Photo-inactivation ofNeisseria gonorrhoeae: A paradigm changing approach for combating antibiotic-resistant gonococcal infection

Photo-inactivation of Neisseria gonorrhoeae: A paradigm changing approach for combating antibiotic-resistant gonococcal infection | 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 Photo-inactivation of Neisseria gonorrhoeae : A paradigm changing approach for combating antibiotic-resistant gonococcal infection Ying Wang , Raquel Ferrer-Espada , Yan Baglo , Xueping S. Goh , Kathryn D. Held , View ORCID Profile Yonatan H. Grad , Ying Gu , Jeffrey A. Gelfand , View ORCID Profile Tianhong Dai doi: https://doi.org/10.1101/422634 Ying Wang 1 Wellman Center for Photomedicine, Massachusetts General Hospital , Harvard Medical School, Boston, MA 02114, USA . 2 Department of Laser Medicine, Chinese PLA General Hospital , Beijing 100853, China . Find this author on Google Scholar Find this author on PubMed Search for this author on this site Raquel Ferrer-Espada 1 Wellman Center for Photomedicine, Massachusetts General Hospital , Harvard Medical School, Boston, MA 02114, USA . Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yan Baglo 1 Wellman Center for Photomedicine, Massachusetts General Hospital , Harvard Medical School, Boston, MA 02114, USA . Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xueping S. Goh 1 Wellman Center for Photomedicine, Massachusetts General Hospital , Harvard Medical School, Boston, MA 02114, USA . Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kathryn D. Held 3 Department of Radiation Oncology, Massachusetts General Hospital , Harvard Medical School, Boston, MA 02114, USA . Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yonatan H. Grad 4 Department of Immunology and Infectious Diseases , Harvard T.H. Chan School of Public Health, Boston, MA 02115, USA . Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yonatan H. Grad Ying Gu 2 Department of Laser Medicine, Chinese PLA General Hospital , Beijing 100853, China . Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeffrey A. Gelfand 5 Department of Medicine, Massachusetts General Hospital , Harvard Medical School, Boston, MA 02114, USA . Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tianhong Dai 1 Wellman Center for Photomedicine, Massachusetts General Hospital , Harvard Medical School, Boston, MA 02114, USA . Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tianhong Dai For correspondence: tdai{at}mgh.harvard.edu Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Neisseria gonorrhoeae is the causative pathogen of the sexually transmitted disease gonorrhea, a disease at risk of becoming untreatable due to increasing antibiotic resistance. There is a critical need for the development of new anti-gonococcal therapies. In this study, we investigated the effectiveness of antimicrobial blue light (aBL), an innovative non-antibiotic approach, for the inactivation of antibiotic-resistant N . gonorrhoeae . Our findings indicated that aBL at 405 nm preferentially inactivated antibiotic-resistant N. gonorrhoeae over the vaginal epithelial cells. Furthermore, no genotoxicity of aBL to the vaginal epithelial cells was observed at the exposure for inactivating N. gonorrhoeae . aBL also effectively inactivated N. gonorrhoeae that had invaded into the vaginal epithelial cells. No gonococcal resistance to aBL developed after 15 successive cycles of sub-therapeutic inactivation. Endogenous aBL-active photosensitizing chromophores (porphyrins and flavins) in N. gonorrhoeae were identified and quantified using ultra performance liquid chromatography, with coproporphyrin being the most abundant endogenous porphyrin species. Taken together, aBL at 405 nm represents a potent potential treatment for gonococcal infections. One Sentence Summary aBL selectively inactivated antibiotic-resistant Neisseria gonorrhoeaen over normal vaginal epithelial cells. INTRODUCTION Neisseria gonorrhoeae is a human-specific bacterial pathogen and the cause of gonococcal infection (gonorrhea) ( 1 , 2 ). If not appropriately treated, gonorrhea can result in severe sequelae such as pelvic inflammatory disease, infertility, ectopic pregnancy, first trimester abortion, neonatal conjunctivitis leading to blindness and, less frequently, male infertility and disseminated gonococcal infections ( 3 ). The CDC estimates that 820,000 new gonococcal infections occur each year in the United States ( 4 ), with the estimated total annual economic cost for the treatment of gonorrhea approaching $5 billion ( 5 ). Furthermore, gonorrhea facilitates the transmission and acquisition of HIV (6-8). In the absence of a gonococcal vaccine, public health control of gonorrhea relies primarily on the availability of effective antibiotic treatment ( 3 ). However, N . gonorrhoeae has become resistant to each of the first-line antibiotics that have been used to treat it ( 9 , 10 ). The antibiotic combination of azithromycin and ceftriaxone is the current CDC recommendation for treating gonorrhea ( 11 ). Worryingly, the susceptibility to ceftriaxone has been decreasing globally in recent years, resistance to azithromycin is prevalent and emerges rapidly in settings where this drug is frequently used, and N . gonorrhoeae strains with decreased susceptibility or resistance to ceftriaxone and concomitant azithromycin resistance are already circulating globally ( 12 ). For example, a cluster of cases was recently reported in Hawaii that was resistant to both azithromycin and ceftriaxone ( 13 , 14 ). The CDC flagged multidrug-resistant N . gonorrhoeae as an urgent threat to human health ( 15 ), the highest level of concern. As the use of antibiotics drives the emergence and spread