Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates
Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Lorenzo M. Lazzaro [verfasserIn] Marta Cassisi [verfasserIn] Stefania Stefani [verfasserIn] Floriana Campanile [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2022 |
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| Schlagwörter: |
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| Übergeordnetes Werk: |
In: Frontiers in Cellular and Infection Microbiology - Frontiers Media S.A., 2016, 11(2022) |
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| Übergeordnetes Werk: |
volume:11 ; year:2022 |
| Links: |
|---|
| DOI / URN: |
10.3389/fcimb.2021.816657 |
|---|
| Katalog-ID: |
DOAJ074500759 |
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| 520 | |a Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427motif I; L475Q, 7 aa upstream of the 482SDN484motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes. | ||
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10.3389/fcimb.2021.816657 doi (DE-627)DOAJ074500759 (DE-599)DOAJ59364d367a284381a48426bf1dd0e990 DE-627 ger DE-627 rakwb eng QR1-502 Lorenzo M. Lazzaro verfasserin aut Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427motif I; L475Q, 7 aa upstream of the 482SDN484motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes. Enterococcus faecalis time-kill curve assays PBP4 ceftobiprole pbp4 gene expression Microbiology Marta Cassisi verfasserin aut Stefania Stefani verfasserin aut Floriana Campanile verfasserin aut In Frontiers in Cellular and Infection Microbiology Frontiers Media S.A., 2016 11(2022) (DE-627)664968554 (DE-600)2619676-1 22352988 nnns volume:11 year:2022 https://doi.org/10.3389/fcimb.2021.816657 kostenfrei https://doaj.org/article/59364d367a284381a48426bf1dd0e990 kostenfrei https://www.frontiersin.org/articles/10.3389/fcimb.2021.816657/full kostenfrei https://doaj.org/toc/2235-2988 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 |
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10.3389/fcimb.2021.816657 doi (DE-627)DOAJ074500759 (DE-599)DOAJ59364d367a284381a48426bf1dd0e990 DE-627 ger DE-627 rakwb eng QR1-502 Lorenzo M. Lazzaro verfasserin aut Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427motif I; L475Q, 7 aa upstream of the 482SDN484motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes. Enterococcus faecalis time-kill curve assays PBP4 ceftobiprole pbp4 gene expression Microbiology Marta Cassisi verfasserin aut Stefania Stefani verfasserin aut Floriana Campanile verfasserin aut In Frontiers in Cellular and Infection Microbiology Frontiers Media S.A., 2016 11(2022) (DE-627)664968554 (DE-600)2619676-1 22352988 nnns volume:11 year:2022 https://doi.org/10.3389/fcimb.2021.816657 kostenfrei https://doaj.org/article/59364d367a284381a48426bf1dd0e990 kostenfrei https://www.frontiersin.org/articles/10.3389/fcimb.2021.816657/full kostenfrei https://doaj.org/toc/2235-2988 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 |
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10.3389/fcimb.2021.816657 doi (DE-627)DOAJ074500759 (DE-599)DOAJ59364d367a284381a48426bf1dd0e990 DE-627 ger DE-627 rakwb eng QR1-502 Lorenzo M. Lazzaro verfasserin aut Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427motif I; L475Q, 7 aa upstream of the 482SDN484motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes. Enterococcus faecalis time-kill curve assays PBP4 ceftobiprole pbp4 gene expression Microbiology Marta Cassisi verfasserin aut Stefania Stefani verfasserin aut Floriana Campanile verfasserin aut In Frontiers in Cellular and Infection Microbiology Frontiers Media S.A., 2016 11(2022) (DE-627)664968554 (DE-600)2619676-1 22352988 nnns volume:11 year:2022 https://doi.org/10.3389/fcimb.2021.816657 kostenfrei https://doaj.org/article/59364d367a284381a48426bf1dd0e990 kostenfrei https://www.frontiersin.org/articles/10.3389/fcimb.2021.816657/full kostenfrei https://doaj.org/toc/2235-2988 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 |
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10.3389/fcimb.2021.816657 doi (DE-627)DOAJ074500759 (DE-599)DOAJ59364d367a284381a48426bf1dd0e990 DE-627 ger DE-627 rakwb eng QR1-502 Lorenzo M. Lazzaro verfasserin aut Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427motif I; L475Q, 7 aa upstream of the 482SDN484motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes. Enterococcus faecalis time-kill curve assays PBP4 ceftobiprole pbp4 gene expression Microbiology Marta Cassisi verfasserin aut Stefania Stefani verfasserin aut Floriana Campanile verfasserin aut In Frontiers in Cellular and Infection Microbiology Frontiers Media S.A., 2016 11(2022) (DE-627)664968554 (DE-600)2619676-1 22352988 nnns volume:11 year:2022 https://doi.org/10.3389/fcimb.2021.816657 kostenfrei https://doaj.org/article/59364d367a284381a48426bf1dd0e990 kostenfrei https://www.frontiersin.org/articles/10.3389/fcimb.2021.816657/full kostenfrei https://doaj.org/toc/2235-2988 Journal toc kostenfrei GBV_USEFLAG_A SYSFLAG_A GBV_DOAJ GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_73 GBV_ILN_74 GBV_ILN_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_206 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_602 GBV_ILN_2003 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 11 2022 |
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Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates |
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Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427motif I; L475Q, 7 aa upstream of the 482SDN484motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes. |
| abstractGer |
Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427motif I; L475Q, 7 aa upstream of the 482SDN484motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes. |
| abstract_unstemmed |
Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427motif I; L475Q, 7 aa upstream of the 482SDN484motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes. |
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| title_short |
Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates |
| url |
https://doi.org/10.3389/fcimb.2021.816657 https://doaj.org/article/59364d367a284381a48426bf1dd0e990 https://www.frontiersin.org/articles/10.3389/fcimb.2021.816657/full https://doaj.org/toc/2235-2988 |
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Marta Cassisi Stefania Stefani Floriana Campanile |
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