Emerging therapies for multidrug resistant Acinetobacter baumannii

1 2 3 Emerging therapies for multidrug resistant Acinetobacter baumannii 4 5 6 7 Meritxell García-Quintanilla*, Marina R. Pulido*, Rafael López-Rojas, Jerónimo 8 Pachón and Michael J. McConnell 9 10 11 Unit of Infectious Disease, Microbiology, and Preventive Medicine, Institute of 12 Biomedicine of Sevilla (IBiS), University Hospital Virgen del Rocío/CSIC/University 13 of Sevilla, 41013, Sevilla, Spain. 14 15 16 17 *MGQ and MRP contributed equally to this work 18 19 20 Corresponding author: Michael J. McConnell (mcconnell.mi[email protected]) 21 22 23 24 25 Key Words: Acinetobacter baumannii, antibiotic resistance, phage therapy, vaccine, 26 antimicrobial peptides, iron chelation therapy 27 28 29 2 Abstract 30 The global emergence of multidrug resistant Acinetobacter baumannii has 31 reduced the number of clinically-available antibiotics that retain activity against this 32 pathogen. For this reason, the development of novel prevention and treatment strategies 33 for infections caused by A. baumannii is necessary. A number of studies have begun to 34 characterize non-antibiotic approaches that utilize novel mechanisms of action to 35 achieve antibacterial activity. Recent advances in phage therapy, iron chelation therapy, 36 antimicrobial peptides, prophylactic vaccination, photodynamic therapy and nitric 37 oxide-based therapies have all been shown to have activity against A. baumannii. 38 However, before these approaches can be used clinically there are still limitations and 39 remaining questions that must be addressed. 40 3 Acinetobacter baumannii and antibiotic resistance 41 Acinetobacter baumannii has emerged as an important cause of nosocomial 42 infections, most notably ventilator-associated pneumonia and bacteremia, and less 43 frequently meningitis, skin and soft tissue infections, urinary tract infections, and 44 endocarditis [1]. A. baumannii pneumonia and bacteraemia are typically acquired in the 45 hospital setting and are associated with high mortality [2, 3]. Whereas thirty years ago 46 infections caused by A. baumannii could be effectively treated with traditionally used 47 antibiotics, the global spread of multidrug resistant strains with resistance to 48 antimicrobials from multiple antibiotic classes has dramatically reduced the number of 49 drugs that retain activity against this pathogen. These trends have necessitated the use 50 of new antibiotic treatment strategies that include the use of tigecycline, the first of a 51 new class of antibiotics termed glycylcyclines, and colistin, an ‘old’ polymixin 52 antibiotic that was introduced into clinical practice over 50 years ago. Unfortunately, 53 infections caused by strains with resistance to these antibiotics have already been 54 described [4-6], demonstrating the ability of A. baumannii to rapidly acquire resistance. 55 Of particular concern are pan-drug resistant strains with resistance to all clinically-used 56 antibiotics [7, 8]. The increased difficulty of clinically managing infections caused A. 57 baumannii due to a lack of active antimicrobials has prompted the development of novel 58 strategies for managing these infections. In this review, recent advances in non-59 antibiotic approaches that are currently being explored for prevention and treatment of 60 A. baumannii infections are described. 61 62 Phage therapy 63 Bacteriophages, or phages, are viruses that infect, and in some cases lyse, 64 bacterial cells. The potential use of bacteriophages as antibacterial agents was 65 4 recognized at almost the same time as their discovery nearly a century ago [9]. 66 However, the dawn of the antibiotic era slowed interest in this area in Western counties. 67 In the present context of infections caused by multidrug resistant bacteria for which 68 there are a decreasing number of active antimicrobials, research exploring the use of 69 phage therapy as an alternative treatment has been renewed. In 2010, the first two 70 reports characterizing lytic phages specific for A. baumannii, phages AB1 and AB2, 71 were published [10, 11]. Since then, numerous additional phages with lytic activity 72 against A. baumannii clinical isolates have been described [12-17], and one of them has 73 been fully sequenced (Phage AB1 accession number HM368260) [18]. In general, the 74 phages were isolated from sewage water, patient sputum, clinical samples or 75 marine/pond samples and all have shown to be highly specific for A. baumannii (Table 76 1). 