of multidrug-resistant N . gonorrhoeae (3, 16-18), there is a critical need for the development of new treatment strategies ( 19 , 20 ). An innovative non-antibiotic therapy, antimicrobial blue light (aBL), has attracted increasing attention due to its intrinsic antimicrobial properties without the involvement of exogenous photosensitizers ( 21 , 22 ). Although the mechanism of action of aBL is still not fully understood, a common hypothesis is that aBL excites naturally occurring endogenous photosensitizers (e.g., iron-free porphyrins or/and flavins) within bacterial cells and subsequently leads to the production of cytotoxic oxidative species ( 23 , 24 ). The advantages of aBL over traditional antibiotics include rapid action and equal inactivation effectiveness independent of antibiotic resistance ( 21 , 22 ). It is also currently thought that bacteria are less able to develop resistance to aBL than to traditional antibiotics because of the multi-target characteristic of aBL (21, 22, 25). In comparison to traditional antimicrobial photodynamic therapy, aBL is appealing in that it inactivates bacteria (renders bacteria unable to multiply) without the involvement of exogenous photosensitizers (26-32). In addition, it is well accepted that aBL is much less detrimental to the host cells than germicidal ultraviolet irradiation ( 33 , 34 ). aBL has never been studied as a treatment for gonorrhea ( 35 ). In this study, we evaluated the effectiveness of aBL in rendering antibiotic-resistant N . gonorrhoeae unable to multiply (termed inactivation), the toxicity of aBL to the epithelial cells, and the potential development of gonococcal resistance to aBL. In addition, we determined and quantified the presence of aBL-active endogenous photosensitizing chromophores in N. gonorrhoeae . RESULTS Effectiveness of aBL inactivation of N. gonorrhoeae in planktonic suspensions To achieve 3-log 10 CFU inactivation of N. gonorrhoeae in suspensions, aBL exposures of 25 J/cm 2 (for ATCC700825; ∽7 min illumination) to 59 J/cm 2 (for Strain #199; ∽16 min illumination) were used ( Fig. 1A ). To inactivate all the bacterial CFU in suspensions (> 6-log 10 CFU inactivation), aBL exposures of 45 J/cm 2 (for ATCC700825; 12.5 min illumination) to 108 J/cm 2 (for Strain #199; 30 min illumination) were applied ( Fig. 1A ). Download figure Open in new tab Fig. 1. Effectiveness of aBL inactivation of N. gonorrhoeae at different wavelengths. ( A ) 405-nm wavelength. ATCC 700825 and clinical isolates 174, 179, 181, 199 were studied. (B) 470 nm wavelength. ATCC 700825 was studied. Bars: standard error of the means. The anti-gonococcal effect of 470-nm aBL was tested on ATCC 700825, and was much less effective than 405-nm aBL ( Fig. 1B ). An aBL exposure of 170 J/cm 2 (∽ 47 min illumination) was required to produce 3-log 10 CFU inactivation. According to this finding, we employed 405-nm wavelength for the studies following. Transmission electron microscopy (TEM) images taken after varying aBL exposures revealed aBL-induced morphological changes in N. gonorrhoeae cells, such as formation of bulges (black arrows), breakage of cell walls accompanied by cytoplasmic release (blue arrows), loss of outer membranes (white arrows) and cellular disintegration (blue stars) ( Fig. 2 ). Decreased density of cytoplasm was observed in some cells accompanied by increased cellular size, suggesting that aBL induced damage to the cell wall of N. gonorrhoeae . Download figure Open in new tab Fig. 2. Transmission electron microscopy (TEM) images of untreated and 405-nm aBL treated N. gonorrhoeae (ATCC700825). Panels A – D are the images of cultures exposed to 0 (no aBL), 9, 18, and 27 J/cm 2 aBL, respectively. Images were taken immediately after each aBL exposure. Black arrows: bulges formation; blue arrows: breakage of cell wall accompanied by cytoplasmic release; white arrows: loss of outer membrane; blue stars: cellular disintegration. Scale bar: 100 nm . Toxicity of 405-nm aBL to normal vaginal epithelial cells As shown in Fig.1A, a 405-nm aBL exposure of 6-log 10 CFU inactivation). In contrast, the toxicity study of aBL using normal vaginal epithelial cells showed that no statistically significant loss of viability of VK2/E6E7 cells was observed when aBL exposures of up to 108 J/cm 2 were applied ( P =0.6676) ( Fig. 3A ). Comet assay results showed that no aBL induced DNA damage occurred in the VK2/E6E7 cells at aBL exposures up to 216 J/cm 2 ( Fig. 3B ). Download figure Open in new tab Fig. 3. Toxicity of 405-nm aBL to normal vaginal epithelial cells. (A) Change of the viability of normal VK2/E6E7 cells in response to 405-nm aBL at different exposures. Viability of VK2/E6E7 cells: 0 J/cm 2 vs. 54 J/cm 2 : P = 0.3474, 0 J/cm 2 vs. 108: P = 0.6676; 0 J/cm 2 vs. 162 J/cm 2 : P < 0.0001. Bars denote standard errors. ( B ) Comet assay images of normal VK2/E6E7 cells with and without 405-nm aBL exposure. The 0 J/cm 2 , 54 J/cm 2 , 108 J/cm 2 , and 216 J/cm 2 images show the cultures exposed to 0 J/cm 2 , 9 J/cm 2 , 18 J/cm 2 , and 27 J/cm 2 aBL, respectively. Positive control is treated with H 2 O 2 . Presence and quantity of endogenous porphyrins and flavins in N. gonorrhoeae The UPLC chromatograms identified the presence of several types of endogenous porphyrins in N. gonorrhoeae , including uroporphyrin, 7-carboxylporphyrin, 6-carboxylporphyrin, 5-carboxylporphyrin and coproporphyrin ( Fig. 4 ). No PpIX was detected in any of the five N. gonorrhoeae strains studied. Quantitative analysis revealed that the most abundant type of endogenous porphyrin species in N. gonorrhoeae was coproporphyrin, with concentrations ranging from 7.93 to 19.16 nmol/g (protein weight) in the five