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Abstract
Discussion Forum (0)
Abstract number: 3110

Session Type: 1-hour Mini Oral Flash

Session Title: 1-hour Mini Oral Flash

Authors(s): F. Kunisch (1, 2), S. Yildirim (3), C. Schaudinn (4), J. Wagemans (5), R. Lavigne (5), A. Trampuz (2), M. Gonzalez Moreno (2)

Authors Affiliations(s): (1) Westfälische Wilhelms-Universität Münster, Germany, (2) Charité - Universitätsmedizin Berlin, Germany, (3) Technische Universität Berlin, Germany, (4) Robert Koch Institute, Germany, (5) KU Leuven - University of Leuven, Belgium

Background:

Emergence and worldwide spread of multidrug-resistant (MDR) Pseudomonas aeruginosa strains represent a public health threat. Moreover, its ability to colonize surfaces forming biofilms makes antibiotic treatment challenging. Phage therapy appears as an alternative strategy to treat biofilm-forming infections caused by MDR bacteria. Here, the antibiofilm properties of novel bacteriophages active against MDR P. aeruginosa were improved through in-vitro directed evolution by a serial passage assay.

Methods:

Bacteriophages isolated from hospital sewage were genome sequenced and their host range among 101 international MDR isolates screened by spot assay on soft agar overlays. Four bacteriophages were selected for directed evolution (n=30 rounds) (Fig. 1). Within each 24h round, dilutions of the bacteriophage mixture were exposed to pre-established biofilms of eight P. aeruginosa strains. The heat (J) produced by each sample was monitored by isothermal microcalorimetry. All bacteriophages from samples showing >75% heat reduction compared to the control samples (without bacteriophages) were pooled and included into the next round. Evolved bacteriophages were isolated from round 15 and 30 to be further characterized (genetic- and phenotypically). Antimicrobial activity of parental and evolved bacteriophages against pre-established biofilms was analyzed by isothermal microcalorimetry.

Results:

48% of the tested strains were susceptible to at least one of the four parental bacteriophages selected for in-vitro evolution. Throughout the evolution, within the first 8h of incubation, a steady reduction in the heat produced by bacteriophage-containing samples compared to control samples was evidenced by the dilution factors: lower titers of the bacteriophage mixture correlated with heat reductions comparable to higher titers in earlier rounds (Fig. 2). Bacterial regrowth after about 8h of co-incubation with bacteriophages was observed in all strains except Paer09, possibly due to the emergence of bacteria resistant to bacteriophages. Evolved bacteriophages isolated at round 15 and 30 revealed a substantial improvement in activity against pre-stablished biofilms compared to the parental bacteriophage (Fig. 3).

Conclusions:

Directed evolution of novel bacteriophages against biofilms of clinical MDR P. aeruginosa strains improved their antimicrobial activity compared to parental non-evolved bacteriophages. Combination therapies of evolved bacteriophages with antibiotics and in-vivo studies should be further investigated.

Keyword(s): MDR Pseudomonas aeruginosa, Bacteriophage evolution, Antibiofilm therapy

Abstract number: 3110

Session Type: 1-hour Mini Oral Flash

Session Title: 1-hour Mini Oral Flash

Authors(s): F. Kunisch (1, 2), S. Yildirim (3), C. Schaudinn (4), J. Wagemans (5), R. Lavigne (5), A. Trampuz (2), M. Gonzalez Moreno (2)

Authors Affiliations(s): (1) Westfälische Wilhelms-Universität Münster, Germany, (2) Charité - Universitätsmedizin Berlin, Germany, (3) Technische Universität Berlin, Germany, (4) Robert Koch Institute, Germany, (5) KU Leuven - University of Leuven, Belgium

Background:

Emergence and worldwide spread of multidrug-resistant (MDR) Pseudomonas aeruginosa strains represent a public health threat. Moreover, its ability to colonize surfaces forming biofilms makes antibiotic treatment challenging. Phage therapy appears as an alternative strategy to treat biofilm-forming infections caused by MDR bacteria. Here, the antibiofilm properties of novel bacteriophages active against MDR P. aeruginosa were improved through in-vitro directed evolution by a serial passage assay.