77 The majority of studies characterizing A. baumannii phages have consisted of in 78 vitro studies assessing the ability of the phages to lyse clinical isolates of A. baumannii. 79 Phage AB1 was lytic for only one of five clinical multidrug resistant A. baumannii 80 isolates tested [11], whereas AB2 was capable of infecting ATCC 17978, ATCC 19606 81 and 25 of 125 (20%) of clinical isolates [10]. Phage Abp53 was able to infect and lyse 82 the ATCC 19606 strain and 7 of 26 (27%) multidrug resistant A. baumannii strains [13] 83 while bacteriophage ZZ1 infected only 3 of 23 (13%) multidrug resistant clinical 84 isolates [12]. Phages identified more recently have shown a broader host range, with 85 phages AB7-IBB1, AB7-IBB2 and KM18p lysing 23 of 39 (59%) and 19 of 39 (49%) 86 and 15 of 34 (44%) of clinical isolates tested, respectively [15-17]. In 2012 the A. 87 baumannii phage AP22 was shown to lyse 89 of 130 clinical A. baumannii strains 88 (68%), the broadest host range against A. baumannii observed to date [14]. These 89 studies illustrate that a potential limitation of phage therapy is the limited host range of 90 5 A. baumannii phages. However the narrow spectrum of individual phages was 91 overcome in a study by Lin et al. in which a cocktail of phages was able to lyse 113 of 92 127 (89%) A. baumannii strains [10]. Only one report has evaluated the use of an A. 93 baumannii specific phage for treatment in an animal model of infections [19]. In this 94 study, the phage BS46 was able to protect mice infected intraperitoneally with five 95 times the LD50 of a highly virulent A. baumannii strain. Clearly, additional studies in 96 animal models are needed in order to characterize the potential therapeutic efficacy of 97 phages specific for A. baumannii. 98 In addition to their potential for therapeutic use, phages could also be used for 99 removing A. baumannii from colonized surfaces in the hospital environment. A. 100 baumannii can form robust biofilms, which protect the bacteria from environmental 101 insults and allow it to persist on environmental surfaces [20]. It was recently shown 102 that the A. baumannii specific phages AB7-IBBI and AB7-ABB2 can inhibit biofilm 103 formation, and also remove approximately 75% of preformed biofilms [15, 16], 104 demonstrating the possible utility of these phages in environmental biocontrol. 105 Taken together, the studies addressing the use phage therapy for A. baumannii 106 infections suggest that the phages isolated to date have lytic potential against clinical 107 isolates of A. baumannii and have high host range specificity, an issue of crucial 108 importance as it would be ideal to avoid secondary effects on normal human flora. 109 However, two concerns that have been raised in studies using phages against non-110 Acinetobacter bacteria are the rapid clearance of phages by macrophages and the 111 induction of antiphage antibodies [21-23]. Additional issues such as the appearance of 112 resistant strains and the inflammatory response in humans after administration of phage 113 therapy will also need to be addressed before phage therapy can be considered for 114 clinical use. 115 6 Iron chelation and gallium based therapies 116 Iron is an essential cofactor for many bacterial processes, raising the possibility 117 of using iron chelators and iron competitors as antibacterial agents. While a number of 118 studies have demonstrated the antibacterial activity of various iron chelators [24-31], it 119 is only recently that this approach has been applied to A. baumannii. Figure 1 illustrates 120 the structure of the iron chelators that have been used to-date with A. baumannii. 