N. gonorrhoeae strains. The concentrations and content percentages of each porphyrin species in the N. gonorrhoeae strains are summarized in Table 1 . View this table: View inline View popup Download powerpoint Table 1. Concentrations of porphyrins in the extracts of five N. gonorrhoeae strains Download figure Open in new tab Fig. 4. Chromatograms from the UPLC analysis. A: standard porphyrin and PPIX. UP: uroporphyrin I; 7P: 7-carboxylporphyrin I; 6P: 6-carboxylporphyrin I; 5P: 5-carboxylporphyrin I; CP: coproporphyrin; MPIX: mesoporphyrin IX; PPIX: protoporphyrin IX. B, C, D, E, and F: extracts of ATCC700825, #174, #179, #181, #199, respectively. G: standard flavins. H, I, J, K, and L: G: extracts of ATCC700825, #174, #179, #181, #199, respectively. UPLC results also indicated the presence of flavins in N. gonorrhoeae ( Fig. 5 ). For all the strains except #179, the concentrations of FAD and FMN were higher than that riboflavin ( Fig. 5 ). Download figure Open in new tab Fig. 5. Concentrations of FAD, FMN, and Riboflavin in the extracts of N. gonorrhoeae and VK2/E6E7 cells Presence and quantity of endogenous porphyrins and flavins in normal vaginal epithelial cells Quantitative analysis by UPLC showed that the total content of endogenous porphyrins in VK2/E6E7 cells is 0.0176 nmol/g (protein weight) ( Table 2 ), which is hundreds of times lower than the total content of porphyrins in N. gonorrhoeae (9.34 to 22.26 nmol/g; Table 1 ). The content of flavins in VK2/E6E7 cells is also significantly lower than that in the N. gonorrhoeae ( Fig. 5 ). View this table: View inline View popup Download powerpoint Table 2. Concentrations of endogenous porphyrins in VK2/E6E7 cells Potential development of gonococcal resistance to aBL Fig. 8 shows the log 10 -CFU reduction of ATCC 700825 in response to 15 successive cycles of sub-therapeutic aBL exposures at 405 nm. Correlation analysis found a weak tendency of increased aBL inactivation of N. gonorrhoeae with the number of cycles (correlation coefficient =0.24). However, the correlation is not statistically significant ( P =0.40), indicating that development of gonococcal resistance to aBL did not occur after 15 successive cycles of sub-therapeutic aBL exposures. Effect of bacterium-host cell interaction on the tolerance of the vaginal epithelial cells to aBL and the efficacy of aBL inactivation of N. gonorrhoeae Fig. 7 displays the viability changes of VK2/E6E7 cells and N. gonorrhoeae cells in the co-cultures of VK2/E6E7 cells and N. gonorrhoeae in response to 405-nm aBL. In comparison with the result of non-infected VK2/E6E7 cells shown in Fig. 3 , it can be seen that infected VK2/E6E7 cells are more susceptible to aBL inactivation than non-infected VK2/E6E7 cells. Download figure Open in new tab Fig. 6. Efficacy of 405-nm aBL inactivation of N. gonorrhoeae (ATCC 700725) of 15 successive cycles of sub-therapeutic exposures. Bars: Standard error. Download figure Open in new tab Fig. 7. Viability changes of infected human vaginal epithelial cells (VK2/E6E7) by N. gonorrhoeae and intracellular N. gonorrhoeae in response to 405-nm aBL. (A) Infected VK2/E6E7 cells ; (B) Intracellular N. gonorrhoeae . Bars: Standard errors. The viability loss in the infected VK2/E6E7 cells was 11.3% (0.05-log 10 CFU) for 54 J/cm 2 ( P =0.146) and 24.9% (0.12-log 10 CFU) for 108 J/cm 2 ( P =0.0011), respectively ( Fig. 7A ). Under the same aBL exposures, 4.18- and 5.07-log 10 CFU reduction of N. gonorrhoeae cells were observed, respectively ( Fig. 7B ), indicating that N. gonorrhoeae cells were still much more susceptible to aBL inactivation than VK2/E6E7 cells in the co-cultures. Confocal microscope imaging ascertained the invasion of VK2/E6E7 cells by N. gonorrhoeae ( Fig. 8A ). Viable VK2/E6E7 cells and N. gonorrhoeae were stained green by SYTO9, and nonviable VK2/E6E7 cells and N. gonorrhoeae were stained red by propidium iodide (PI). A few spontaneously dead VK2/E6E7 cells and N. gonorrhoeae cells were observed in the culture without aBL irradiation ( Fig. 8A ). Download figure Open in new tab Download figure Open in new tab Fig. 8. Confocal images of the vaginal epithelial cells (VK2/E6E7) infected with N. gonorrhoeae (ATCC700825). ( A ) Low magnification. aBL: treated with 405-nm aBL. 60mW/cm 2 , 108J/cm 2 . NT: no treatment. PI:excitation wavelength 559 nm, emission wavelength 603 nm; SYTO9: excitation wavelength 488nm, emission wavelength 520 nm; Alexa Fluor 647-soybean lectin: excitation wavelength 635nm, emission wavelength 668 nm. Scale bar: 5 µm. ( B ) High magnification. NT: no treatment. aBL: treated with 405-nm aBL (60 mW/cm 2 , 108 J/cm 2 ). + saponin: infected cells incubated with 0.05% saponin for 15 min. PI penetrated into the vaginal epithelial cells. - saponin: infected cells not incubated with saponin. External nonviable bacteria exhibit purple + red fluorescence, appear red only; external viable bacteria exhibit purple +green fluorescence; and intracellular viable bacteria exhibit green fluorescence only. Solid white arrows: viable intracellular N. gonorrhoeae . Hollow white arrows: Nonviable intracellular N. gonorrhoeae . Scale bar: 5 µm. Confocal microscope imaging also revealed that, after an aBL exposure of 108 J/cm 2 , there was a marked increase in the number of nonviable N. gonorrhoeae cells adherent to the VK2/E6E7 cells ( Fig. 8A ). In contrast, no obvious increase in the number of nonviable VK2/E6E7 cells was observed after aBL ( Fig. 8A ). For N. gonorrhoeae cells, not all of them appeared nonviable immediately after aBL as shown in Fig. 8A . This did not seem consistent with the inactivation results of N. gonorrhoeae from colony forming assay, which showed over 5-log 10 CFU