Phage

mariechavignon·January 22, 2025

Description

Data from the article published in Front. Cell. Infect. Microbiol. 12:1060825. doi: 10.3389/fcimb.2022.1060825

Summary


The data available here are part of the article named Bacteriophage-based decontamination to control environmental colonization by Staphylococcus capitis in neonatal intensive care units: An in vitro proof-of-concept (Front. Cell. Infect. Microbiol. 12:1060825. doi: 10.3389/fcimb.2022.1060825). Data are presented if xlsx files.

Abstarct of the article:

Introduction: In neonatal intensive care units (NICUs), the standard chemical-based disinfection procedures do not allow a complete eradication of pathogens from environmental surfaces. In particular, the clone Staphylococcus capitis NRCS-A, a significant pathogen in neonates, was shown to colonize neonatal incubators. The aim of this study was to evaluate the in vitro effect of a bacteriophage cocktail on NRCS-A eradication. Methods: Three bacteriophages were isolated, genetically characterized and assessed for their host range using a collection of representative clinical strains (n=31) belonging to the clone NRCS-A. The efficacy of a cocktail including these three bacteriophages to eradicate the reference strain S. capitis NRCS-A CR01 was determined in comparison or in combination with the chemical disinfectant Surfanios Premium on either dry inoculum or biofilm-embedded bacteria. The emergence of bacterial resistance against the bacteriophages alone or in cocktail was evaluated by growth kinetics. Results: The three bacteriophages belonged to two families and genera, namely Herelleviridae/Kayvirus for V1SC01 and V1SC04 and Rountreeviridae/Andhravirus for V1SC05. They were active against 17, 25 and 16 of the 31 tested strains respectively. Bacteriophage cocktails decreased the bacterial inoculum of both dry spots and biofilms, with a dose dependent effect. The sequential treatment with bacteriophages then Surfanios Premium did not show enhanced efficacy. No bacterial resistance was observed when using the bacteriophage cocktail. Discussion: This study established a proof-of-concept for the use of bacteriophages to fight against S. capitis NRCS-A. Further investigations are needed using a larger bacterial collection and in real-life conditions before being able to use such technology in NICUs.

>The document entitled "Efficacy_of-bacteriophages_and_surfanios-premium_on_NRCS-A" contains the results of the experiments described below.

Activity of a bacteriophage cocktail on dry spots and pre-formed biofilm of S. capitis NRCS-A

The effect of a bacteriophage cocktail was tested on the reference strain CR01 in comparison with the disinfectant Surfanios Premium (SP) (composed of N-(3-aminopropyl)-N-dodécylpropane-1,3-diamine and chlorure de didécyldiméthylammonium) (ANIOS, Lezennes, France). Both treatments were tested on dry spots and on pre-formed biofilms using a previously published method with some modifications (Stachler et al., 2021). For bacteriophage cocktails preparation, the three bacteriophages were associated by diluting each of them in equal measure in either TSB or Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA) to obtain cocktails at three different final concentrations (3.104 PFU/mL, 3.106 PFU/mL or 3.108 PFU/mL). Bacteria were exposed to the bacteriophage cocktail for 6 h in wet chamber at 37°C. The chemical treatment consisted in exposition to SP at 0.25 % in water at room temperature for 20 minutes, corresponding to the classical treatment used in NICUs for incubator disinfection (Butin et al., 2019). Untreated positive controls were treated with either TSB or PBS (controls for bacteriophage treatments) or water (control for SP treatment) alone.

Dry spots were obtained by inoculating 10 µL drops of a 1.107 CFU/mL bacterial PBS suspension in a 96 well microplate (Greiner Bio-One, Cap Horn, France). The drops were dried at room temperature during 4.5 h under microbiological safety workstation. The dry spots treatment consisted of covering the adhered bacteria with 20 µL of bacteriophages cocktail, SP or control medium. Then, 100 µL of Dey Engley Neutralizing broth (Merk, Darmstadt, Germany) was applied during 5 min to inactivate the SP action. This same neutralising treatment was applied to dry spots exposed to bacteriophages to avoid experimental variations. Finally, the neutralising broth was discarded and the surviving bacteria were suspended in 100 µL of PBS by scraping and enumerated.

Biofilm was obtained by inoculating 100 µL of a 1.107CFU/mL bacterial suspension in TSB in a 96 well microplate that was incubated 24 h at 37°C. After incubation, the biofilm was first rinsed using the steam method as previously described (Tasse et al., 2018) before being exposed to 150 µL of bacteriophage cocktails or SP 0.25 % as described above. After incubation, the supernatant was carefully removed and 200 µL of Dey Engley Neutralizing broth was applied for 5 minutes. The biofilm was then rinsed with the steam method, suspended in 200 µL of PBS by scraping and enumerated.