121 Deferoxamine is a bacterial siderophore that has been shown to have low antibacterial 122 activity against a number of bacterial pathogens, likely due to the fact that many 123 bacteria have receptors for deferoxamine and thus are able to acquire deferoxamine-124 bound iron [24]. In agreement with these studies, deferoxamine did not have 125 antibacterial activity against A. baumannii grown in a number of different liquid media 126 [24, 32, 33]. Deferiprone is a synthetic bidentate iron chelator that has antimicrobial 127 activity against a number of bacterial species, however showed only modest activity 128 against A. baumannii [24]. The same study also evaluated the activity of the iron 129 chelators Apo6619, VK28 dihydrochloride and 2,2- bipyridyl (DIP). The authors 130 observed that if bacteria were grown in Mueller-Hinton broth, DIP showed the highest 131 antimicrobial activity against A. baumannii [minimum inhibitory concentration (MIC) = 132 64 µg/ml] while Apo6619 and VK28 had somewhat less activity (MIC ≥ 128). 133 Interestingly, the antibacterial effect appeared to be dependent on the medium used as 134 bacteria grown in RPMI demonstrated lower MIC values [24]. 135 Gallium (Ga+3) is a transition metal with a similar atomic radius and valence to 136 iron (Fe+3), allowing it to compete with Fe+3 for binding to iron-requiring enzymes, 137 proteins and microbial siderophores. However, because gallium cannot undergo 138 oxidation-reduction cycles it acts as an inhibitor to these bacterial components upon 139 binding. Similar to the results seen with iron chelators, the effect of gallium nitrate on 140 7 A. baumannii growth depended on the growth medium used [32]. The antimicrobial 141 activity of gallium was increased in serum compared to iron-rich growth media, likely 142 due to the presence of transferrin which sequesters free iron. This study also showed 143 that gallium treatment resulted in lower bacterial loads in the lungs of mice infected 144 intranasally with A. baumannii, indicating that gallium maintains activity in vivo. 145 Antunes et al. recently demonstrated that low concentration of gallium could inhibit 146 growth of A. baumannii isolates in human serum and reduce mortality in a Galleria 147 mellonella infection model [34]. Importantly, this study also observed a significant in 148 vitro synergy between colistin and gallium for both colistin-sensitive and colistin-149 resistant A. baumannii isolates suggesting that the use of gallium in combination with 150 clinically-used antibiotics may be a viable treatment option for multidrug resistant 151 strains. In summary, iron chelation and gallium based therapies show good activity in 152 vitro, but additional work is needed to characterize their in vivo effiicacy. 153 154 Antimicrobial peptides 155 Antimicrobial peptides are key elements of the immune system of higher 156 organisms, making them potential candidates for use as antibacterial agents [35]. 157 Although numerous distinct antimicrobial peptides have been developed, most of them 158 share a cationic character and fold into amphipathic conformations that allow them to 159 act as bacterial membrane disruptors, although some peptides have also been shown to 160 exert intracellular mechanisms of action [35, 36]. 161 Several antibacterial peptides have been studied in vitro and in vivo against A. 162 baumannii. The cecropin A-melittin hybrid peptides demonstrated in vitro bactericidal 163 activity against multidrug resistant and colistin-resistant A. baumannii strains [37, 38], 164 and showed local efficacy in an experimental model of peritoneal sepsis caused by a 165 8 pan-drug resistant strain [39]. However, the peptide exhibited a short half-life after 166 administration, limiting its systemic efficacy. Several peptides derived from frog and 167 toad skin and their analogs have also been studied. The brevinin-2-related peptide [40] 168 and six cationic a-helical frog skin-derived peptides [41] showed good activity (MIC 4 – 169 128 µg/ml) against multidrug resistant strains of A. baumannii and low hemolytic 170 activity. One of the frog skin peptides, Alyteserin-2a, presented relatively weak 171 antimicrobial activity and produced hemolysis. Interestingly, however, its analog 172 containing D-lysines at positions 7 and 11 showed higher potency against multidrug 173 resistant A. baumannii and lower hemolytic activity and cytotoxicity [42]. A3-APO is a 174 proline-rich antibacterial peptide that, despite high MIC values (32 - >64 µg/ml), 175 showed improved in vivo efficacy compared with imipenem in mouse models of 176 carbapenem-resistant A. baumannii infection, when used either intravenously or 177 intramuscularly [43]. Moreover, in an A. baumannii blast injury model, A3-APO 178 improved survival and reduced bacterial loads in the blood and in wounds, and 179 improved wound appearance significantly more than treatment with imipenem or 180 colistin [44]. 