reduction of N. gonorrhoeae . A possible reason of this inconsistence is that in addition to cell membrane damage of bacteria, other mechanisms (e.g., oxidative DNA damage) were involved in bacterial cell inactivation without necessarily damaging the cell membrane ( 36 , 37 ), which were not detectable by PI staining. Without aBL, under higher magnification ( Fig. 8B ), viable adherent N. gonorrhoeae cells are visible, depicted as green ˝dots˝ by SYTO9. Because N. gonorrhoeae are only about 1/10 the size of the vaginal epithelial cells, intracellular viable bacteria could only be identified in certain cross sections of the vaginal epithelial cells (solid white arrows). In untreated VK2/E6E7 cells, nonviable intracellular N. gonorrhoeae cells were rarely observed after permeabilizing the membranes of VK2/E6E7 cells by saponin, as shown by the confocal image ( Fig. 8B ). When VK2/E6E7 cells (without being permeabilized) were treated with aBL, increased numbers of nonviable adherent N. gonorrhoeae cells were observed after aBL exposure. After permeabilizing, nonviable N. gonorrhoeae cells (hollow white arrows) were also observed in VK2/E6E7 cells. DISCUSSION In this study, we explored the potential use of aBL for the treatment of gonococcal infection, a disease that is on the rise worldwide, poses an urgent threat to human health, and is becoming untreatable through standard antibiotic strategies. Our findings demonstrated that N . gonorrhoeae , including antibiotic-resistant strains, were highly susceptible to 405-nm aBL. TEM imaging data suggested that aBL induced damage to the bacterial cell wall and subsequently led to bacterial death. Many studies reported that, in contrast to most other bacterial species, N. gonorrhoeae contains no superoxide dismutase (SOD), which is the major antioxidant defense system ( 38 ). This might explain the high susceptibility of N. gonorrhoeae to aBL inactivation, which is dominantly mediated by singlet oxygen. Unpublished data in our laboratory showed that N . gonorrhoeae is on average 7.32-, 5.87-, and 28.0-fold more susceptible to aBL than N Pseudomonas aeruginosa , Acinetobacter baumannii , and Escherichia coli, respectively. We also tested the effectiveness of aBL at another wavelength, 470-nm, for the inactivation of N . gonorrhea . In contrast to wavelength 405 nm, which targets porphyrins, wavelength 470 nm targets flavins. We found that the anti-gonococcal efficacy of 470-nm aBL was significantly lower than 405-nm aBL. A possible reason for this is the lower singlet oxygen quantum yield of flavins compared with porphyrins upon aBL irradiation ( 39 ). The UPLC analysis in this report confirmed the presence of both endogenous porphyrins and flavins in N. gonorrhoeae . The UPLC results also showed that the most abundant type of porphyrin in N. gonorrhoeae was coproporphyrin, which is a highly efficient singlet oxygen generator. The singlet oxygen quantum yields was determined to be 0.37 for coproporphyrin III ( 40 ). Interestingly, PpIX, which is commonly present in pathogenic bacteria, was not detected in any of the five N. gonorrhoeae isolates studied in this report. The biosynthesis of photosensitizing porphyrins in bacteria occurs in the pathway of the synthesis of heme ( 41 ). It is generally believed that the heme synthesis pathway of most bacteria begins with charged glutamyl-tRNA Glu to form the universal precursor ALA, and porphyrins are formed through a series of conserved enzymatic steps. PpIX was believed to be the classical pathway. However, recent studies reported that there is a divergent noncanonical pathway that produces heme through the coproporphyrin III intermediate ( 42 , 43 ). The lack of endogenous PpIX and rich of endogenous coproporphyrin in N. gonorrhoeae indicates that, unlike other Gram-negative bacteria, N. gonorrhoeae synthesizes heme via the coproporphyrin III pathway. In addition to synthesizing its own heme, N. gonorrhoeae is able to internalize and utilize exogenous heme within host epithelial cells for growth ( 44 ). The comparison of the aBL-susceptibility between N. gonorrhoeae and human vaginal epithelial cells suggested that there is a therapeutic window where N. gonorrhoeae could be selectively inactivated while normal vaginal epithelial cells are preserved. This can be explained by our UPLC findings that the content of aBL-active endogenous photosensitizers in the vaginal epithelial cells is much lower than that in N. gonorrhoeae . N . gonorrhoeae has evolved mechanisms to infect a variety of host cells and subvert clearance by the host immune response. Infection of genital mucosa by N . gonorrhoeae involves adherence to and invasion of epithelial cells. Therefore, to be an effective therapeutic, aBL must also be able to inactivate intracellular N. gonorrhoeae . Our results in this study demonstrated that aBL inactivated N. gonorrhoeae both adherent to and inside the vaginal epithelial cells. Although the infection of the vaginal epithelial cells by N. gonorrhoeae slightly reduced the tolerance of the vaginal epithelial cells to aBL, we showed that there still exited a therapeutic window of aBL for preferentially inactivate N. gonorrhoeae over the vaginal epithelial cells. The hypothesis that bacteria are unlikely to develop resistance to aBL is supported by the findings in the present study. It is well accepted that photo-oxidative stress reacts with several cellular macromolecules including proteins, lipids, DNA, and RNA, and subsequently results in cell damage ( 45 ). This multi-target feature of aBL minimizes the potential of aBL-resistance development by bacteria ( 21 , 22 ). We experimentally investigated the potential development of aBL-resistance