selekce (genetika) MeSH
• sériové pasážování MeSH
• substituce aminokyselin * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Alterations in PBP2a have been recognized in cefotaxime-resistant laboratory mutants and β-lactam-resistant clinical isolates of Streptococcus pneumoniae. DNA sequencing revealed fundamental differences between these two settings. Internal stop codons in pbp2a occurred in all three laboratory mutants analyzed, caused by a mutation in pbp2a of mutant C604, and tandem duplications within pbp2a resulting in premature stop codons in another two mutants C403 and C406. In contrast, mosaic PBP2a genes were observed in several penicillin-resistant clinical isolates from South Africa, the Czech Republic, Hungary, and in the clone Poland23F-16, with sequence blocks diverging from sensitive strains by over 4%. Most of these pbp2a variants except pbp2a from the South African strain contained sequences related to pbp2a of Streptococcus mitis B6, confirming that this species serves as reservoir for penicillin-resistance determinants.

• MeSH
• bakteriální geny genetika   MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamy farmakologie   MeSH
• DNA bakterií genetika   MeSH
• lidé MeSH
• mikrobiální testy citlivosti metody   MeSH
• mutace genetika   MeSH
• peniciliny farmakologie   MeSH
• peptidsynthasy genetika   MeSH
• proteiny vázající penicilin genetika   MeSH
• rezistence na penicilin genetika   MeSH
• Streptococcus pneumoniae účinky léků   genetika   MeSH
• transportní proteiny genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Jihoafrická republika MeSH
• Maďarsko MeSH

D-alanyl-D-alanine carboxypeptidase, product of dacD gene in Francisella, belongs to penicillin binding proteins (PBPs) and is involved in remodeling of newly synthetized peptidoglycan. In E. coli, PBPs are synthetized in various growth phases and they are able to substitute each other to a certain extent. The DacD protein was found to be accumulated in fraction enriched in membrane proteins from severely attenuated dsbA deletion mutant strain. It has been presumed that the DsbA is not a virulence factor by itself but that its substrates, whose correct folding and topology are dependent on the DsbA oxidoreductase and/or isomerase activities, are the primary virulence factors. Here we demonstrate that Francisella DacD is required for intracellular replication and virulence in mice. The dacD insertion mutant strain showed higher sensitivity to acidic pH, high temperature and high osmolarity when compared to the wild-type. Eventually, transmission electron microscopy revealed differences in mutant bacteria in both the size and defects in outer membrane underlying its SDS and serum sensitivity. Taken together these results suggest DacD plays an important role in Francisella pathogenicity.

• MeSH
• antibakteriální látky farmakologie   MeSH
• buněčná stěna metabolismus   MeSH
• DD-karboxypeptidasa genetika   metabolismus   MeSH
• Francisella tularensis účinky léků   růst a vývoj   patogenita   MeSH
• kultivované buňky MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidoglykan biosyntéza   MeSH
• proteindisulfidisomerasy genetika   MeSH
• proteiny vázající penicilin genetika   metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• tularemie mikrobiologie   patologie   MeSH
• virulence genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

GpsB regulatory protein and StkP protein kinase have been proposed as molecular switches that balance septal and peripheral (side-wall like) peptidoglycan (PG) synthesis in Streptococcus pneumoniae (pneumococcus); yet, mechanisms of this switching remain unknown. We report that ΔdivIVA mutations are not epistatic to ΔgpsB division-protein mutations in progenitor D39 and related genetic backgrounds; nor is GpsB required for StkP localization or FDAA labeling at septal division rings. However, we confirm that reduction of GpsB amount leads to decreased protein phosphorylation by StkP and report that the essentiality of ΔgpsB mutations is suppressed by inactivation of PhpP protein phosphatase, which concomitantly restores protein phosphorylation levels. ΔgpsB mutations are also suppressed by other classes of mutations, including one that eliminates protein phosphorylation and may alter division. Moreover, ΔgpsB mutations are synthetically lethal with Δpbp1a, but not Δpbp2a or Δpbp1b mutations, suggesting GpsB activation of PBP2a activity. Consistent with this result, co-IP experiments showed that GpsB complexes with EzrA, StkP, PBP2a, PBP2b and MreC in pneumococcal cells. Furthermore, depletion of GpsB prevents PBP2x migration to septal centers. These results support a model in which GpsB negatively regulates peripheral PG synthesis by PBP2b and positively regulates septal ring closure through its interactions with StkP-PBP2x.

• MeSH
• aminoacyltransferasy genetika   metabolismus   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčná stěna metabolismus   MeSH
• buněčné dělení genetika   fyziologie   MeSH
• faktory virulence genetika   metabolismus   MeSH
• fosforylace MeSH
• membránové proteiny genetika   metabolismus   MeSH
• mutace genetika   MeSH
• peptidoglykan biosyntéza   MeSH
• proteiny vázající penicilin genetika   metabolismus   MeSH
• Streptococcus pneumoniae genetika   metabolismus   MeSH
• zastoupení bazí genetika   MeSH
• Publikační typ
• časopisecké články MeSH