In addition, a sequential treatment with a bacteriophage cocktail at the highest bacteriophages concentration (3.108PFU/mL) followed by a SP treatment was also evaluated on bacterial biofilm. For this purpose, after the step of bacteriophage treatment as described above, an additional steam rinse was performed and then SP was applied followed by inactivation as previously described.

Log10 reduction values (LRV) for each treatment were calculated as follows: Log10(N/N0) with N corresponding to the number of bacteria in the treated well and N0 corresponding to the number of bacteria in the untreated control well. The experiments were performed three times in triplicate for the simple treatments and three times in sextuplicate for the sequential treatment. Bacteriophage cocktails titres were checked each time.

>The document entitled "Growth kinetic data" contains the results of the experiments described below.

Evaluation of bacterial resistance emergence during bacteriophage treatment

Growth kinetics were performed to determine if bacterial resistance occurred following the treatment with one bacteriophage alone compared to cocktails of the three bacteriophages. Briefly, CR01 was incubated in TSB until the exponential growth phase and then diluted in TSB to reach a concentration of 1.107 CFU/mL. A 96 well microplate (Thermo Fisher Scientific, Waltham, MA, USA) was then inoculated with 1.106CFU of bacteria and either 1.106 PFU, 1.107 PFU or 1.108PFU of each bacteriophage alone or in cocktail (to reach a MOI of respectively 1, 10 or 100) in a final TSB volume of 200 µL. Bacterial growth alone without bacteriophage addition and absence of bacterial contamination in bacteriophage suspensions and in TSB were also controlled in the microplate. The outlines of the microplate were filled with water to prevent evaporation then the microplate was incubated 24 h in a microplate reader Tecan infinite® 200 PRO (Tecan, Männedorf, Swiss) under agitation at 37°C with OD600 nm (optical density) measurement every 30 min. Emergence of bacterial resistance was considered when the OD600 nm exceeded the threshold of 0.01 (after TSB control media OD600 nm subtraction) that corresponded to the threshold of bacterial growth. The experiments were performed three times in technical triplicates and both bacteriophage titres and bacterial inoculum were verified each time.

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Phage

mariechavignon·January 22, 2025

Description

Data from the article published in Front. Cell. Infect. Microbiol. 12:1060825. doi: 10.3389/fcimb.2022.1060825

Summary


The data available here are part of the article named Bacteriophage-based decontamination to control environmental colonization by Staphylococcus capitis in neonatal intensive care units: An in vitro proof-of-concept (Front. Cell. Infect. Microbiol. 12:1060825. doi: 10.3389/fcimb.2022.1060825). Data are presented if xlsx files.

Abstarct of the article:

Introduction: In neonatal intensive care units (NICUs), the standard chemical-based disinfection procedures do not allow a complete eradication of pathogens from environmental surfaces. In particular, the clone Staphylococcus capitis NRCS-A, a significant pathogen in neonates, was shown to colonize neonatal incubators. The aim of this study was to evaluate the in vitro effect of a bacteriophage cocktail on NRCS-A eradication. Methods: Three bacteriophages were isolated, genetically characterized and assessed for their host range using a collection of representative clinical strains (n=31) belonging to the clone NRCS-A. The efficacy of a cocktail including these three bacteriophages to eradicate the reference strain S. capitis NRCS-A CR01 was determined in comparison or in combination with the chemical disinfectant Surfanios Premium on either dry inoculum or biofilm-embedded bacteria. The emergence of bacterial resistance against the bacteriophages alone or in cocktail was evaluated by growth kinetics. Results: The three bacteriophages belonged to two families and genera, namely Herelleviridae/Kayvirus for V1SC01 and V1SC04 and Rountreeviridae/Andhravirus for V1SC05. They were active against 17, 25 and 16 of the 31 tested strains respectively. Bacteriophage cocktails decreased the bacterial inoculum of both dry spots and biofilms, with a dose dependent effect. The sequential treatment with bacteriophages then Surfanios Premium did not show enhanced efficacy. No bacterial resistance was observed when using the bacteriophage cocktail. Discussion: This study established a proof-of-concept for the use of bacteriophages to fight against S. capitis NRCS-A. Further investigations are needed using a larger bacterial collection and in real-life conditions before being able to use such technology in NICUs.

>The document entitled "Efficacy_of-bacteriophages_and_surfanios-premium_on_NRCS-A" contains the results of the experiments described below.