Methods:

Bacteriophages isolated from hospital sewage were genome sequenced and their host range among 101 international MDR isolates screened by spot assay on soft agar overlays. Four bacteriophages were selected for directed evolution (n=30 rounds) (Fig. 1). Within each 24h round, dilutions of the bacteriophage mixture were exposed to pre-established biofilms of eight P. aeruginosa strains. The heat (J) produced by each sample was monitored by isothermal microcalorimetry. All bacteriophages from samples showing >75% heat reduction compared to the control samples (without bacteriophages) were pooled and included into the next round. Evolved bacteriophages were isolated from round 15 and 30 to be further characterized (genetic- and phenotypically). Antimicrobial activity of parental and evolved bacteriophages against pre-established biofilms was analyzed by isothermal microcalorimetry.

Results:

48% of the tested strains were susceptible to at least one of the four parental bacteriophages selected for in-vitro evolution. Throughout the evolution, within the first 8h of incubation, a steady reduction in the heat produced by bacteriophage-containing samples compared to control samples was evidenced by the dilution factors: lower titers of the bacteriophage mixture correlated with heat reductions comparable to higher titers in earlier rounds (Fig. 2). Bacterial regrowth after about 8h of co-incubation with bacteriophages was observed in all strains except Paer09, possibly due to the emergence of bacteria resistant to bacteriophages. Evolved bacteriophages isolated at round 15 and 30 revealed a substantial improvement in activity against pre-stablished biofilms compared to the parental bacteriophage (Fig. 3).

Conclusions:

Directed evolution of novel bacteriophages against biofilms of clinical MDR P. aeruginosa strains improved their antimicrobial activity compared to parental non-evolved bacteriophages. Combination therapies of evolved bacteriophages with antibiotics and in-vivo studies should be further investigated.

Keyword(s): MDR Pseudomonas aeruginosa, Bacteriophage evolution, Antibiofilm therapy

Directed evolution of bacteriophages to improve their antibiofilm efficacy against clinical multidrug-resistant Pseudomonas aeruginosa strains
Fabian Kunisch
Fabian Kunisch
ESCMID eAcademy. Kunisch F. 07/09/2021; 332747; 3110
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Fabian Kunisch
Abstract
Discussion Forum (0)
Abstract number: 3110

Session Type: 1-hour Mini Oral Flash

Session Title: 1-hour Mini Oral Flash

Authors(s): F. Kunisch (1, 2), S. Yildirim (3), C. Schaudinn (4), J. Wagemans (5), R. Lavigne (5), A. Trampuz (2), M. Gonzalez Moreno (2)

Authors Affiliations(s): (1) Westfälische Wilhelms-Universität Münster, Germany, (2) Charité - Universitätsmedizin Berlin, Germany, (3) Technische Universität Berlin, Germany, (4) Robert Koch Institute, Germany, (5) KU Leuven - University of Leuven, Belgium

Background:

Emergence and worldwide spread of multidrug-resistant (MDR) Pseudomonas aeruginosa strains represent a public health threat. Moreover, its ability to colonize surfaces forming biofilms makes antibiotic treatment challenging. Phage therapy appears as an alternative strategy to treat biofilm-forming infections caused by MDR bacteria. Here, the antibiofilm properties of novel bacteriophages active against MDR P. aeruginosa were improved through in-vitro directed evolution by a serial passage assay.

Methods:

Bacteriophages isolated from hospital sewage were genome sequenced and their host range among 101 international MDR isolates screened by spot assay on soft agar overlays. Four bacteriophages were selected for directed evolution (n=30 rounds) (Fig. 1). Within each 24h round, dilutions of the bacteriophage mixture were exposed to pre-established biofilms of eight P. aeruginosa strains. The heat (J) produced by each sample was monitored by isothermal microcalorimetry. All bacteriophages from samples showing >75% heat reduction compared to the control samples (without bacteriophages) were pooled and included into the next round. Evolved bacteriophages were isolated from round 15 and 30 to be further characterized (genetic- and phenotypically). Antimicrobial activity of parental and evolved bacteriophages against pre-established biofilms was analyzed by isothermal microcalorimetry.

