Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review

Wonder drug penicillin started the era of antibiotics in 1928 and since then it has tremendously developed modern medicine. Persistent use of antibiotics, self-medication, and exposure to infections in hospitals has provoked the emergence of multidrug resistant (MDR) bacteria responsible for 15.5% Hospital Acquired Infection (HAIs) in the world (Rice, 2008 ; Allegranzi et al., 2011 ; Ibrahim et al., 2012 ; Pendleton et al., 2013 ). The term “ESKAPE” encompasses six such pathogens with growing multidrug resistance and virulence: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa , and Enterobacter spp. (Rice, 2008 ). ESKAPE pathogens are responsible for majority of nosocomial infections and are capable of “escaping” the biocidal action of antimicrobial agents (Rice, 2008 ; Navidinia, 2016 ). A systematic review of clinical and economic impact of antibiotic resistance reveals that, ESKAPE pathogens are associated with the highest risk of mortality thereby resulting in increased health care costs (Founou et al., 2017 ). World Health Organization (WHO) has also recently listed ESKAPE pathogens in the list of 12 bacteria against which new antibiotics are urgently needed (Tacconelli et al., 2018 ). They describe three categories of pathogens namely critical, high and medium priority, according to the urgency of need for new antibiotics. Carbapenem resistant A. baumannii and P. aeruginosa along with extended spectrum β-lactamase (ESBL) or carbapenem resistant K. pneumoniae and Enterobacter spp. are listed in the critical priority list of pathogens; whereas, vancomycin resistant E. faecium (VRE) and methicillin and vancomycin resistant S. aureus (MRSA and VRSA) are in the list of high priority group. The mechanisms of multidrug resistance exhibited by ESKAPE are broadly grouped into three categories namely, drug inactivation commonly by an irreversible cleavage catalyzed by an enzyme, modification of the target site where the antibiotic may bind, reduced accumulation of drug either due to reduced permeability or by increased efflux of the drug (Santajit and Indrawattana, 2016 ). They are also able to form biofilms that physically prevent the immune response cells of host as well as antibiotics to inhibit the pathogen. Moreover, biofilms protect specialized dormant cells called persister cells that are tolerant to antibiotics which cause difficult-to-treat recalcitrant infections (Lewis, 2007 ). The general antimicrobial therapy to effectively treat infections involves the use of antibiotics either singly or in combination. With every passing year, the overall number of antibiotics effective against ESKAPE is declining, which is predisposing us toward a future with antibiotics that are ineffective. Analysis of the antibiotic lists recommended in the Clinical & Laboratory Standards Institute (CLSI) guidelines revealed that many antibiotics suggested against ESKAPE since 2010 have been deleted with addition of relatively few antibiotics/antibiotic combinations. Furthermore, there are incidences of resistance reported against some of these newly added antibiotics ( Table 1 ). It is, therefore, imperative to find alternative ways to treat infections especially those caused by ESKAPE pathogens. Table 1 Antibiotics added/revised and eliminated against ESKAPE from CLSI document M100 since 2010. Antibiotics added/with revised breakpoint Tested against pathogen Ef S K A P E Amikacin, Amoxicillin-clavulanate, Ampicillin-sulbactam, Cefaclor, Cefamandole, Cefdinir, Cefmetazole, Cefonicid, Cefotetan, Cefpodoxime, Cefprozil, Cefuroxime, Kanamycin, Loracarbef, Netilmicin, Oxacillin, Tobramycin 0 Aztreonam 1 1 Cefazolin, Cefepime, Ceftazidime 0 1 1 Cefoperazone, Moxalactam 0 0 Cefotaxime, Ceftizoxime, Ceftriaxone 0 1 0 1 Ceftaroline 1 1 1 Ceftazidime-avibactam, Ceftolozane-tazobactam 1 1 Cephalothin 0 0 0 Colistin, Piperacillin 1 Dalbavancin, Telavancin, Oritavancin, Tedizolid 1 1 Doripenem, Imipenem, Meropenem 0 1 1 1 1 Ertapenem 0 1 1 1 Mezlocillin 0 Nalidixic acid 0 0 Piperacillin-tazobactam, Ticarcillin-clavulanate 0 1 Ticarcillin 0 0 0 0 Please note that the table includes ONLY those antibiotics which are deleted or newly added since 2010 . = Antibiotics deleted from CLSI guidelines between 2010 and 2018; 1, New antibiotics added in the CLSI guidelines since 2010 ; = No resistance reported till date ; = Resistance reported; Ef, E. faecium; S, S. aureus; K, K. pneumoniae; A, A. baumannii; P, P. aeruginosa; E= Enterobacter spp . S = (Long et al., 2014 ; Chan et al., 2015 ; Nigo et al., 2017 ); K = (Zowawi et al., 2015 ; Vuotto et al., 2017 ; Stanley et al., 2018 ); A = (Göttig et al., 2014 ; Goic-Barisic et al., 2017 ; Nowak et al., 2017 ; Caio et al., 2018 ; Chuang and Ratnayake, 2018 ); P = (Prakash et al., 2014 ; Gill et al., 2016 ; Alipour et al., 2017 ; Mohapatra et al., 2018 ; Palavutitotai et al., 2018 ) ; E = (Lee et al., 2015 ; Babouee Flury et al., 20