181 Although antimicrobial peptides have shown promising results in in vitro and in 182 animal models, a number of issues still remain to be addressed. A major technical 183 challenge that must be resolved before antimicrobial peptides can be used systemically 184 will be the design of peptides that maintain antibacterial activity but are resistant to 185 proteolytic degradation in serum. This limitation, however, is not problematic when the 186 peptides are administered topically, as demonstrated by a recent clinical trial [45]. In 187 addition, the hemolytic activity that is seen with some peptides will need to be reduced 188 in order to prevent toxicity upon systemic administration. Studies aimed at developing 189 9 peptide analogs through the introduction of structural changes that modify the peptide’s 190 stability and toxicity profiles are addressing these limitations. 191 192 Prophylactic vaccines 193 The increased prevalence of infections caused by A. baumannii, as well as the 194 identification of factors associated with higher risk for acquiring these infections, has 195 prompted research aimed at developing a prophylactic vaccine. Populations that would 196 potentially benefit from prophylactic vaccination for A. baumannii could include 197 patients at high risk for requiring mechanical ventilation, patients with extensive burn 198 injury, and members of the military at high risk for sustaining war-related trauma. The 199 ideal vaccine would target bacterial components that are highly conserved among 200 strains within the A. baumannii species. In addition, the targeted antigen(s) should be 201 highly expressed during infection and would be present on the bacterial surface to allow 202 for interaction between the antigen and the antibodies produced by vaccination. 203 The first reports describing the development of vaccines for A. baumannii were 204 based on formulations that incorporated multiple bacterial antigens into the vaccine. An 205 inactivated whole cell vaccine was highly immunogenic and elicited antibodies against 206 multiple bacterial antigens [46]. Tissue bacterial loads were approximately 5 log10 207 lower in vaccinated mice compared to control mice 12 hours post-infection, and serum 208 pro-inflammatory cytokine levels were lower in vaccinated mice. Importantly, the 209 inactivated whole cell vaccine was able to protect mice from infection with multiple A. 210 baumannii strains, including a pan-resistant clinical isolate. Similar results were 211 obtained in independent studies with vaccines based on A. baumannii outer membrane 212 vesicles and outer membrane fractions [47, 48]. Although these vaccines were highly 213 effective in mouse models, their use in humans may be complicated by the high levels 214 16 References 332 1 McConnell, M.J., et al. (2012) Acinetobacter baumannii: human infections, factors 333 contributing to pathogenesis and animal models. FEMS Microbiol Rev doi: 334 10.1111/j.1574-6976.2012.00344.x 335 2 Vila, J. and Pachón, J. (2008) Therapeutic options for Acinetobacter baumannii 336 infections. Expert Opin Pharmacother 9, 587-599 337 3 Wisplinghoff, H., et al. (2000) Nosocomial bloodstream infections caused by 338 Acinetobacter species in United States hospitals: clinical features, molecular 339 epidemiology, and antimicrobial susceptibility. Clin Infect Dis 31, 690-697 340 4 Chen, Y.H., et al. (2012) Trends in the susceptibility of clinically important resistant 341 bacteria to tigecycline: results from the Tigecycline In Vitro Surveillance in Taiwan 342 study, 2006 to 2010. Antimicrob Agents Chemother 56, 1452-1457 343 5 López-Rojas, R., et al. (2011) Acinetobacter baumannii resistant to colistin alters its 344 antibiotic resistance profile: a case report from Spain. J Infect Dis 204, 1147-1148 345 6 Rolain, J.M., et al. (2011) Acinetobacter baumannii resistant to colistin with impaired 346 virulence: a case report from France. J Infect Dis 204, 1146-1147 347 7 Taccone, F.S., et al. (2006) Successful treatment of septic shock due to pan-resistant 348 Acinetobacter baumannii using combined antimicrobial therapy including tigecycline. 