by N. gonorrhea by carrying out 15 successive cycles of subtherapeutic aBL inactivation. No development of aBL resistance by N. gonorrhea was observed. Our finding in this regard is also consistent with that of a recent study, showing that 15 repeated sub-lethal exposures of Staphylococcus aureus suspension did not affect the susceptibility of the organism to 405-nm light ( 25 ). In conclusion, this study provides novel fundamental information regarding the use of aBL for treating gonococcal infection. aBL at 405-nm inactivates antibiotic-resistant N. gonorrhoeae while preserving the host cells. Endogenous blue light active photosensitizers (porphyrins and flavins) are present in N. gonorrhoeae cells. The development of gonococcal resistance to aBL is unlikely. Taken together, 405-nm aBL is potentially a promising strategy for combating otherwise untreatable multidrug-resistant gonococcal infection. Further studies are warranted to investigate whether other vaginal flora or biofilms might impact the utility of aBL, optimize the aBL parameters, assess the effectiveness and safety of aBL therapy for gonococcal infection in animal models, and explore potential clinical applications. MATERIALS AND METHODS Blue light sources Two light-emitting diodes with peak emissions at 405 nm (M405L2, Thorlabs, New Jersey, USA) and 470 nm (M470L2), which are the optimal wavelengths targeting porphyrins ( 46 ) and flavins ( 47 ), respectively, were used for irradiation. The irradiance of 60 mW/cm 2 , which was optimized based on log 10 CFU inactivation of bacteria per unit radiant exposure of aBL (J/cm 2 ) in a preliminary study, was employed in the study. Light irradiance was measured using a PM100D power/energy meter (Thorlabs, New Jersey, USA). N. gonorrhoeae strains and culture conditions ATCC700825 (FA 1090) and four clinical antibiotic-resistant N. gonorrhoeae isolates were studied. ATCC700825 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA), and the four additional isolates were obtained through the CDC & FDA Antibiotic Resistance Isolate Bank. Bacteria were routinely grown on gonococcal medium base GC (Remel, Lenexa, KS, USA) agar plates containing GCHI enrichment (Remel, Lenexa, KS, USA) and hemoglobin at 37 °C and 5% CO2. Human vaginal epithelial cells and growth conditions Human vaginal epithelial cells, VK2/E6E7 (ATCC ® CRL-2616), were purchased from the ATCC (Rockville, MD, USA). Cells were cultured in keratinocyte serum-free medium (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor, 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA), and 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C and 5% CO 2 . All experiments were performed in the exponential growth phase of VK2/E6E7 cells (48 h after plating). aBL inactivation of N. gonorrhoeae in suspensions Overnight N. gonorrhoeae cultures were collected from GC agar plates, washed using PBS, and then re-suspended in PBS to OD 600nm =0.3 (∽10 8 CFU/mL). Three ( 3 ) mL bacterial suspension was added into a 35-mm petri dish prior to aBL exposure. After varying aBL exposures (9, 18, 27, 36, 45, 54, 72, 90, and 108 J/cm 2 ) had been delivered, 30-µL samples of the bacterial suspension were taken and N. gonorrhoeae CFUs were measured using colony-forming assay. The experiment was performed in 3 independent replicates for each condition. In addition, bacterial suspensions without being exposed to aBL served as the negative controls. Evaluation of aBL toxicity to human vaginal epithelial cells To test the toxicity of aBL to normal vaginal epithelial cells, VK2/E6E7 cells were exposed to aBL at varying exposures of 54 J/cm 2 , 108 J/cm 2 , and 162 J/cm 2 . The viability of VK2/E6E7 cells was measured using MTT assay 24h after aBL exposure. The experiment was performed in 6 independent replicates for each condition. In addition, aBL-induced DNA damage in VK2/E6E7 cells was evaluated using a CometAssay kit (Trevigen Inc., USA). The experiments were performed based on the Trevigen standard protocol for single cell gel electrophoresis. Briefly, aBL-treated and untreated VK2/E6E7 cells were detached using trypsin. The cell pellets were resuspended at 37 °C with molten LMAgarose after centrifugation and then loaded onto a pre-coated CometSlide ™ slide (50µL/well), which was immediately cooled on ice. The embedded cells were lysed in cold Trevigen Lysis Solution overnight at 4 °C and then electrophoresed in cold alkaline buffer (pH 13.3). DNA strands/fragments stained with SYBR Gold were visualized under an Olympus FV-1000 confocal microscope at 496/522 nm. Ultra-Performance Liquid Chromatography (UPLC) analysis of endogenous aBL-absorptive photosensitizers in N. gonorrhoeae and human vaginal epithelial cells UPLC was used to separate and quantify the endogenous aBL-absorptive photosensitizers (porphyrins and flavins) in N. gonorrhoeae and VK2/E6E7 cells. To extract the endogenous photosensitizers from the cells, overnight cultures were washed using PBS. The cell pellets were collected after centrifugation (at 13,500 × g for 6 min), re-suspended in 1.0 mL of extraction solvent (ratio of ethanol to dimethyl sulfoxide to acetic acid, 80:20:1 [vol/vol/vol]), and then stored at -80°C for 24 h. The cell walls were then disrupted by sonication for 20 min. After centrifugation (at 13,500 × g for 6 min), the supernatant was collected as the whole-cell lysates. The protein content in each sample was measured using BCA protein assay. Standard porphyrins (Chromatographic Marker Kit, Product # CMK-1A, Frontier Scientific, Logan, Utah; and PpIX, Sigma Aldrich) and standard flavins (Sigma Aldrich) were used as the reference compounds. The Chromatographic Marker kit is composed of