Pulsed blue light and phage therapy: A novel synergistic bactericide

Pulsed blue light and phage therapy: A novel synergistic bactericide | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Pulsed blue light and phage therapy: A novel synergistic bactericide View ORCID Profile Amit Rimon , View ORCID Profile Jonathan Belin , View ORCID Profile Ortal Yerushalmy , Sivan Alkalay-Oren , Yonatan Eavri , Anatoly Shapochnikov , View ORCID Profile Shunit Coppenhagen-Glazer , View ORCID Profile Ronen Hazan , View ORCID Profile Lilach Gavish doi: https://doi.org/10.1101/2024.07.29.605651 Amit Rimon 1 Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel 2 Tzameret, The Military Track of Medicine, The Hebrew University-Hadassah Medical School; Jerusalem , Israel 3 The Israeli Phage Center (IPTC) of the Hebrew University and Hadassah Medical Center; Jerusalem , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Amit Rimon Jonathan Belin 1 Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jonathan Belin Ortal Yerushalmy 1 Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel 3 The Israeli Phage Center (IPTC) of the Hebrew University and Hadassah Medical Center; Jerusalem , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ortal Yerushalmy Sivan Alkalay-Oren 1 Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel 3 The Israeli Phage Center (IPTC) of the Hebrew University and Hadassah Medical Center; Jerusalem , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yonatan Eavri 4 Institute for Research in Military Medicine (IRMM), Faculty of Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anatoly Shapochnikov 5 The Department of Medical Neurobiology, Institute for Medical Research (IMRIC), Faculty of Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site Shunit Coppenhagen-Glazer 1 Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel 3 The Israeli Phage Center (IPTC) of the Hebrew University and Hadassah Medical Center; Jerusalem , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shunit Coppenhagen-Glazer Ronen Hazan 1 Institute of Biomedical and Oral Research (IBOR), Faculty of Dental Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel 3 The Israeli Phage Center (IPTC) of the Hebrew University and Hadassah Medical Center; Jerusalem , Israel Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ronen Hazan Lilach Gavish 4 Institute for Research in Military Medicine (IRMM), Faculty of Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel 5 The Department of Medical Neurobiology, Institute for Medical Research (IMRIC), Faculty of Medicine, The Hebrew University of Jerusalem; Jerusalem , Israel 6 The Saul and Joyce Brandman Hub for Cardiovascular Research Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lilach Gavish For correspondence: lilachg{at}ekmd.huji.ac.il Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Antibiotic-resistant Pseudomonas aeruginosa (PA) is a critical health threat. Novel treatment approaches are urgently required in this post-antibiotic era. In the current study, we investigated the bactericidal combinatorial potential of two non-antibiotic alternative approaches: phage therapy and pulsed blue light (PBL). Bacteriophages (phages), are viruses that specifically infect and lyse bacteria without harming eukaryotic cells. Pulsed blue light (PBL) alters bacterial membranes and was clinically shown to be innocuous to the skin in low doses. Here, using a low dose 457nm, 33KHz PBL combined with specific PA targeting phages, we demonstrated a synergistic effect that achieved complete inhibition of planktonic bacteria and a 40% reduction in formed biofilms. As part of this study, we also developed a user-friendly python-based tool for extraction of growth curve outcomes. In vivo studies are warranted for further validation of this combinatorial treatment. This approach may lead to a novel, antibiotic complementary modality to help patients suffering from difficult-to-treat antibiotic-resistant infections. One Sentence Summary Low-dose pulsed blue light and phage therapy have a synergistic bactericidal effect on Pseudomonas aeruginosa planktonic cultures and formed biofilm 1 INTRODUCTION 1.1 Antibiotic-Resistant Bacteria and Biofilms The rise in prevalence and propagation of antibiotic-resistant bacteria is a critical health concern, with a direct association of 1.27 million deaths in 2019 alone 1 . The genetic plasticity of bacterial populations, facilitated by selective pressures from antibiotic over-usage in clinical, agricultural, and environmental applications, results in rapid