349 Eur J Clin Microbiol Infect Dis 25, 257-260 350 8 Valencia, R., et al. (2009) Nosocomial outbreak of infection with pan-drug-resistant 351 Acinetobacter baumannii in a tertiary care university hospital. Infect Control Hosp 352 Epidemiol 30, 257-263 353 9 Haq, I.U., et al. (2012) Bacteriophages and their implications on future biotechnology: 354 a review. Virol J 9, 9 355 17 10 Lin, N.T., et al. (2010) Isolation and characterization of phi AB2: a novel 356 bacteriophage of Acinetobacter baumannii. Res Microbiol 161, 308-314 357 11 Yang, H., et al. (2012) Isolation and characterization of a virulent bacteriophage 358 AB1 of Acinetobacter baumannii. BMC Microbiol 10, 131 359 12 Jin, J., et al. (2012) Isolation and characterization of ZZ1, a novel lytic phage that 360 infects Acinetobacter baumannii clinical isolates. BMC Microbiol 12, 156 361 13 Lee, C.N., et al. (2011) Lytic myophage Abp53 encodes several proteins similar to 362 those encoded by host Acinetobacter baumannii and phage phiKO2. Appl Environ 363 Microbiol 77, 6755-6762 364 14 Popova, A.V., et al. (2012) Isolation and characterization of wide host range lytic 365 bacteriophage AP22 infecting Acinetobacter baumannii. FEMS Microbiol Lett 332, 40-366 46 367 15 Thawal, N.D., et al. (2012) Effect of a novel podophage AB7-IBB2 on 368 Acinetobacter baumannii biofilm. Curr Microbiol 65, 66-72 369 16 Yele, A.B., et al. (2012) Novel lytic bacteriophage AB7-IBB1 of Acinetobacter 370 baumannii: isolation, characterization and its effect on biofilm. Arch Virol 157, 1441-371 1450 372 17 Shen, G.H., et al. (2012) Isolation and Characterization of phikm18p, a Novel Lytic 373 Phage with Therapeutic Potential against Extensively Drug Resistant Acinetobacter 374 baumannii. PLoS One 7, e46537 375 18 Li, P., et al. (2012) Bioinformatic analysis of the Acinetobacter baumannii phage 376 AB1 genome. Gene 507, 125-134 377 19 Soothill, J.S. (1992) Treatment of experimental infections of mice with 378 bacteriophages. J Med Microbiol 37, 258-261 379 18 20 Espinal, P., et al. (2012) Effect of biofilm formation on the survival of Acinetobacter 380 baumannii on dry surfaces. J Hosp Infect 80, 56-60 381 21 Carlton, R.M. (1999) Phage therapy: past history and future prospects. Arch 382 Immunol Ther Exp (Warsz) 47, 267-274 383 22 Merril, C.R., et al. (1996) Long-circulating bacteriophage as antibacterial agents. 384 Proc Natl Acad Sci U S A 93, 3188-3192 385 23 Smith, H.W., et al. (1987) Factors influencing the survival and multiplication of 386 bacteriophages in calves and in their environment. J Gen Microbiol 133, 1127-1135 387 24 Thompson, M.G., et al. (2012) Antibacterial Activities of Iron Chelators against 388 Common Nosocomial Pathogens. Antimicrob Agents Chemother 56, 5419-5421 389 25 Neupane, G.P. and Kim, D.M. (2009) Comparison of the effects of deferasirox, 390 deferiprone, and deferoxamine on the growth and virulence of Vibrio vulnificus. 391 Transfusion 49, 1762-1769 392 26 Moreau-Marquis, S., et al. (2009) Tobramycin and FDA-approved iron chelators 393 eliminate Pseudomonas aeruginosa biofilms on cystic fibrosis cells. Am J Respir Cell 394 Mol Biol 41, 305-313 395 27 Ibrahim, A.S., et al. (2011) Combination therapy of murine mucormycosis or 396 aspergillosis with iron chelation, polyenes, and echinocandins. Antimicrob Agents 397 Chemother 55, 1768-1770 398 28 Ibrahim, A.S., et al. (2007) The iron chelator deferasirox protects mice from 399 mucormycosis through iron starvation. J Clin Invest 117, 2649-2657 400 29 Fothergill, J.L., et al. (2012) Novel therapeutic strategies to counter Pseudomonas 401 aeruginosa infections. Expert Rev Anti Infect Ther 10, 219-235 402 19 30 Fernandes, S.S., et al. (2010) Identification of a new hexadentate iron chelator 403 capable of restricting the intramacrophagic growth of Mycobacterium avium. Microbes 404 Infect 12, 287-294 405 31 Denny, B.J., et al. (2005) Antimicrobial activity of a series of 1-alkyl-2-(4-406 pyridyl)pyridinium bromides against Gram-Positive and Gram-Negative bacteria. Med 407 Princ Pract 14, 