uroporphyrin I, 7-carboxylporphyrin I, 6-carboxylporphyrin I, 5-carboxylporphyrin I, coproporphyrin, mesoporphyrin IX, mesoporphyrin IX dihydrochloride; and the standard flavins included Flavin mononucleotide(FMN), flavin adenine dinucleotide (FAD), and riboflavin. UPLC separation and quantitation of porphyrins and flavins in N. gonorrhoeae and VK2/E6E7 cells were carried out using a Waters ® Acquity UPLC™ System. The system is composed of a binary solvent manager, sample manager, fluorescence detector, column heater and an Acquity UPLC BEH C18, 1.7 µM, 2.1 x 100 mm column. For detecting porphyrins, the excitation was set at 404 nm wavelength and emission at 618 nm; and for detecting flavins, the excitation and emission were set at 260 nm and 470 nm, respectively. TEM examination of morphological changes of N. gonorrhoeae cells induced by aBL To examine the morphological changes of N. gonorrhoeae cells induced by aBL, untreated and aBL treated ATCC700825 N. gonorrhoeae cells were fixed in 1% paraformaldehyde and 1.25% glutaraldehyde immediately after aBL exposures [9 J/cm 2 (2.5 min illumination), 18 J/cm 2 (5 min illumination), and 27 J /cm 2 (7.5 min illumination)] and stored at 4°C for 2 h. The N. gonorrhoeae cells were then washed three times with 0.1 M sodium cacodylate buffer after spinning down (1200 rpm, 10 min) and decanting the fixative. Cell pellets were subsequently processed for TEM imaging. Assessment of potential development of gonococcal resistance to aBL To assess if N. gonorrhoeae has the potential to develop resistance to aBL, suspensions of ATCC 700825 in PBS were subject to 15 repeated cycles of sub-therapeutic aBL exposures. In the 1st cycle, the aBL exposure was adjusted to produce ∽ 4.0-log 10 CFU inactivation of N. gonorrhoeae , and the same exposure level of aBL was then used throughout the successive cycles. In each cycle, the surviving bacterial cells after aBL exposure were collected, sub-cultured, and grown overnight for the next cycle of aBL inactivation. Infection of human vaginal epithelial cells by N. gonorrhoeae N. gonorrhoeae have evolved the mechanism to invade into human vaginal epithelial cells to overcome host defense barrier ( 48 ). To assess the effectiveness of 405-nm aBL inactivation of intracellular N. gonorrhoeae , VK2/E6E7 cells were seeded into a 35-mm petri dish (Transwell ® , Costar, NY, USA) at a cell density of 2 × 10 5 cells/dish. The VK2/E6E7 cells were incubated in 2 mL keratinocyte serum-free medium for 48 h at 37 °C. The supernatant was then discarded and 1 mL suspension of ATCC 700825 was added. A multiplicity of infection (M.O.I.) of 50 (bacteria: VK2/E6E7 cells) was used according to the optimized M.O.I. results of a previous study ( 49 ). The co-cultures of VK2/E6E7 cells and N. gonorrhoeae were incubated at 37 °C with 5% CO 2 for 4 h. The invasion of N. gonorrhoeae cells into VK2/E6E7 cells was examined by confocal microscope (Olympus, FV 1000-MPE Confocal) using the method described previously ( 50 ). aBL inactivation of intracellular N. gonorrhoeae Four identical cultures (A, B, C, and D) of VK2/E6E7 cells infected with N. gonorrhoeae were prepared for each experiment. Culture A was treated with aBL followed by incubation with 0.05% saponin for 20 min to lyse the VK2/E6E7 cells and liberate the bacteria. The viability of N. gonorrhoeae was then determined using colony forming assay. Culture B was treated with the same aBL exposure, and the viability of VK2/E6E7 cells was assessed using MTT assay 24 h after aBL exposure. Cultures C and D were not exposed to aBL. The viability of N. gonorrhoeae in Culture C was measured using colony forming assay and the viability of VK2/E6E7 cells in Culture D was measured using MTT assay. aBL exposures of 54 J/cm 2 and 108 J/cm 2 were tested. The experiment was performed in 6 independent replicates for each condition. Fluorescence microscopy for determining the viability of intracellular N. gonorrhoeae For fluorescence microscopy imaging, fluorescent dyes PI and SYTO9 (Invitrogen™) were used to assess the viability of N. gonorrhoeae as well as the vaginal epithelial cells immediately after aBL exposure. To discriminate the viability of intracellular bacteria and the bacteria adherent to the vaginal epithelial cells, N. gonorrhoeae -infected human vaginal epithelial cells were incubated with Alexa Fluor 647-coupled soybean lectin (Invitrogen™, false-colored purple) for 10 min before adding PI and SYTO9. In the VK2/E6E7 cells with intact cell membranes, intracellular nonviable N. gonorrhoeae cells could not be stained by PI because the cell membranes of VK2/E6E7 cells are impermeable to PI. To stain intracellular nonviable N. gonorrhoeae using PI, we used 0.05% saponin to permeabilize VK2/E6E7 cells. when adding PI and SYTO9. Under confocal microscopy, nonviable bacteria adherent to the vaginal epithelial cells exhibited purple and red fluorescence, intracellular nonviable bacteria red only, adherent viable bacteria purple and green, and intracellular viable bacteria green only. Statistical Analysis Data were presented as the mean ± standard error. The differences between different conditions were analyzed using one-way analysis of variance (ANOVA). P values of <0.05 were considered statistically significant. Acknowledgements Funding This study was supported in part by the National Institute Health (R01AI123312 to T.D.) and the U.S. Department of Defense (FA9550-17-1-0277 to T.D.). Y.W. was supported by National Natural Science Foundation of China (61575222) and China Scholarship Council (201603170004). Author contributions Y.W., R.F.E., Y.B., K.D.H., Y.H.G., Y.G., J.A.F., and T.D. designed the experiments. Y.W., R.F.E., Y.B., and X.S.G. performed the experiments. Y.W., Y.B., and T.D. analyzed the data and wrote the manuscript. R.F.E., K.D.H., Y.H.G. and J.A.F. reviewed the manuscript. Competing interests The authors declare that they have no competing interests. Data and materials availability REFERENCES AND NOTES 1. ↵ The STD Aware . 