bacterial adaptation and resistance mechanisms 2 – 4 . This phenomenon drastically curtails the efficacy of conventional antibiotic therapies particularly in biofilms, structured bacterial communities encased in a self-produced extracellular matrix. These microbial aggregates foster increased resistance to antimicrobial agents and host immune responses 5 , 6 and pose substantial challenges in clinical and industrial settings 7 – 9 . Pseudomonas aeruginosa (PA), a gram-negative rod, is one of the 6 leading pathogens for death associated with antibiotic resistance 1 , and appears in a biofilm form, particularly in burns and other wounds 10 . Thus, novel treatments, approaches, or combinations of approaches are needed at this time against it. In this study, we focus on two such approaches and demonstrate their synergism against PA. 1.2 Phage Therapy A rising reinvigorated approach against resistant bacteria is the use of bacteriophages (phages 11 , viruses that kill bacteria without harming eukaryotic cells and which are the most prominent biological entity with estimated 10 31 particles worldwide 12 . Unlike antibiotics, phages target specific bacterial strains, while largely sparing other microbiota 12 . Among the advantages of using phages as therapeutic agents is their ability to penetrate biofilm structures 13 and most importantly, bacterial phage resistance can be handled through novel phage isolation, phage engineering, or naturally by evolutionary changes 14 . Phage therapy has proven effective against several antibiotic-resistant pathogens including Pseudomonas aeruginosa 15 – 17 , Staphylococcus aureus 18 , and Cutibacterium acnes 19 – 21 , particularly in skin-related infections. Moreover, phage therapy has been successfully used in compassionate cases and case series 15 , 22 – 25 including by our team in the Israeli Phage Therapy Center (IPTC), of non-healing infections caused by antibiotic-resistant bacteria 26 , 27 . 1.3 Pulsed Blue Light (PBL) Another promising approach to combat resistant bacteria is antimicrobial blue light, that was shown to be effective against both gram- positive and gram-negative bacteria notably against antibiotic resistant PA strains 28 – 30 . The blue light photons were shown to be absorbed by bacterial chromophores such as porphyrins resulting in excessive reactive oxygen species (ROS) that alter the bacterial membranes 31 . Blue light in low doses was determined clinically to be innocuous to the skin 32 . Pulsed blue light (PBL) was shown to have greater bactericidal efficacy compared to continuous blue light in much lower power intensity and energy doses in planktonic cultures 33 and was shown to partially disrupt and disassemble biofilm structures 34 . 1.4 Study Rationale and Objective In view of the increase in antibiotic-resistant bacteria and fewer available new antibiotics 35 coupled with the fact that PBL and phages eradicate bacteria through different mechanisms, we hypothesize that a combination of these interventions may culminate in a synergistic bactericidal effect. The objective of the current study was to determine the efficacy of this novel approach on planktonic and biofilm PA cultures. 2 RESULTS 2.1 Selected phages for the study This study used two phages specific for PA, lytic (PASA16) and lysogenic (PAShipCat1). The phage PASA16 was previously isolated and characterized in our lab 36 and used in various clinical compassionate treatments 15 . As described previously 36 , PASA16 has a 66,127 bp long genome (GenBank accession number MT933737.1 ) with a GC content of 55.6% and contains 90 coding DNA sequences (CDS). PAShipCat1 is a novel lysogenic phage isolated and characterized in this work. This phage has a 50602 bp long genome (GenBank accession number PP067092 ), with a GC content of 59.3% and 82 coding DNA sequences (CDS). According to its genome sequence, PAShipCat1 belongs to the Caudoviricetes class ( Figure 3 ). Since the genome of PAShipCat1 is found as a prophage in many PA bacteria and encodes a C-like Repressor, it probably has lysogenic abilities 37 . These two phages were selected deliberately due to their moderate effects, which allowed us to observe synergistic effects with the PBL. The phage PAShipCat1 was used in the planktonic-culture experiment ( Figure 4A ). In the biofilm experiment, we used PASA16, which had a milder efficacy against biofilm ( Figure 4B ), although it is highly active against planktonic cultures ( Fig 4A ). Download figure Open in new tab Fig. 1. Experimental Design and Outcomes (A) PAShipCat1 phage was added 