377-381 408 32 de Leseleuc, L., et al. (2012) In Vitro and In Vivo Biological Activities of Iron 409 Chelators and Gallium Nitrate against Acinetobacter baumannii. Antimicrob Agents 410 Chemother 56, 5397-5400 411 33 Fecteau, M.E., et al. (2012) Antimicrobial activity of gallium nitrate against 412 Mycobacterium avium subsp. paratuberculosis in neonatal calves. J Vet Intern Med 25, 413 1152-1155 414 34 Antunes, L.C., et al. (2012) In Vitro and In Vivo Antimicrobial Activities of Gallium 415 Nitrate against Multidrug-Resistant Acinetobacter baumannii. Antimicrob Agents 416 Chemother 56, 5961-5970 417 35 Fjell, C.D., et al. (2011) Designing antimicrobial peptides: form follows function. 418 Nat Rev Drug Discov 11, 37-51 419 36 Vila-Farres, X., et al. (2012) Update of peptides with antibacterial activity. Curr 420 Med Chem doi: 421 37 Giacometti, A., et al. (2003) Comparative activities of cecropin A, melittin, and 422 cecropin A-melittin peptide CA(1-7)M(2-9)NH2 against multidrug-resistant nosocomial 423 isolates of Acinetobacter baumannii. Peptides 24, 1315-1318 424 38 Rodríguez-Hernández, M.J., et al. (2006) Studies on the antimicrobial activity of 425 cecropin A-melittin hybrid peptides in colistin-resistant clinical isolates of 426 Acinetobacter baumannii. J Antimicrob Chemother 58, 95-100 427 20 39 López-Rojas, R., et al. (2011) Efficacy of cecropin A-melittin peptides on a sepsis 428 model of infection by pan-resistant Acinetobacter baumannii. Eur J Clin Microbiol 429 Infect Dis 30, 1391-1398 430 40 Conlon, J.M., et al. (2009) Antimicrobial properties of brevinin-2-related peptide 431 and its analogs: Efficacy against multidrug-resistant Acinetobacter baumannii. Chem 432 Biol Drug Des 74, 488-493 433 41 Conlon, J.M., et al. (2012) Efficacy of six frog skin-derived antimicrobial peptides 434 against colistin-resistant strains of the Acinetobacter baumannii group. Int J Antimicrob 435 Agents 39, 317-320 436 42 Conlon, J.M., et al. (2012) Analogues of the frog skin peptide alyteserin-2a with 437 enhanced antimicrobial activities against Gram-negative bacteria. J Pept Sci 18, 270-438 275 439 43 Ostorhazi, E., et al. (2011) Intramuscularly administered peptide A3-APO is 440 effective against carbapenem-resistant Acinetobacter baumannii in mouse models of 441 systemic infections. Biopolymers 96, 126-129 442 44 Ostorhazi, E., et al. (2011) Preclinical advantages of intramuscularly administered 443 peptide A3-APO over existing therapies in Acinetobacter baumannii wound infections. 444 J Antimicrob Chemother 65, 2416-2422 445 45 Lipsky, B.A., et al. (2008) Topical versus systemic antimicrobial therapy for treating 446 mildly infected diabetic foot ulcers: a randomized, controlled, double-blinded, 447 multicenter trial of pexiganan cream. Clin Infect Dis 47, 1537-1545 448 46 McConnell, M.J. and Pachón, J. (2010) Active and passive immunization against 449 Acinetobacter baumannii using an inactivated whole cell vaccine. Vaccine 29, 1-5 450 21 47 McConnell, M.J., et al. (2011) Vaccination with outer membrane complexes elicits 451 rapid protective immunity to multidrug-resistant Acinetobacter baumannii. Infect 452 Immun 79, 518-526 453 48 McConnell, M.J., et al. (2011) Outer membrane vesicles as an acellular vaccine 454 against Acinetobacter baumannii. Vaccine 29, 5705-5710 455 49 Fattahian, Y., et al. (2011) Protection against Acinetobacter baumannii infection via 456 its functional deprivation of biofilm associated protein (Bap). Microb Pathog 51, 402-457 406 458 50 Luo, G., et al. (2012) Active and passive immunization protects against lethal, 459 extreme drug resistant-Acinetobacter baumannii infection. PLoS One 7, e29446 460 51 Bentancor, L.V., et al. (2012)Evaluation of the Trimeric Autotransporter Ata as a 461 Vaccine Candidate against Acinetobacter baumannii Infections. Infect Immun 80, 3381-462 3388 463 52 Bentancor, L.V., et al. (2012) Poly-N-acetyl-beta-(1-6)-glucosamine is a target for 464 protective immunity against Acinetobacter baumannii infections. Infect Immun 80, 651-465 656 466 53 Ashkenazi, H., et al. (2003) Photodynamic effects of antioxidant substituted 467 porphyrin photosensitizers on gram-positive and -negative bacterial. Photochem 468 Photobiol 77, 186-191 469 54 Nitzan, Y., et