2017 . The history of gonorrhea . STDAware Blog . 2. ↵ Mandal A. Nov 28 , 2012 2012. Gonorrhea history, on AZoNetwork . https://www.news-medical.net/health/Gonorrhea-History.aspx# . Accessed 3. ↵ Unemo M. 2015 . Current and future antimicrobial treatment of gonorrhoea - the rapidly evolving Neisseria gonorrhoeae continues to challenge . BMC Infect Dis 15 : 364 . OpenUrl CrossRef PubMed 4. ↵ Centers for Disease Control and Prevention (CDC) . January 4, 2018 2018 . Gonococcal infections . https://www.cdc.gov/std/tg2015/gonorrhea.htm . Accessed 5. ↵ Macaluso M , TJ Wright-Schnapp , Chandra A , Johnson R , CL Satterwhite , Pulver A , SM Berman , RY Wang , SL Farr , LA Pollack . 2010 . A public health focus on infertility prevention, detection, and management . Fertil Steril 93 : 16.e11-10 . OpenUrl 6. MS Cohen , IF Hoffman , RA Royce , Kazembe P , JR Dyer , CC Daly , Zimba D , PL Vernazza , Maida M , SA Fiscus , JJ Eron , Jr. 1997 . Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP Malawi Research Group . Lancet 349 : 1868 – 1873 . OpenUrl CrossRef PubMed Web of Science 7. Ward H , Rönn M. 2010 . The contribution of STIs to the sexual transmission of HIV . Current opinion in HIV and AIDS 5 : 305 – 310 . OpenUrl CrossRef 8. MS Cohen . 1998 . Sexually transmitted diseases enhance HIV transmission: no longer a hypothesis . Lancet 351 Suppl 3 : 5 – 7 . OpenUrl CrossRef PubMed Web of Science 9. ↵ Centers for Disease Control and Prevention (CDC) . March 28, 2018 2018 . Antibiotic-Resistant Gonorrhea Basic Information . https://www.cdc.gov/std/gonorrhea/arg/basic.htm . Accessed 10. ↵ PA Rice , WM Shafer , Ram S , AE Jerse . 2017 . Neisseria gonorrhoeae: Drug resistance, mouse models, and vaccine development . Annu Rev Microbiol 71 : 665 – 686 . OpenUrl CrossRef 11. ↵ Centers for Disease Control and Prevention (CDC) . 2012 . Update to CDC’s Sexually transmitted diseases treatment guidelines, 2010: toral cephalosporins no longer a recommended treatment for gonococcal infections . MMWR Morb Mortal Wkly Rep 61 : 590 – 594 . OpenUrl PubMed 12. ↵ RD Kirkcaldy , Harvey A , JR Papp , Del Rio C , OO Soge , KK Holmes , EW Hook , 3rd, Kubin G , Riedel S , Zenilman J , Pettus K , Sanders T , Sharpe S , Torrone E. 2016 . Neisseria gonorrhoeae Antimicrobial Susceptibility Surveillance - The Gonococcal Isolate Surveillance Project, 27 Sites, United States, 2014 . MMWR Surveill Summ 65 : 1 – 19 . OpenUrl CrossRef PubMed 13. ↵ ZR Balsara , SS Ross , PC Dolber , JS Wiener , Tang Y , PC Seed . 2013 . Enhanced susceptibility to urinary tract infection in the spinal cord-injured host with neurogenic bladder . Infect Immun 81 : 3018 – 3026 . OpenUrl Abstract / FREE Full Text 14. ↵ Wappes J. September 21 , 2016 2016 . CDC reports first highly resistant gonorrhea in US, on University of Minnesota . http://www.cidrap.umn.edu/news-perspective/2016/09/cdc-reports-first-highly-resistant-gonorrhea-us . Accessed OpenUrl 15. ↵ Centers for Disease Control and Prevention (CDC) . 2013. Antibiotic Resistance Threats in the United States , 2013 . http://www.cdc.gov/drugresistance/threat-report-2013/ . xAccessed 05/04/2015 . 16. Unemo M , RA Nicholas . 2012 . Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea . Future Microbiol 7 : 1401 – 1422 . OpenUrl CrossRef PubMed Web of Science 17. Unemo M , Del Rio C , WM Shafer . 2016 . Antimicrobial resistance expressed by Neisseria gonorrhoeae: A major global public health problem in the 21st Century . Microbiol Spectr 4 . 18. CM Kahler . 2018 . Multidrug-resistant Neisseria gonorrhoeae: future therapeutic options . Future Microbiol 13 : 499 – 501 . OpenUrl 19. ↵ Dvorsky G. July 17 , 2017 2017 . Experts sound the alarm as drug resistant gonorrhea goes global, on Gizmodo . https://gizmodo.com/experts-sound-the-alarm-as-drug-resistant-gonorrhea-goe-1796710964 . Accessed 20. ↵ Alirol E , TE Wi , Bala M , ML Bazzo , XS Chen , Deal C , JR Dillon , Kularatne R , Heim J , EW Hooft van Huijsduijnen R , Hook , MM Lahra , DA Lewis , Ndowa F , WM Shafer , Tayler L , Workowski K , Unemo M , Balasegaram M. 2017 . Multidrug-resistant gonorrhea: A research and development roadmap to discover new medicines . PLoS Med 14 : e1002366 . OpenUrl CrossRef 21. ↵ Wang Y , Wang Y , Wang Y , CK Murray , MR Hamblin , DC Hooper , Dai T. 2017 . Antimicrobial blue light inactivation of pathogenic microbes: State of the art . Drug Resist Updat 33-35 : 1 – 22 . OpenUrl 22. ↵ Dai T , Gupta A , CK Murray , MS Vrahas , GP Tegos , MR Hamblin . 2012 . Blue light for infectious diseases: Propionibacterium acnes, Helicobacter pylori, and beyond? Drug Resistance Updates 15 : 223 – 236 . OpenUrl CrossRef PubMed 23. ↵ Dai T , Gupta A , CK Murray , MS Vrahas , GP Tegos , MR Hamblin . 2012 . Blue light for infectious diseases: Propionibacterium acnes, Helicobacter pylori, and beyond? Drug Resist Updat 15 : 223 – 236 . OpenUrl CrossRef PubMed 24. ↵ MR Hamblin , Viveiros J , Yang C , Ahmadi A , RA Ganz , MJ Tolkoff . 2005 . Helicobacter pylori accumulates photoactive porphyrins and is killed by visible light . Antimicrob Agents Chemother 49 : 2822 – 2827 . OpenUrl Abstract / FREE Full Text 25. ↵ RM Tomb , Maclean M , JE Coia , SJ MacGregor , JG Anderson . 