10, 90, or 150 minutes after 30 minutes of pulsed blue light (PBL) treatment to planktonic P. aeruginosa (PA14). The bacteria were transferred to a plate reader for 24 hours to produce growth curves. (B) Measures of bactericidal effect were extracted from the growth curves including ‘Lag time’ (time to threshold) and ‘Area under the curve at 20 hours (AUC 20). (C) Biofilms of PA14 were formed and PASA16 phage was added either 24 hours or 10 minutes prior to PBL or 10 minutes after. PBL included multiple 30-minute irradiation (blue line) with 30 minute intervals (dotted line). Crystal violet or live-dead stain were used to evaluate the biofilm biomass and viability. This figure was created with Biorender.com Download figure Open in new tab Fig. 2. Pulsed Blue Light Source Photograph of the pulsed blue light source used in this study designed to homogenously irradiate a 96-well plate at a distance of 5 cm within a humid incubator. A total of 153 royal blue LEDs (457nm) were attached to an aluminum surface and electrically interconnected. Power was supplied via an LED driver with a 555 pulse generator module. Waveform and frequency were measured with an oscilloscope connected to a light detector. Pulses were generated at a frequency of 33kHz with a 50% duty cycle and their waveforms were triangular and rectangular at 4.7 and 7.2 mW/cm² respectively. Download figure Open in new tab Fig. 3: PAShipCat1 Sequence and Structure The anti- Pseudomonas aeruginosa phage, PAShipCat1. (A) TEM imaging reveals a typical structure of Caudoviricetes class-phage with an icosahedral head and a tail, in agreement with the genomic data (B) which also placed it within the Caudoviricetes family taxonomically. Due to its possession of a c-like repressor and phage homologs detected in other bacterial genomes, it has most probably lysogenic characteristics. Download figure Open in new tab Fig. 4: Comparative Phage Bactericidal Efficacy and Stability under PBL (A) Bactericidal efficacy variation among phages: PASA16 demonstrated potent elimination of PA in planktonic culture, while PAShipCat1 exhibited a moderate effect. (B) In a biofilm setting, PASA16 outperformed PAShipCat1, showcasing higher efficiency. (C-D) Effect of Pulsed Blue Light (PBL) on Phage Stability: Viable counts of planktonic P. aeruginosa infected with PAShipCat1 or PASA16 phages pre-exposed to 7.2 mW/cm² pulsed blue light for up to 3 hours (78 J/cm²). Bar graphs represent mean ± SD, n=5 biological repeats per dose (average of triplicates). Note that the phages significantly reduced the bacterial count, while PBL did not influence viral count or the bactericidal effect of the phage. *p<0.001 by ANOVA Tukey as post hoc. 2.2 PBL has no direct effect on phage number or activity Initially, we assessed the effect of PBL on the phages by irradiating them followed by quantification of phage titer. The titer of PAShipCat1 or PASA16 phages measured in PFU/ml was not reduced in up to 3 hours of PBL irradiation (p>0.5 by 1-way ANOVA for both phages). The bactericidal activity of these irradiated phages was not impaired as determined by CFU (p<0.001 for all PBL doses) ( Figure 4C and D and supplemental Table 1). 2.3 Timing of Administration and Dose/Concentration Response After determining that 457nm 33kHz PBL irradiation does not directly affect phage activity, we proceeded to evaluate PBL/Phage combination treatments. The timing of phage addition to planktonic P. aeruginosa (10 6 CFU) in relation to PBL was tested by adding PAShipCat1 phage (10 6 PFU) at 10, 90, or 150 minutes post the 30’ PBL treatment (8.5 J/cm 2 ) ( Figure 1A ). While PBL and phage alone, significantly reduced the area under the curve (AUC) at 20h post treatment compared to the controls by 28% and 51% respectively, their combination reduced it by 73% and the best timing was determined at 10-minutes post PBL ( Figure 5A and supplemental Table 2). Download figure Open in new tab Fig. 5: PBL and Phage Synergism is Dependent on the Interval Duration between Interventions, phage concentration, and PBL dose (A) The bactericidal effect in response to 8.5 J/cm 2 PBL followed by addition of 10 6 PFU Phage 10, 90, or 150 minutes later. Bar=mean ± SD area under growth curve (AUC) at 20 hours, n=4 (each biological repeat was an average of triplicate technical repeats). Addition of phage at 10 minutes was the most effective. *, #, † p<0.001 from control, PBL, phage respectively by 2-way ANOVA with Tukey as post-hoc test. (B) The bactericidal effect in response to different combinations of PBL dose × phage concentration: Bacteria were irradiated for 30 minutes with 0, 4.7, 6, or 7.2 mW/cm 2 PBL (0, 8.5, 10.8, or 13 J/cm 2 ) and 10 minutes later, phages were added at 0, 10 2 , 10 4 , 10 6 , or 10 8 PFU/ml. Bar=mean ± SD lag time [hours] over control. Bactericidal effects were directly associated with dose and concentration. p 20% over expected). (C-D) Growth curves by OD 600 : PA14 or antibiotic resistant PATZ2 were treated with PAShipCat1 or PASA16 at 10 PFU/ml respectively with or without 30 minutes PBL 7.2 mW/cm 2 both showing synergistic bactericidal effect. n =5 (each an average of triplicate technical repeats). Using the above protocol, we tested the effect of different phage concentrations combined with different PBL power intensities on bacterial growth curve lag phase. Phage alone significantly extended the lag phase as did PBL alone (p<0.001 for all concentrations and power intensities). Combining PBL and phages resulted in an additive effect for 10 2 /10 4 PFU/ml and up to 144% synergistic effect for greater concentrations ( Figure 5B ) with no bacterial growth observed at the highest dose/concentration combination ( Figure 5C ). This combination reduced the CFU by 9 logs in PA14 and achieved significant results also when applied to the 2 μg/ml Ceftazidime-resistant PATZ2 ( Figure 5D and supplemental Table 3). 2.4 Biofilm Viability and Biomass Next, we assessed the effect of the PBL-phage combination in a more challenging setting, formed biofilm. Initially, we tested the timing of phage addition in relation to the PBL doses ( Figure 1C ). Using crystal violet stain, we found that both PBL and phage reduced biofilm biomass with the most efficient protocol being phage added immediately before PBL regardless of the dose (p<0.001 across doses, presence of phage, and timing of phage addition by 3-way ANOVA) ( Figure 6A ). Based on these results we chose to add the phage immediately before a 26J/cm 2 PBL treatment for the next stage. Using live/dead stain we found that all treatments significantly decreased the biofilm viability (p<0.001 vs controls). While phage alone decreased biofilm viability by 50% of control, addition of PBL further reduced the biofilm viability to 19% (p=0.012 PBL&phage vs phage alone) ( Figure 6B and supplemental Table 4). Download figure Open in new tab Fig. 6: Biofilm Viability and Biomass is Effectively Reduced Following PBL and Phage Treatment. (A) Graph showing the reduction in biofilm biomass in response to PBL dose immediately before or 1 day after addition of phage by crystal violet staining. Biofilms were irradiated with 0-52 J/cm 2 PBL (see figure 2C for detailed experimental design). Data points = mean+SD %reduction in crystal violet stain levels (normalized to control). Lines added for visualization. The most effective combination was a PBL dose of 26 J/cm 2 immediately after phage administration. n=5 biological repeats per dose (each an average of 3 technical repeats). (B) Representative live-dead fluorescently stained biofilm samples from each experimental group visualized at ×4 magnification with %Live of total determined by the method of Robertson et al 67 . Maximal intensity projection of the Z-stack constructed by confocal microscopy was extracted from 17 sections in 12.5μm intervals. Note that the samples treated by PBL exhibit significantly higher red staining demonstrating a reduction in biofilm viability. 3 DISCUSSION The results of this study show that both planktonic and biofilm cultures of PA are highly susceptible to phage therapy combined with pulsed blue light. The synergistic effect was found to result in 99% reduction of planktonic cultures and 40% of preformed biofilms with the best bactericidal effects obtained at a dose of 26J/cm 2 with 10 8 PFU/ml phage added within 10 minutes of PBL administration. This bactericidal combination of PA-targeted phage therapy and non-risk low-dose pulsed blue light, can be used against superficial wound infections such as burns, acne, diabetic foot ulcers, cellulitis and others that pose an escalating challenge due to antibiotic resistance 1 , 38 . Moreover, this combined approach can provide a novel avenue for further enhancing biofilm management 7 , 39 . 