Results:

48% of the tested strains were susceptible to at least one of the four parental bacteriophages selected for in-vitro evolution. Throughout the evolution, within the first 8h of incubation, a steady reduction in the heat produced by bacteriophage-containing samples compared to control samples was evidenced by the dilution factors: lower titers of the bacteriophage mixture correlated with heat reductions comparable to higher titers in earlier rounds (Fig. 2). Bacterial regrowth after about 8h of co-incubation with bacteriophages was observed in all strains except Paer09, possibly due to the emergence of bacteria resistant to bacteriophages. Evolved bacteriophages isolated at round 15 and 30 revealed a substantial improvement in activity against pre-stablished biofilms compared to the parental bacteriophage (Fig. 3).

Conclusions:

Directed evolution of novel bacteriophages against biofilms of clinical MDR P. aeruginosa strains improved their antimicrobial activity compared to parental non-evolved bacteriophages. Combination therapies of evolved bacteriophages with antibiotics and in-vivo studies should be further investigated.

Keyword(s): MDR Pseudomonas aeruginosa, Bacteriophage evolution, Antibiofilm therapy

Abstract number: 3110

Session Type: 1-hour Mini Oral Flash

Session Title: 1-hour Mini Oral Flash

Authors(s): F. Kunisch (1, 2), S. Yildirim (3), C. Schaudinn (4), J. Wagemans (5), R. Lavigne (5), A. Trampuz (2), M. Gonzalez Moreno (2)

Authors Affiliations(s): (1) Westfälische Wilhelms-Universität Münster, Germany, (2) Charité - Universitätsmedizin Berlin, Germany, (3) Technische Universität Berlin, Germany, (4) Robert Koch Institute, Germany, (5) KU Leuven - University of Leuven, Belgium

Background:

Emergence and worldwide spread of multidrug-resistant (MDR) Pseudomonas aeruginosa strains represent a public health threat. Moreover, its ability to colonize surfaces forming biofilms makes antibiotic treatment challenging. Phage therapy appears as an alternative strategy to treat biofilm-forming infections caused by MDR bacteria. Here, the antibiofilm properties of novel bacteriophages active against MDR P. aeruginosa were improved through in-vitro directed evolution by a serial passage assay.

Methods:

Bacteriophages isolated from hospital sewage were genome sequenced and their host range among 101 international MDR isolates screened by spot assay on soft agar overlays. Four bacteriophages were selected for directed evolution (n=30 rounds) (Fig. 1). Within each 24h round, dilutions of the bacteriophage mixture were exposed to pre-established biofilms of eight P. aeruginosa strains. The heat (J) produced by each sample was monitored by isothermal microcalorimetry. All bacteriophages from samples showing >75% heat reduction compared to the control samples (without bacteriophages) were pooled and included into the next round. Evolved bacteriophages were isolated from round 15 and 30 to be further characterized (genetic- and phenotypically). Antimicrobial activity of parental and evolved bacteriophages against pre-established biofilms was analyzed by isothermal microcalorimetry.

Results:

48% of the tested strains were susceptible to at least one of the four parental bacteriophages selected for in-vitro evolution. Throughout the evolution, within the first 8h of incubation, a steady reduction in the heat produced by bacteriophage-containing samples compared to control samples was evidenced by the dilution factors: lower titers of the bacteriophage mixture correlated with heat reductions comparable to higher titers in earlier rounds (Fig. 2). Bacterial regrowth after about 8h of co-incubation with bacteriophages was observed in all strains except Paer09, possibly due to the emergence of bacteria resistant to bacteriophages. Evolved bacteriophages isolated at round 15 and 30 revealed a substantial improvement in activity against pre-stablished biofilms compared to the parental bacteriophage (Fig. 3).

Conclusions:

Directed evolution of novel bacteriophages against biofilms of clinical MDR P. aeruginosa strains improved their antimicrobial activity compared to parental non-evolved bacteriophages. Combination therapies of evolved bacteriophages with antibiotics and in-vivo studies should be further investigated.

Keyword(s): MDR Pseudomonas aeruginosa, Bacteriophage evolution, Antibiofilm therapy

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