Phages are century old therapeutic agents that were used for the treatment of bacterial infections. The discovery of antibiotics was an influential factor in side-lining this ambition (Mann, 2005 ). The focus on phage therapy has sharpened ever since antimicrobial resistance has been on a dramatic rise. Lytic phages against ESKAPE pathogens have been isolated from hospital wastewater, making them easily available therapeutic agents (Latz et al., 2016 ). Bacteriophages used for therapy present many advantages such as high host specificity (prevent damage to normal flora, do not infect the eukaryotic cells), low dosages for treatment, rapid proliferation inside the host bacteria, etc. that make them ideal candidates to treat bacterial infections (Domingo-Calap and Delgado-Martínez, 2018 ). Unlike antibiotics, the advantage of using phages is that, they develop new infectivity and regain an upper hand over bacteria as they mutate alongside their host (Pirnay et al., 2018 ). Several studies carried out in vitro have proven phages to be effective as antibacterial agents against biofilm and planktonic cells of ESKAPE (Pallavali et al., 2017 ; Dvořáčková et al., 2018 ; Jamal et al., 2019 ). Table 2 gives information of phage therapy studied in various animal models as well as recent case studies and case reports of patients infected with ESKAPE pathogens. Phage therapy carried out in animal wound infection models have shown reduced mortality and enhanced wound healing. Additional studies carried out in vivo have also demonstrated efficacy and safety (non-toxic with reduced inflammatory responses) of phages used in treatment of bacterial infections. Phage therapy, though promising, comes with some limitations. It can, however, be overcome by appropriate modifications (Wittebole et al., 2014 ; El-Shibiny and El-Sahhar, 2017 ; Domingo-Calap and Delgado-Martínez, 2018 ). High specificity of the phages can be considered as both advantageous and a limiting factor. Monophage therapy involves the need to check the efficacy of the phage by testing it in vitro against the disease-causing bacteria before applying it to a patient which can be a difficult process. The use of phage cocktails, comprising of a combination of phages acting against different bacterial species or strains, can avoid these problems (Chan et al., 2013 ). International experts believe that an ideal phage cocktail should be prepared using phages belonging to different families or groups such as having broad host range, high adsorption ability to the highly conserved cell wall structures in bacteria. Using such phage cocktails may reduce the emergence of phage resistant bacterial population. However, others advocate strategies wherein individual active phages are applied sequentially to the patient. In clinical practice, however, it appears to be difficult (Rohde et al., 2018 ). Genomic characterization of phages is very important so as to predict their “safety” in therapeutic applications as demonstrated by several experts in this field. Phages can be vectors for horizontal gene transfer in bacteria, sometimes being involved in exchange of virulence or antibiotic resistance genes making a microbe more pathogenic or resistant to an antibiotic (Chen and Novick, 2009 ). Phages reported for therapeutic applications should not harbor virulence or antibiotic resistance genes as well as integrases, site-specific recombinases, and repressors of the lytic cycle that may accelerate the transfer/integration of these genes in the host bacterial genome. Algorithms that can be used for predicting lifestyle of a phage, and its virulent traits are available but their database needs to be updated with more genome sequences of phages (Mcnair et al., 2012 ). Two recent reviews excellently describe the work flow to select ideal phage candidates for therapeutic purposes (Casey et al., 2018 ; Philipson et al., 2018 ). Recent studies demonstrating in vivo efficacy of phages against ESKAPE infections have used fully characterized phages that show no virulence or antibiotic resistance genes, are considered safe as they do not exhibit any allergic or immune response, and are also reported to remain stable at varied pH and temperature which make them ideal candidates for therapy (Fish et al., 2016 ; Kishor et al., 2016 ; Wang et al., 2016 ; Zhou et al., 2018 ). Similarly, it has also been reported that the bacterial strains used for phage production should ideally be free of functional prophages. These prophages may get induced and contaminate the phage preparation. However, a recent report discusses the risk benefit evaluation that needs to be done in highly experimental treatments of patients infected with MDR pathogens such as ESKAPE (Rohde et al., 2018 ). Another limitation reported is the stability of phages and their proper administration in order to reach the site of action. Phage formulations are ingested orally, administered nasally or applied topically (Malik et al., 201

