PBP2a
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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
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
Resistance of staphylococci to methicillin is important especially in the case of Staphylococcus aureus isolates. Its impact in veterinary medicine is not exactly specified in coagulase-negative staphylococci; however, these staphylococci may represent an important reservoir of resistance genes. The study aimed at detecting resistance to methicillin in coagulase-negative staphylococci from raw materials and foodstuffs of animal origin and assessing the tests frequently used to determine this resistance. Coagulase-negative staphylococci (198 isolates of 12 species) were tested. Resistance to methicillin was determined by the disk diffusion method using oxacillin and cefoxitin disks, microdilution method, detection of PBP2a and the mecA gene. Of the tested isolates, 109 (55.1%) were classified as resistant by the diffusion test with oxacillin, 32 isolates (16.2%) by the test with cefoxitin and 50 isolates (25.3%) on the basis of oxacillin minimum inhibitory concentration (MIC). No resistant isolates were incorrectly identified as susceptible when using the disk diffusion method with oxacillin (sensitivity of 100%). However, apart from 22 correctly classified resistant isolates, another 87 isolates were incorrectly identified as resistant as well (specificity of 50.6%). The test with cefoxitin showed the lowest (45.5%) sensitivity in determination of resistant isolates. By contrast, this test was the most precise in classification of resistant isolates (specificity of 87.5%). When using the microdilution method, resistant strains were identified with the sensitivity and specificity of 68.2% and 80.1%, respectively. The results revealed substantial variability of methicillin-resistant isolates ranging from 16.2% to 55.1%, depending on the phenotyping methods and recommended interpretation criteria used. Therefore, it is advisable to reconsider the current interpretation criteria in the case of coagulasenegative staphylococci of animal origin (with the exception of S. epidermidis).
- Klíčová slova
- gen mecA, koaguláza negativní stafylokoky,
- MeSH
- bakteriální léková rezistence * genetika MeSH
- cefoxitin MeSH
- mikrobiální testy citlivosti klasifikace metody MeSH
- mléčné výrobky mikrobiologie MeSH
- mléko mikrobiologie MeSH
- oxacilin MeSH
- proteiny vázající penicilin izolace a purifikace MeSH
- rezistence na methicilin * MeSH
- senzitivita a specificita MeSH
- Staphylococcus * genetika klasifikace účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by β-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan, and a noncovalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A prerequisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall cross-linking reaction.
- MeSH
- biokatalýza MeSH
- buněčná stěna metabolismus MeSH
- katalytická doména MeSH
- krystalografie rentgenová MeSH
- peptidoglykan metabolismus MeSH
- proteiny vázající penicilin metabolismus MeSH
- simulace molekulární dynamiky MeSH
- Streptococcus pneumoniae enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Seventy-two penicillin-resistant Streptococcus pneumoniae isolates collected from clinical specimens in the Czech and Slovakian Republics between 1990 and 1992 were analyzed by a variety of molecular techniques. Most of the highly resistant isolates (40/72) (penicillin MIC between 1 up to 16 micrograms/ml) were represented by two distinct pneumococcal clones, and most of these isolates (35/40) were also resistant to at least two other antibiotics (tetracycline plus chloramphenicol or erythromycin). All 17 isolates belonging to the first clone were of serotype 14, had very high penicillin MICs (8-12 micrograms/ml), shared a common, abnormal penicillin-binding protein (PBP) pattern and one of two related pulsed-field gel electrophoretic (PFGE) patterns. The 15 isolates belonging to the second clone were all of serotype 19A, had penicillin MICs between 1 and 4 micrograms/ml, shared a unique, abnormal PBP pattern, and could be divided into two subgroups on the basis of PFGE patterns, one of which was indistinguishable from the PFGE pattern of a multiresistant capsular type 19A clone of S. pneumoniae already identified earlier in Hungary. Thirty-two of the 72 pneumococcal isolates had lower penicillin MICs (0.1-0.5 microgram/ml), and these isolates differed from the more highly resistant ones in several respects: They belonged to seven different serotypes, showed large variation in PFGE patterns (20 patterns in 32 isolates) and most of them (21/32) were resistant to penicillin only. Tentative explanations for these findings, in terms of epidemiological and molecular mechanisms, are considered.
- MeSH
- bakteriální proteiny * MeSH
- hexosyltransferasy * MeSH
- karboxypeptidasatranspeptidasa genetika MeSH
- lidé MeSH
- peptidyltransferasy * MeSH
- pneumokokové infekce epidemiologie mikrobiologie MeSH
- proteiny vázající penicilin MeSH
- rezistence na penicilin * MeSH
- Streptococcus pneumoniae genetika izolace a purifikace účinky léků MeSH
- transportní proteiny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
- Geografické názvy
- Česká republika MeSH
- Slovenská republika MeSH
Seventy-two penicillin-resistant Streptococcus pneumoniae isolates collected from clinical specimens in the Czech and Slovakian Republics between 1990 and 1992 were analyzed by a variety of molecular techniques. Most of the highly resistant isolates (40/72) (penicillin MIC between 1 up to 16 micrograms/ml) were represented by two distinct pneumococcal clones, and most of these isolates (35/40) were also resistant to at least two other antibiotics (tetracycline plus chloramphenicol or erythromycin). All 17 isolates belonging to the first clone were of serotype 14, had very high penicillin MICs (8-12 micrograms/ml), shared a common, abnormal penicillin-binding protein (PBP) pattern and one of two related pulsed-field gel electrophoretic (PFGE) patterns. The 15 isolates belonging to the second clone were all of serotype 19A, had penicillin MICs between 1 and 4 micrograms/ml, shared a unique, abnormal PBP pattern, and could be divided into two subgroups on the basis of PFGE patterns, one of which was indistinguishable from the PFGE pattern of a multiresistant capsular type 19A clone of S. pneumoniae already identified earlier in Hungary. Thirty-two of the 72 pneumococcal isolates had lower penicillin MICs (0.1-0.5 microgram/ml), and these isolates differed from the more highly resistant ones in several respects: They belonged to seven different serotypes, showed large variation in PFGE patterns (20 patterns in 32 isolates) and most of them (21/32) were resistant to penicillin only. Tentative explanations for these findings, in terms of epidemiological and molecular mechanisms, are considered.