3.1 Phages and antibiotics Currently, phage therapy is usually combined with antibiotic treatment 40 . Lin et al. reported that the eradication level is dependent on the PA strain and the antibiotic used. For example, phage PEV20 achieved complete inhibition in planktonic cultures when combined with ciprofloxacin but not with amikacin and colistin 41 . However, Knezevic et al., reported that combination with ciprofloxacin leads to only a 3.3 log reduction in PA 42 . Similarly, in this study we demonstrated complete inhibition of planktonic PA14 but partial inhibition of PATZ2. Interestingly, antibiotics alone were ineffective in reducing Pseudomonas biofilm biomass 5 , 43 . However, Chaudhry et al. demonstrated that combining 10 µg/ml ciprofloxacin with phages resulted in a 47-74% biomass reduction in a clinical cystic fibrosis P. aeruginosa strain originated biofilm, higher than the 40% synergistic effect found in the current study 43 , 44 . Although additional PBL dose did not increase the level of biomass eradication ( Figure 6A ), increasing the power intensity may result in increased eradication efficacy ( Figure 5B ) which will be the focus of future studies. 3.2 Blue Light While blue light may be harmful to the eyes, skin exposure was clinically determined to be innocuous 32 . In a clinical study, healthy volunteers were exposed for 5 consecutive days to a daily dose of 100J/cm 2 of 420 nm blue light. No indication of DNA damage, inflammation, or apoptosis were detected in their skin biopsies, although transient keratinocyte melanogenesis and vacuolization was observed 45 . However, in vitro, blue light at wavelength of 453nm, exerted cytotoxic effects on human keratinocytes in doses higher than 500 J/cm 2 46 – 20 times higher than the doses used in this study. In another cell culture study, blue light of 470nm was reported to slightly decrease cell count and viability in fibroblasts using a power of 100mW/cm 2 culminating to a dose of 110J/cm 2 47 – 12 times the power and 4 times the dose used here. These studies indicate that the wavelength and energetic parameters used in the current study are not expected to be harmful to the skin. The bactericidal effect of continuous blue light was extensively studied with special attention to the tradeoff between power and dose to avoid a photothermal effect: Rupel et al used 455nm blue light, irradiance of 300mW/cm 2 and dose of 60 or 120 J/cm 2 to irradiated planktonic cultures of PA and achieved inhibition for 24h 48 . In the current study we achieved the same by using a similar wavelength, 7.2mW/cm 2 , and dose of 13J/cm 2 . Pulsed light allows using higher peak power densities compared to those that could be used in continuous waves, without exposing the skin to excessive tissue heating 49 . Indeed, PBL in very low average irradiances and doses was determined to be superior to continuous wave. Using multiple 30-minute sessions of 450nm PBL at doses of 7.6 J/cm 2 or less, Bumah et al reported reduction of 7 log CFU/ml of planktonic C. acne and MRSA and determined that PBL was superior to continuous wave in similar doses. Additionally, they reported that PBL suppressed C.acnes and MRSA biofilms by 64% and 53% respectively 34 , 50 . In the current study, addition of phage to PBL in similar wavelength and energetic parameters achieved 40% suppression of pre-formed PA biofilms. 3.3 Direct effect of PBL on phages We found that pulsed blue light did not affect viral count or activity in neither PASA16 or PAShipCat1, even when irradiated for extended durations and high dose (up to 3 hours, 7.2mW/cm 2 , and 39 J/cm 2 ). However, previous reports indicated that several single stranded RNA viruses can be inactivated. Enwemeka et al reported that 405-450 nm PBL with 12 mW/cm 2 (130 J/cm 2 ) 51 can effectively inactivate strains of coronavirus. Tomb et al and Richardson et al reported that continuous blue light can inactivate feline calicivirus 52 and murine leukemia virus 53 respectively. It was found that light did not directly affect the viral envelope or viral genome but rather impaired the reverse transcription complex 53 . Both PASA16 or PAShipCat1 have a double stranded DNA genome, thereby they do not include the RT complex required for multiplication in RNA viruses. This lack of RT complex and the much lower power level and total dose applied, may explain why these phages were not susceptible to pulsed blue light used in the current study. 3.4 Practical considerations We found that the most effective timing is PBL immediately after phage administration in biofilm cultures ( Figure 5 - 6 ) and that the synergistic effect was still prominent even when phage was administered 24h before PBL such as in the case of intravenous, or oral phage delivery 54 , 55 . Our study employed clinically applicable pulsed blue light power density and pulse frequency 34 , 47 , 56 , making our findings relevant to potential clinical applications. Notably, one of the two phages utilized, PASA16, has been previously involved in various clinical compassionate treatments 15 . 