Activity of a bacteriophage cocktail on dry spots and pre-formed biofilm of S. capitis NRCS-A

The effect of a bacteriophage cocktail was tested on the reference strain CR01 in comparison with the disinfectant Surfanios Premium (SP) (composed of N-(3-aminopropyl)-N-dodécylpropane-1,3-diamine and chlorure de didécyldiméthylammonium) (ANIOS, Lezennes, France). Both treatments were tested on dry spots and on pre-formed biofilms using a previously published method with some modifications (Stachler et al., 2021). For bacteriophage cocktails preparation, the three bacteriophages were associated by diluting each of them in equal measure in either TSB or Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, Waltham, MA, USA) to obtain cocktails at three different final concentrations (3.104 PFU/mL, 3.106 PFU/mL or 3.108 PFU/mL). Bacteria were exposed to the bacteriophage cocktail for 6 h in wet chamber at 37°C. The chemical treatment consisted in exposition to SP at 0.25 % in water at room temperature for 20 minutes, corresponding to the classical treatment used in NICUs for incubator disinfection (Butin et al., 2019). Untreated positive controls were treated with either TSB or PBS (controls for bacteriophage treatments) or water (control for SP treatment) alone.

Dry spots were obtained by inoculating 10 µL drops of a 1.107 CFU/mL bacterial PBS suspension in a 96 well microplate (Greiner Bio-One, Cap Horn, France). The drops were dried at room temperature during 4.5 h under microbiological safety workstation. The dry spots treatment consisted of covering the adhered bacteria with 20 µL of bacteriophages cocktail, SP or control medium. Then, 100 µL of Dey Engley Neutralizing broth (Merk, Darmstadt, Germany) was applied during 5 min to inactivate the SP action. This same neutralising treatment was applied to dry spots exposed to bacteriophages to avoid experimental variations. Finally, the neutralising broth was discarded and the surviving bacteria were suspended in 100 µL of PBS by scraping and enumerated.

Biofilm was obtained by inoculating 100 µL of a 1.107CFU/mL bacterial suspension in TSB in a 96 well microplate that was incubated 24 h at 37°C. After incubation, the biofilm was first rinsed using the steam method as previously described (Tasse et al., 2018) before being exposed to 150 µL of bacteriophage cocktails or SP 0.25 % as described above. After incubation, the supernatant was carefully removed and 200 µL of Dey Engley Neutralizing broth was applied for 5 minutes. The biofilm was then rinsed with the steam method, suspended in 200 µL of PBS by scraping and enumerated.

In addition, a sequential treatment with a bacteriophage cocktail at the highest bacteriophages concentration (3.108PFU/mL) followed by a SP treatment was also evaluated on bacterial biofilm. For this purpose, after the step of bacteriophage treatment as described above, an additional steam rinse was performed and then SP was applied followed by inactivation as previously described.

Log10 reduction values (LRV) for each treatment were calculated as follows: Log10(N/N0) with N corresponding to the number of bacteria in the treated well and N0 corresponding to the number of bacteria in the untreated control well. The experiments were performed three times in triplicate for the simple treatments and three times in sextuplicate for the sequential treatment. Bacteriophage cocktails titres were checked each time.

>The document entitled "Growth kinetic data" contains the results of the experiments described below.

Evaluation of bacterial resistance emergence during bacteriophage treatment

Growth kinetics were performed to determine if bacterial resistance occurred following the treatment with one bacteriophage alone compared to cocktails of the three bacteriophages. Briefly, CR01 was incubated in TSB until the exponential growth phase and then diluted in TSB to reach a concentration of 1.107 CFU/mL. A 96 well microplate (Thermo Fisher Scientific, Waltham, MA, USA) was then inoculated with 1.106CFU of bacteria and either 1.106 PFU, 1.107 PFU or 1.108PFU of each bacteriophage alone or in cocktail (to reach a MOI of respectively 1, 10 or 100) in a final TSB volume of 200 µL. Bacterial growth alone without bacteriophage addition and absence of bacterial contamination in bacteriophage suspensions and in TSB were also controlled in the microplate. The outlines of the microplate were filled with water to prevent evaporation then the microplate was incubated 24 h in a microplate reader Tecan infinite® 200 PRO (Tecan, Männedorf, Swiss) under agitation at 37°C with OD600 nm (optical density) measurement every 30 min. Emergence of bacterial resistance was considered when the OD600 nm exceeded the threshold of 0.01 (after TSB control media OD600 nm subtraction) that corresponded to the threshold of bacterial growth. The experiments were performed three times in technical triplicates and both bacteriophage titres and bacterial inoculum were verified each time.

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