Antimicrobial light therapy, either alone or combined with a photosensitizer (PS), results in a photooxidative stress response that leads to microbial death. Excitation of PS with light of an appropriate wavelength leads to formation of an excited triplet state. An excited PS can transfer electrons or energy to biomolecules or molecular oxygen, resulting in the formation of reactive oxygen species (ROS) or singlet oxygen radicals, which are toxic to cellular targets such as nucleic acids, proteins and lipids (Mai et al., 2017 ; Yang M.-Y. et al., 2018 ). Some of the most frequently used PSs include phenothiazinium derivatives (methylene blue, toluidine blue), xanthine derivatives (rose bengal), porphyrin, chlorin, or fullerene derivatives amongst many others (Abrahamse and Hamblin, 2016 ; Cieplik et al., 2018 ). Antimicrobial photodynamic therapy is widely used for treating dental, skin, and soft tissue infections. For a more detailed description of the current state and future prospects of light therapy with respect to the various photosensitisers, light sources, and methods used, mechanism of antimicrobial action or antibiofilm potential, the reader may be referred to the excellent reviews published recently (Cieplik et al., 2018 ; Hu et al., 2018 ; Tomb et al., 2018 ; Wozniak and Grinholc, 2018 ). However, none of these reviews have especially focused on in vivo studies of aPDT against ESKAPE pathogens. There has been extensive research on designing the PSs so as to improve their pharmaceutical potential. An ideal PS used for antimicrobial therapy should have greater permeability to cross the microbial cell wall/cell membrane, selective toxicity toward the microbial cell with minimal or no damage to the host tissue and an absorption coefficient appropriate for effective penetration at the site of action. The PS chosen should not have a long half-life which causes prolonged photosensitization in the host cells even after the infection is cured. It should also not be effluxed out by the microbial efflux systems (Cieplik et al., 2018 ; Hu et al., 2018 ; Tomb et al., 2018 ; Wozniak and Grinholc, 2018 ). Efficacy of aPDT also depends on the light fluence, PS concentration and treatment time (Tomb et al., 2017 ; Sueoka et al., 2018 ; Ullah et al., 2018 ). PSs chosen preferably have a large absorption coefficient in the visible spectrum, especially in the long wavelength (red near infrared) region, to allow effective penetration of light in the infected tissue ( Table 2 ). Many researchers have attempted to improve the availability of PS by potentiating or functionalizing it with other molecules including galactose, amino acids, efflux pump inhibitors, potassium iodide, EDTA etc. A variety of PSs functionalized with addends are used to target ESKAPE pathogens. A boron-dipyrrolemethene (BODIPY)-based polygalactose, named pGEMA-I (7.3 kDa) with increased water solubility was used to demonstrate antibacterial and antibiofilm activity against P. aeruginosa , without much affecting the viability of normal cells. It was demonstrated that the selective recognition of the pathogen was due its carbohydrate binding lectin protein (LecA) which interacted with the galactose moiety of the PS (Zhao et al., 2018 ). C60-fullerene (LC16) bearing deca-quaternary chain and deca-tertiary-amino groups facilitates electron-transfer reactions via the photoexcited fullerene for antimicrobial effect studied in A. baumannii and S. aureus (Huang et al., 2014 ; Zhang et al., 2015 ). Another drawback of aPDT is that the ROS generation may cease after the light irradiation is turned off thus allowing un-killed bacteria to re-grow. Potentiating aPDT with potassium iodide allows the formation of iodine/tri-iodide that may remain active in the wound for a longer duration sufficient enough to prevent bacterial re-growth (Zhang et al., 2015 ; Wen et al., 2017 ). In vitro studies have shown that blue light (aBL) has a broad spectrum antibacterial and antibiofilm activity against all six ESKAPE members (Halstead et al., 2016 ). In vivo data also corroborated this finding and further confirmed that using a low penetrating blue light of 415 ± 10 nm should be a preferred choice of treatment in case of topical wound infections as it causes minimal damage to the uninfected tissue cells below (Amin et al., 2016 ; Wang et al., 2017 ; Katayama et al., 2018 ). Some studies additionally report that an exogenous PS may not be required (Amin et al., 2016 ; Wang et al., 2017 ). Their finding was supported by experimental data showing that the endogenous porphyrins present in the bacterial cell membrane play a role in triggering the photoxidative response (Amin et al., 2016 ). aBL using 5-aminolevulinic acid with disodium EDTA (ALA-EDTA/2Na) had antibacterial and antibiofilm potential thus showing significant wound healing of P. aeruginosa infected cutaneous ulcers in mice model (Katayama et al., 2018 ). However, the role of EDTA in increasing the antibacterial