3.5 Mechanism The proposed synergism between phage therapy and pulsed blue light could involve at least four possible mechanisms. Firstly, similar to UV, PBL might induce lysogen activation, triggering dormant phages within bacterial cells 57 , thereby enhancing overall phage activity. Secondly, a significant change in the Multiplicity of Infection (MOI), the amount of phages for each bacterium, could illustrate the association between phage activity and bacterial numbers 58 , with lower initial counts leading to more efficient phage activity. A third potential scenario involves occupying all of the cell’s DNA repair mechanisms, which could impede bacterial recovery and proliferation, limiting the spread of the infection. Finally, membrane instability induced by pulsed blue light 31 , 56 might facilitate the activity of phage lytic enzymes, promoting the rapid destruction of bacterial cells. Further studies are warranted to explore which mechanism(s) is responsible to this synergistic effect. 3.6 Limitations Our study’s limitations stem from the exclusive focus on the PA strains PA14 and PATZ2 with only two phages, limiting the generalizability of our findings. Prior research highlighted variable phage effectiveness in reducing Pseudomonas cultures or biofilm biomass, emphasizing the specificity of strains and phages 59 , 60 . Another limitation is the 30-minute treatment duration in our study, potentially being lengthy. Subsequent experiments should explore shorter durations and higher power settings. Additionally, we limited ourselves to the use of 457nm wavelength although shorter wavelengths were shown to be more effective, to preclude safety concerns which arise with UV wavelengths 61 . Nevertheless, these results are promising enough to pursue combinations of PBL and phages as a novel therapeutic to superficial infections. In conclusion, we present a bactericidal novel synergistic combination of phage therapy and low-dose pulsed blue light demonstrating effectiveness in both planktonic and pre-grown biofilm of Pseudomonas aeruginosa . Future research should include diverse bacterial strains, additional phages, and a comprehensive exploration of underlying mechanisms. Finally, clinical trials should assess the clinical implications of the treatment suggested herein for patients inflicted with antibiotic resistant superficial infectious conditions. 4 MATERIALS AND METHODS 4.1 Study Design In this study we used a combinatorial dose-response design (phage concentration × PBL power density) to achieve a bactericidal effect over P . aeruginosa planktonic cultures in the logarithmic phase and pre-formed biofilms ( Figure 1A-C ). 4.2 Bacteria 4.2.1 Bacterial strains The bacteria used here was the reference Pseudomonas aeruginosa strain PA14 62 , obtained from our laboratory stock collection and PATZ2 that was isolated from a clinical phage therapy case in our institution 15 and was determined to be resistant to 2μg/ml Ceftazidime (Panpharma Z.I, Luitre, France). 4.2.2 Planktonic bacteria Bacteria were grown, from a -80°C stock, overnight in Luria Bertani (LB) medium at 37°C under conditions of continuous agitation, diluted 1:1000, and re-grown in LB. Bacteria grown to mid-logarithmic (OD 600 ∼= 0.5) phase were used for the experiments. 4.2.3 Biofilm Bacteria were grown overnight, and 200 μl of 10 8 CFU/ml per well were transferred into the inner wells of a 96-well plate, and grown for 48h. Outer wells were filled with double distilled water to prevent inhomogeneous evaporation 63 . 4.3 Phage isolation and characterization 4.3.1 Phage isolation Isolation of phages was performed using the standard double-layered agar method 64 . Briefly, an environmental sample was centrifuged (centrifuge 5430R, rotor FA-45-24-11HS; Eppendorf, Hamburg, Germany) at 10,000 g for 10 min, and the supernatant was filtered through 0.22-μm-pore-size filters (Merck Millipore, Rahway, NJ). Exponentially grown PA14 (10 8 CFU/ml) cells were inoculated with a filtered sample, incubated for 24 h at 37°C and then filtered. The lysate was diluted in LB broth and spotted on a PA14 bacterial lawn grown on soft 0.6% agarose and incubated overnight at 37°C. Observed plaques were transferred into broth using a sterile loop and stored in LB at 4°C. 4.3.2 Phage sequencing The phage genome sequencing was performed as previously described 19 . Briefly, the phage DNA isolation kit (Norgen Biotek, Thorold, Canada) and the Illumina Nextera XT DNA kit (Illumina, San Diego, CA) were used to extract DNA and prepare sequence libraries respectively. A flow cell (1 × 150 bp single-end reads) was used for normalization, pooling, and tagging and the Illumina NextSeq 500 platform was used for sequencing. Trimming, quality control, read assembly, and analyses were performed using Geneious Prime® 2023.2.1 and its plugins ( https://www.geneious.com ). The SPAdes plugin of Geneious Prime and the Pharokka ( https://github.com/gbouras13/pharokka ) version 1.6.1 were used to perform assembly and annotation respectively 65 . 