al. (1998) Eradication of Acinetobacter baumannii by photosensitized 470 agents in vitro. J Photochem Photobiol B 42, 211-218 471 55 Carpenter, B.L., et al. (2012) Porphyrin-cellulose nanocrystals: a photobactericidal 472 material that exhibits broad spectrum antimicrobial activity. Photochem Photobiol 88, 473 527-536 474 22 56 Tsai, T., et al. (2011) Chitosan augments photodynamic inactivation of gram-475 positive and gram-negative bacteria. Antimicrob Agents Chemother 55, 1883-1890 476 57 Dai, T., et al. (2009) Photodynamic therapy for Acinetobacter baumannii burn 477 infections in mice. Antimicrob Agents Chemother 53, 3929-3934 478 58 Ragas, X., et al. (2010) Photodynamic inactivation of Acinetobacter baumannii 479 using phenothiazinium dyes: in vitro and in vivo studies. Lasers Surg Med 42, 384-390 480 59 Komerik, N., et al. (2000) The effect of photodynamic action on two virulence 481 factors of gram-negative bacteria. Photochem Photobiol 72, 676-680 482 60 Giuliani, F., et al. (2010) In vitro resistance selection studies of RLP068/Cl, a new 483 Zn(II) phthalocyanine suitable for antimicrobial photodynamic therapy. Antimicrob 484 Agents Chemother 54, 637-642 485 61 Sulemankhil, I., et al. (2012) Prevention and Treatment of Virulent Bacterial 486 Biofilms with an Enzymatic Nitric Oxide-Releasing Dressing. Antimicrob Agents 487 Chemother doi: 488 62 Moller, M.N., et al. (2007) Acceleration of nitric oxide autoxidation and nitrosation 489 by membranes. IUBMB Life 59, 243-248 490 63 Fang, F.C. (1997) Perspectives series: host/pathogen interactions. Mechanisms of 491 nitric oxide-related antimicrobial activity. J Clin Invest 99, 2818-2825 492 64 Fukuto, J.M., J. Y. Cho, and C. H. Switzer. (2000) The Chemical Properties of Nitric 493 Oxide and Related Nitrogen Oxides. In L. J. Ignarro (ed.), Nitric Oxide: Biology and 494 Pathology. Academic Press, San Diego, CA. 495 65 Mihu, M.R., et al. (2010) The use of nitric oxide releasing nanoparticles as a 496 treatment against Acinetobacter baumannii in wound infections. Virulence 1, 62-67 497 498 499 500 23 501 Table 1. Characterized lytic bacteriophages that infect A. baumannii clinical isolates 502 503 Phage Genome Genome size (kb) Tail Lysis of clinical isolates Reference AB1 AB2 Abp53 ZZ1 AB7-IBB1 AB7-IBB2 Km18p AP22 dsDNA dsDNA dsDNA dsDNA DNA ND dsDNA dsDNA 45-47 40 95 166 75 170 45 46 Long, non-contractile Short Contractile Contractile Long, non-contractile Short No tail Contractile 20% (1 of 5) 20% (25 of 125) 27% (7 of 26) 13% (3 of 23) 59% (23 of 39) 49% (19 of 39) 44% (15 of 34) 68% (89 of 130) 11 10 13 12 16 15 17 14 Abbreviations: dsDNA, double-stranded DNA; ND: not determined 504 505 506 24 Box 1. Outstanding questions 507 - Bacteriophages with lytic activity against clinical isolates of A. baumannii have shown 508 limited host range. Are there phages with broader host ranges that are yet to be 509 discovered? Can the use of ‘cocktails’ of phages overcome this limitation? 510 - What is the nature of immune response produced upon the administration of 511 bacteriophages and what side effects could be produced by these reactions? 512 - Can toxicity and stability issues that have been observed with antimicrobial peptides 513 be overcome using peptide derivatives (e.g. by substituting D-amino acids)? 514 - Which populations would benefit from prophylactic vaccination for A. baumannii and 515 when should they be vaccinated? 516 - Can A. baumannii develop resistance to photodynamic therapy and NO-based 517 treatment approaches?518 25 Figure Legends 519 Figure 1. Chemical structure of iron chelators. Chemical structures of the iron 520 chelators that have been tested for activity against A. baumannii. 521 522 Figure 2. Production of reactive oxygen species with photodynamic therapy. The 523 schematic represents the process by which photoactivation (ƒ) of photosensitizers (PS) 524 results in an excited state that reacts with oxygen either through type I or type II 525 reactions in order to produce reactive oxygen species (ROS) which induce cellular 526 damage. 527 528 Figure 3. Cell damage produced by nitric oxide-based therapies. The schematic 529 represents the mechanisms by which nitric oxide (NO) induces damage to cellular 530 membranes, proteins and DNA. 531 532 533