There is an urgent need to restock our armamentarium of antimicrobials in order to stay ahead of the ever rising drug resistant ESKAPE pathogens. There is an insufficiency of effective antibiotic combinations in addition to the dry pipeline of new drugs. Huge efforts have been taken to use antibiotics in combination with adjuvants targeting important metabolic mechanisms/pathways contributing to drug resistance (permeablisers, lactamase inhibitors, efflux pump inhibitors, quorum sensing inhibitors, toxin inhibitors etc.) The modest success received to date with such antibiotic-adjuvant combinations has paved way to explore other alternative strategies to combat drug resistance. There is a significant rise in the interest shown by the scientific community to use novel therapeutic agents such as phages, antimicrobial peptides, metal nanoparticles, and photodynamic light which, although, have some limitations as discussed above. Some of the commonly described limitations of these therapies include stability and toxicity of the therapeutic agent, its targeted delivery at the site of infection, or immune response developed by the host against the therapeutic agent. Ongoing research has therefore led to further develop or modify these novel therapeutic agents or therapies so as to surmount the limitations as well as to overcome the barriers of bacterial resistance. This review summarizes studies that demonstrate potential alternative therapies using in vivo models some of which have extended further to the level of clinical trials. The interest in phage therapy to treat bacterial infections is fast growing leading to development of commercial preparations such as “Stafal,” “Sextaphage,” “PhagoBioDerm,” and “Pyophage” against MDR pathogens. Similarly, use of silver nanoparticles as antibiofilm coatings in surgical implants, antimicrobial agents in topical applications or as formulations in wound dressings has shown promising activities in animal models. Clinical trials using commercially available nanosilver coated dressings (Acticoat TM , Aquacel R ) or catheters (AgTive) is another noteworthy advancement. AMPs have received great attention due to their broad spectrum activity; however, they have shown limited pharmaceutical potential due to their toxicity, stability, and production costs. Photodynamic light therapy which is widely used for cancer therapy has also been demonstrated to be an effective strategy for clearing wound infections. However, additional studies demonstrating the efficacy and safety of these therapeutic agents against ESKAPE infections are desired. Similarly, randomized clinical trials would enable these therapeutic agents to cross the regulatory hurdles and find application in clinical practice. It was observed that, majority of these studies have used animal models infected by S. aureus, A. baumannii , and P. aeruginosa to test the efficacy of the therapeutic agent. The probable reason for this could be that these pathogens mostly cause topical infections (wound, burn and abscess) and because majority of the limitations (targeted delivery, stability, immune response, toxicity etc.) described for each therapy can be minimized though not avoided in such models. It would be important to study the effect of these therapeutic agents against systemic infections caused by ESKAPE members. It was also observed that, the methods used for estimating efficacy of any therapeutic agent were not uniform. Table 2 reveals that, the in vivo efficacy of various therapies is given either in terms of log or percent reduction of microbial load or as percent survival of the infected host (animal model). The methods followed to estimate the reduced pathogen loads as well as dosages used for treatment also vary. It is therefore not appropriate to compare these studies to identify the best therapeutic agent/therapy against any ESKAPE member. In addition to the growing concern in searching and evaluating the clinical potential of the above discussed alternative therapies, research on combinatorial approach, based on the synergistic action of two or more therapies is also gaining attention. Most commonly studied combinations involve use of a therapeutic agent/ therapy (phage, aPDT, AMP, or AgNP) in combination with antibiotic/s or in some cases with an efflux pump inhibitor or quorum sensing inhibitor. Another interesting option used was the combination of the therapeutic agents in a conjugate or hybrid (antibiotic-antibiotic, antibiotic-EPI, PS-AMP, PS-EPI etc.) for an increased efficacy against the pathogen. Most of these studies demonstrated that the combinatorial approach helped overcome the limitation caused by individual therapeutic agent. For example, an antibiotic combined with an efflux pump inhibitor or a photosensitizer conjugated with an AMP improved their entry and retention into the target pathogen for enhanced antimicrobial action. Similarly, combinations of antibiotics with nanoparticles or AMPS reduc