2017 . Assessment of the potential for resistance to antimicrobial violet-blue light in Staphylococcus aureus . Antimicrob Resist Infect Control 6 : 100 . OpenUrl 26. CS Enwemeka , Williams D , SK Enwemeka , Hollosi S , Yens D. 2009 . Blue 470-nm light kills methicillin-resistant Staphylococcus aureus (MRSA) in vitro . Photomed Laser Surg 27 : 221 – 226 . OpenUrl CrossRef PubMed 27. CS Enwemeka , Williams D , Hollosi S , Yens D , SK Enwemeka . 2008 . Visible 405 nm SLD light photo-destroys methicillin-resistant Staphylococcus aureus (MRSA) in vitro . Lasers Surg Med 40 : 734 – 737 . OpenUrl CrossRef PubMed 28. Maclean M , SJ MacGregor , JG Anderson , Woolsey G. 2009 . Inactivation of bacterial pathogens following exposure to light from a 405-nanometer light-emitting diode array . Appl Environ Microbiol 75 : 1932 – 1937 . OpenUrl Abstract / FREE Full Text 29. McDonald R , SJ Macgregor , JG Anderson , Maclean M , MH Grant . 2011 . Effect of 405-nm high-intensity narrow-spectrum light on fibroblast-populated collagen lattices: an in vitro model of wound healing . J Biomed Opt 16 : 048003 . OpenUrl CrossRef PubMed 30. Yin R , Dai T , Avci P , AE Jorge , WC de Melo , Vecchio D , YY Huang , Gupta A , MR Hamblin . 2013 . Light based anti-infectives: ultraviolet C irradiation, photodynamic therapy, blue light, and beyond . Curr Opin Pharmacol 13 : 731 – 762 . OpenUrl CrossRef PubMed 31. Dai T , Gupta A , Huang Y , Yin R , CK Murray , MS Vrahas , Sherwood M , GP Tegos , MR Hamblin . 2012 . Blue light rescues mice from potentially fatal Pseudomonas aeruginosa burn infection: efficacy, safety, and mechanism of action . Antimicrob Agents Chemother 57 : 1238 – 1245 . OpenUrl 32. CS Enwemeka . 2013 . Antimicrobial blue light: an emerging alternative to antibiotics . Photomed Laser Surg 31 : 509 – 511 . OpenUrl CrossRef PubMed 33. ↵ MM Kleinpenning , Smits T , MH Frunt , PE van Erp , PC van de Kerkhof , RM Gerritsen . 2010 . Clinical and histological effects of blue light on normal skin . Photodermatol Photoimmunol Photomed 26 : 16 – 21 . OpenUrl CrossRef PubMed 34. ↵ Liebmann J , Born M , Kolb-Bachofen V. 2010 . Blue-light irradiation regulates proliferation and differentiation in human skin cells . J Invest Dermatol 130 : 259 – 269 . OpenUrl CrossRef PubMed Web of Science 35. ↵ Wang Y , Ferrer-Espada R , Gu Y , Dai T. 2017 . Antimicrobial blue light: An alternative therapeutic for multidrug-resistant gonococcal infections? Open Acc J Photoen 1 : 00009 . OpenUrl 36. ↵ VV Bumah , Aboualizadeh E , DS Masson-Meyers , JT Eells , CS Enwemeka , CJ Hirschmugl . 2017 . Spectrally resolved infrared microscopy and chemometric tools to reveal the interaction between blue light (470nm) and methicillin-resistant Staphylococcus aureus . Journal of Photochemistry and Photobiology B: Biology 167 : 150 – 157 . OpenUrl 37. ↵ Yoshida A , Sasaki H , Toyama T , Araki M , Fujioka J , Tsukiyama K , Hamada N , Yoshino F. 2017 . Antimicrobial effect of blue light using Porphyromonas gingivalis pigment . Scientific Reports 7 : 5225 . OpenUrl 38. ↵ FS Archibald , MN Duong . 1986 . Superoxide dismutase and oxygen toxicity defenses in the genus Neisseria . Infect Immun 51 : 631 – 641 . OpenUrl Abstract / FREE Full Text 39. ↵ Baier J , Maisch T , Maier M , Engel E , Landthaler M , Baumler W. 2006 . Singlet oxygen generation by UVA light exposure of endogenous photosensitizers . Biophys J 91 : 1452 – 1459 . OpenUrl CrossRef PubMed Web of Science 40. ↵ Tanielian C , Schweitzer C , Mechin R , Wolff C. 2001 . Quantum yield of singlet oxygen production by monomeric and aggregated forms of hematoporphyrin derivative . Free Radic Biol Med 30 : 208 – 212 . OpenUrl CrossRef PubMed 41. ↵ G.N. C. 2014 . Biosynthesis of the Tetrapyrrole Ring System , p 539 – 553 , Microbial Biochemistry third ed doi: 10.1007/978-94-017-8908-0 . OpenUrl CrossRef 42. ↵ JE Choby , EP Skaar . 2016 . Heme Synthesis and Acquisition in Bacterial Pathogens . J Mol Biol 428 : 3408 – 3428 . OpenUrl CrossRef 43. ↵ Bali S , DJ Palmer , Schroeder S , SJ Ferguson , MJ Warren . 2014 . Recent advances in the biosynthesis of modified tetrapyrroles: the discovery of an alternative pathway for the formation of heme and heme d 1 . Cell Mol Life Sci 71 : 2837 – 2863 . OpenUrl CrossRef PubMed 44. ↵ PC Turner , CE Thomas , Elkins C , Clary S , PF Sparling . 1998 . Neisseria gonorrhoeae heme biosynthetic mutants utilize heme and hemoglobin as a heme source but fail to grow within epithelial cells . Infect Immun 66 : 5215 – 5223 . OpenUrl Abstract / FREE Full Text 45. ↵ Bocking T , KD Barrow , AG Netting , TC Chilcott , HG Coster , Hofer M. 2000 . Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae . Eur J Biochem 267 : 1607 – 1618 . OpenUrl CrossRef PubMed 46. ↵ Rimington C. 1960 . Spectral-absorption coefficients of some porphyrins in the Soret-band region . Biochem J 75 : 620 – 623 . OpenUrl FREE Full Text 47. ↵ Weber S , CW Kay , Mogling H , Mobius K , Hitomi K , Todo T. 2002 . Photoactivation of the flavin cofactor in Xenopus laevis (6 - 4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance . Proc Natl Acad Sci U S A 99 : 1319 – 1322 . OpenUrl Abstract / FREE Full Text 48. ↵ SA Hill , TL Masters , Wachter J. 2016 . Gonorrhea - an evolving disease of the new millennium . Microb Cell 3 : 371 – 389 . OpenUrl 49. ↵ RN Fichorova , PJ Desai , FC Gibson , 3rd, CA Genco . 2001 . Distinct proinflammatory host responses to Neisseria gonorrhoeae infection in immortalized human cervical and vaginal epithelial cells . Infect Immun 69 : 5840 – 5848 . OpenUrl Abstract / FREE Full Text 50. ↵ MB Johnson , AK Criss . 2013 . Fluorescence microscopy methods for determining the viability of bacteria in association with mammalian cells . J Vis Exp doi: 10.3791/50729 . OpenUrl CrossRef Back to top Previous Next Posted September 21, 2018. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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