4.3.3 Phage visualization Phage structure was visualized by Transmission Electron Microscopy (TEM) as described before 19 . Briefly, 1 ml of 10 8 PFU was centrifuged at 20,000 g (centrifuge 5430 R, rotor FA- 45-24-11HS; Eppendorf, Hamburg, Germany) for 2h at room temperature. The pellet was resuspended in 200 μl of 5 mM MgSO 4 , spotted on a 2% uranyl acetate carbon- coated copper grid and incubated for 1 min. Phage visualization was carried out with a TEM 1400 plus (Joel, Tokyo, Japan) and images were captured with a charge-coupled device camera (Gatan Orius 600). 4.4 PBL The light source was designed in-house to allow a uniform irradiation over a 96-well plate inside a humid incubator ( Figure 2 ). The irradiating surface was composed of 460nm blue LED strips (Shenzen Dengsum Optoelectronics Co. Ltd, Shenzen, China) with High Lumens 2835 SMD chips (Epistar corp., Hsinchu, Taiwan) in a density of 120 LEDs/meter. The LEDs wavelength was further measured to be 457nm (FWHM=15) by an authorized laboratory (QCC Hazorea Calibration Technologies, Kibbutz Hazorea, Israel). The device was powered with an LED driver (LCM-40, Mean Well Enterprises CO Ltd, New Taipei City, Taiwan). Pulses, with frequency of 33KHz in 50% duty cycle were achieved with an electronic circuit based on a 555 pulse generator module. Waveform and frequency were measured and visualized with an oscilloscope (Tektronix TBS1064, Beaverton, OR) connected to a light detector. The aluminum plate on which the LED strips were mounted served also to dissipate excess heat. Light intensity was measured in mW/cm 2 at the distance of the 96-well plate with a LaserMate power-meter (Coherent, Auburn group, Coherent-Europe, Utrecht, Holand). The PBL frequency and initial power density were based on published experiments by Enwemeka et al in collaboration with Carewear© 31 , 33 , 34 . 4.5 Quantification Methods 4.5.1 CFU Samples were serially diluted 10-fold on LB agar plates and grown overnight in 37°C. The number of colonies was counted, and the concentration of CFU was calculated. 4.5.2 PFU The lysate was diluted 10-fold in LB broth and spotted over a PA14 bacterial lawn grown on soft 0.6% agar, and incubated overnight at 37°C. Observed plaques were counted, and the concentration of PFU was calculated. 4.5.3 Growth curves Lytic activity was assessed by inoculating logarithmic PA14 (10 7 CFU/ml) with the experimental samples and measuring turbidity with a plate reader (Synergy; BioTek, Winooski, VT) at OD 600nm at 37°C with 5-second shaking every 20 min for 24 hours. The data was smoothed by a 4-point rolling average. The threshold for growth curve detection was based on the measurements of the control group. The ‘lag time’, defined as the time in hours in which the OD increased over the detection threshold, and the area under the curve at 20 hours (‘AUC 20h’) that represents the growth potential ( Figure 1B ) were automatically extracted using the python code ‘growth_curve_outcomes‘ that we have developed for this study and can be easily applied to plate reader growth curve data. The code can be freely accessed in github ( https://github.com/bioimagehuji/growth_curve_outcomes/ ). 4.5.4 Biofilm biomass detection Biofilm biomass was measured using crystal violet stain 66 (Glentham life sciences, Corsham, UK). The liquid containing unattached bacteria was removed by pipetting, wells were rinsed with 100 μl 0.9% NaCl, and dried. A solution of 0.1% crystal violet was added to the wells (125 μl per well), incubated for 15 minutes at room temperature. Dye was removed and the wells were rinsed twice in a distilled water bath. The plate was dried at 37 °C for 1 hour and color was extracted with 30% acetic acid (100 μl per well). The purple colored solution was transferred into a new plate and OD 550nm was determined in a plate reader (Synergy) 66 . 