- MeSH
- bakteriální proteiny * MeSH
- hexosyltransferasy * MeSH
- karboxypeptidasatranspeptidasa genetika MeSH
- lidé MeSH
- peptidyltransferasy * MeSH
- pneumokokové infekce epidemiologie mikrobiologie MeSH
- proteiny vázající penicilin MeSH
- rezistence na penicilin * MeSH
- Streptococcus pneumoniae účinky léků genetika izolace a purifikace MeSH
- transportní proteiny genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
- Geografické názvy
- Česká republika MeSH
- Slovenská republika MeSH
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
BACKGROUND: Data from surveillance on antibiotic resistance have shown an increasing prevalence of non-enzymatic resistance (β-lactamase-negative ampicillin-resistant) to β-lactam antibiotics among H. influenzae strains in the Czech Republic. Aminopenicillins are recommended agents for non-invasive Haemophilus influenzae infections. The phenomenon of non-enzymatic resistance to β-lactams is complicated by the fact that the phenotypic detection of PBP3 with specific amino acid substitutions (rPBP3) is challenging, since rPBP3 isolates have repeatedly been demonstrated to be split by the epidemiological cut-off values (ECOFF) for aminopenicillins defined by EUCAST. OBJECTIVES: We sought to determine whether the penicillin disc has sufficient detection ability to predict the non-enzymatic mechanism; whether other antibiotics can be used for detection; and what is the agreement between the broth microdilution and disc diffusion methods. METHODS: We undertook susceptibility testing of selected antibiotics according to EUCAST of 153 rPBP3 strains, and sequencing of the ftsI gene to determination amino acid substitutions. RESULTS: For a selected set of rPBP strains: (i) the detection capability for penicillin, ampicillin, cefuroxime and amoxicillin/clavulanate was found to be 91.5%, 94.4%, 89.5% and 70.6%, respectively; (ii) the categorical agreement between the disc diffusion method and the MIC for ampicillin and cefuroxime was 71.1% and 83.8%, respectively. CONCLUSIONS: We observed better recognition of rPBP3 strains by the ampicillin disc than by the penicillin disc. There is frequently a discrepancy in the interpretation of susceptibility results between the methods used.
- MeSH
- antibakteriální látky * farmakologie MeSH
- bakteriální proteiny * genetika MeSH
- beta-laktamová rezistence * MeSH
- beta-laktamy * farmakologie MeSH
- fenotyp MeSH
- Haemophilus influenzae * účinky léků genetika izolace a purifikace enzymologie MeSH
- hemofilové infekce mikrobiologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti metody MeSH
- proteiny vázající penicilin * genetika MeSH
- sekvenční analýza DNA MeSH
- substituce aminokyselin * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
The aim of this study was to detect and characterize isolates of methicillin-/oxacillin-resistant Staphylococcus aureus (MRSA) carrying gene mecC (MRSA/mecC) and occurring in the Czech Republic within the period from 2002 to 2017. Altogether, 18 from 3,472 isolates of MRSA were mecC positive (0.52%). The first detection of MRSA/mecC in the Czech Republic is dated to 2004. MRSA/mecC isolates were susceptible to almost all tested antibiotics with few exceptions. Resistances to erythromycin (n = 2), clindamycin (n = 1), trimethoprim-sulfamethoxazole (n = 1), and rifampicin (n = 1) were found in the collection. Multilocus sequence typing and spa typing revealed a genetic heterogeneity of MRSA/mecC strains: three CCs (130, 425, and 2361), five STs (1245, 130, 2361, 425, and a new ST5480), and eight spa types (t843, t978, t1048, t1535, t1736, t6104, t8842, and t17153), which were detected in the study, with the highest prevalence of CC130/t843 lineage (n = 8, 44%). Except for two strains, none from 18 examined isolates harbored genes encoding any of S. aureus toxins: enterotoxins a-u, exfoliative toxins A, B, and D, toxic shock syndrome toxin-1, and the Panton-Valentine leukocidin.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální geny genetika MeSH
- genotyp MeSH
- methicilin rezistentní Staphylococcus aureus genetika MeSH
- mikrobiální testy citlivosti MeSH
- multilokusová sekvenční typizace MeSH
- oxacilin farmakologie MeSH
- polymerázová řetězová reakce MeSH
- proteiny vázající penicilin metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