4.5.5 Live-dead stain Biofilm viability was measured using the Live/Dead cell viability kit (Invitrogen, Waltham, MA) according to the manufacturer’s instructions. Propidium iodide stained the dead cells in red while SYTO9 stained the live cells in green (measured at 630 and 520 nm respectively). The fluorescence emissions of the samples were detected by a Zeiss LSM 410 confocal laser microscope (Carl Zeiss, Oberkochen, Germany) at X4 and X20 magnification. Maximal intensity of the Z-stack of each color was established from multiple horizontal optical sections (17 sections at 12.5-μm intervals for X4 and 9 sections at 10 μm intervals for X20). Three separate fields per sample were measured using ImageJ and the percentage of live bacteria of the total were calculated according to Robertson et al 67 . 4.5.6 Statistics Data is presented as mean±SD or median[interquartile rage] as appropriate. The results were the mean of five independent experiments, each an average of triplicates (unless otherwise stated). The direct effect of PBL over phages was tested by 1-way ANOVA. The role of the time of phage addition or phage concentration in combination with PBL was tested with 2- way ANOVA. The role of order of interventions, dose, and contribution of phage to the bactericidal effect over biofilm biomass was tested by 3-way ANOVA. Tukey’s Honestly- Significant-Difference Test was used for post-hoc pairwise comparisons. The role of the interventions over %Live (ratio of the number of live bacteria out of the total live+dead) was tested by Kruskal-Wallis with Conover-Inman as post-hoc. p-value < 0.05 was considered significant. Statistical analyses were performed with SYSTAT, version 13 (Systat Software, Chicago, USA). List of Supplementary Materials Supplemental Table 1: PBL effect on phage stability Supplemental Table 2: The effect of timing of phage addition Supplemental Table 3: The effect of combinatorial treatment on antibiotic resistance bacteria PATZ2 Supplemental Table 4: The effect of PBL and phage on biofilm viability Funding The Milgrom Family Fund Grant#3015005877(LG, AR, RH) The United States–Israel Binational Science Foundation Grant #2017123 (RH) The Israel Science Foundation IPMP Grant #ISF1349/20 (RH) The Rosetrees Trust Grant A2232 (RH). The Stuart Roden Family (Landsdowne Partners) Research Fund, London, UK (LG). The Alexander Grass Family Research Fund (LG). The Bruce and Baila Waldholtz Research Fund (LG). The Foulks foundation scholarship for MD-PhD students (AR). Author contributions All authors made significant contributions to the preparation of this manuscript. AR, RH, and LG conceptualized and drafted the manuscript, overseeing the funding acquisition process. SCG have edited the manuscript and offered valuable conceptual advice. JB, OY, SAO, and AS were instrumental in executing various methodologies, collaborating closely with AR and LG. Additionally, AR, YE, and LG were responsible in visualizing the data. Supervision throughout the project was provided by RH and LG Competing interests The authors declare that they have no conflict of interest. Data and materials availability Phage genomes were deposited in the https://www.ncbi.nlm.nih.gov/genbank/ , accession numbers are MT933737.1 (PASA16) and PP067092 (PAShipCat1). The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. 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Share Pulsed blue light and phage therapy: A novel synergistic bactericide Amit Rimon , Jonathan Belin , Ortal Yerushalmy , Sivan Alkalay-Oren , Yonatan Eavri , Anatoly Shapochnikov , Shunit Coppenhagen-Glazer , Ronen Hazan , Lilach Gavish bioRxiv 2024.07.29.605651; doi: https://doi.org/10.1101/2024.07.29.605651 Share This Article: Copy Citation Tools Pulsed blue light and phage therapy: A novel synergistic bactericide Amit Rimon , Jonathan Belin , Ortal Yerushalmy , Sivan Alkalay-Oren , Yonatan Eavri , Anatoly Shapochnikov , Shunit Coppenhagen-Glazer , Ronen Hazan , Lilach Gavish bioRxiv 2024.07.29.605651; doi: https://doi.org/10.1101/2024.07.29.605651 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Microbiology Subject Areas All Articles Animal Behavior and Cognition (7644) Biochemistry (17725) Bioengineering (13915) Bioinformatics (42031) Biophysics (21486) Cancer Biology (18635) Cell Biology (25548) Clinical Trials (138) Developmental Biology (13397) Ecology (19937) Epidemiology (2067) Evolutionary Biology (24361) Genetics (15619) Genomics (22538) Immunology (17763) Microbiology (40468) Molecular Biology (17206) Neuroscience (88733) Paleontology (667) Pathology (2842) Pharmacology and Toxicology (4834) Physiology (7657) Plant Biology (15172) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9831) Zoology (2272)