Therapeutic options Staphylococcus

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TherapeuticOptionsandEmergingAlternativesforMultidrugResistantStaphylococcalInfections

ARTICLE·MARCH2015

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2058 Current Pharmaceutical Design, 2015, 21, 2058-2072

Therapeutic Options and Emerging Alternatives for Multidrug Resistant Staphylo-coccal Infections

Maria Magana1, Anastasios Ioannidis

1,2, Emmanouil Magiorkinis

3, Oleg Ursu

4,5, Cristian G. Bologa

4,5,

Stylianos Chatzipanagiotou2, Michael R. Hamblin

6,7,8 and George P. Tegos*

7,8,9

1Department of Nursing, Faculty of Human Movement and Quality of Life Sciences, University of Peloponnese,

Sparta, Greece; 2Department of Clinical Microbiology, Athens Medical School, Aeginition Hospital, Athens,

Greece; 3Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens,

Athens-Goudi, Greece; 4Translational Informatics Division, Department of Internal Medicine, University of New

Mexico Health Sciences Center, Albuquerque, NM, USA; 5Center for Molecular Discovery, University of New

Mexico, Albuquerque, NM, USA; 6Harvard-MIT Division of Health Science and Technology, Cambridge, MA,

USA; 7Department of Dermatology, Harvard Medical School, Boston, MA, USA;

8Wellman Center for Pho-

tomedicine, Massachusetts General Hospital, Boston, MA, USA; 9Torrey Pines Institute for Molecular Studies

11350 SW Village Parkway, Port St. Lucie, FL 34987 USA

Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) remains the single biggest challenge in infectious disease in the civilized world. Moreover, vancomycin resistance is also spreading, leading to fears of untreatable infections as were

common in ancient times. Molecular microbiology and bioinformatics have revealed many of the mechanisms involved in resistance de-velopment. Mobile genetic elements, up-regulated virulence factors and multi-drug efflux pumps have been implicated. A range of ap-

proved antibiotics from the glycopeptide, lipopeptide, pleuromutilin, macrolide, oxazolidinone, lincosamide, aminoglycoside, tetracy-cline, steptogramin, and cephalosporin classes has been employed to treat MRSA infections. The upcoming pipeline of drugs for MRSA

includes some new compounds from the above classes, together with fluoroquinolones, antibacterial peptide mimetics, aminomethylci-clines, porphyrins, peptide deformylase inhibitors, oxadiazoles, and diaminopyrimidines. A range of non-drug alternative approaches has

emerged for MRSA treatment. Bacteriophage-therapy including purified lysins has made a comeback after being discovered in the 1930s. Quorum-sensing inhibitors are under investigation. Small molecule inhibitors of multi-drug efflux pumps may potentiate existing antibi-

otics. The relative failure of staphylococcal vaccines is being revisited by efforts with multi-valent vaccines and improved adjuvants. Photodynamic therapy uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species that can non-

specifically destroy bacteria while preserving host cells. Preparation of nanoparticles can kill bacteria themselves, as well as improve the delivery of anti-bacterial drugs. Anti-MRSA drug discovery remains an exciting field with great promise for the future.

Keywords: MRSA, multidrug resistance, staphylococcal infections, drug discovery.

1. INTRODUCTION: THE THERAPEUTIC CHALLENGE

Antibiotics have been a valuable weapon against bacterial in-fections for over a half century. However, the overuse and misuse of antimicrobials have contributed to the occurrence of multi-antibiotic resistance, a collection of phenomena that constitute one of the leading threats to public health with steadily increasing rates. As a result, each year over 13 million deaths worldwide can be attributed to the emergence of new infections or to the re-emergence of previously well-controlled infectious diseases.

Methicillin-resistant Staphylococcus aureus (MRSA) is a causa-tive agent of high mortality, prolonged hospitalization and increas-ing healthcare costs. MRSA infections account for up to 50% of both nosocomial and community-associated staphylococcal infec-tions [1, 2]. The term "superbugs", often applied to generally resis-tant pathogens, was originally coined to describe MRSA. MRSA incidence rates as well as the epidemiologically-related trends have been monitored very closely during the last decade. Despite the downward trend in mortality in several European countries, MRSA still remains a globally significant public health threat [3, 4]. In addition to MRSA, VRSA (vancomycin resistant S. aureus) and VISA (vancomycin intermediate S. aureus) strains also pose an important threat to second-line treatments for MRSA. The first

*Address correspondence to this author at the Torrey Pines Institute for

Molecular Studies, Port St. Lucie, FL, USA; Wellman Center for Pho-

tomedicine, Massachusetts General Hospital, Boston MA, USA; E-mail: [email protected]

report of VRSA in Europe was published this year from Portugal [5]. Resistance to linezolid and daptomycin has also been docu-mented [6, 7].

Colonization with S. aureus occurs in the anterior nares but it can also occur in the throat, axilla, respiratory tract, skin and soft tissues, perineum and rectum [8-11]. The transmission of S. aureus occurs by “person to person” contact as well as via fomites and colonized surfaces [12-15]. Since S. aureus naturally colonizes the human body, bacteria found to be involved in wound infections usually come from the endogenous flora [16]. Bacterial penetration into tissue is permitted by invasive procedures used in patient care [17, 18]. Foreign body-related infections account for a notable oc-currence rate of MRSA infections in healthcare settings [19]. Medi-cal devices such as catheters or artificial prostheses serve as the major causative factor for the incidence of infections in the major organs of the body that have particularly poor prognosis.

The prevalence of MRSA infections as well as their lethality is frequently attributed to virulence factors that can be classified as both secreted products and as structural features of the bacteria with specific roles in the infection process. The generally accepted belief is that MRSA strains are generated by the insertion of a mobile genetic element called the “Staphylococcal Cassette Chromo-some mec” (SCCmec) through horizontal gene transfer. The mecA gene encodes the penicillin-binding protein 2A (PBP2A) that has a decreased affinity for �-lactam antibiotics. SCCmec is flanked by cassette chromosome recombinase genes (ccrA/ccrB or ccrC) that permit intra- and inter-species transmission of the genetic element.

New Treatments for Resistant Staphylococci Current Pharmaceutical Design, 2015, Vol. 21, No. 16 2059

Resistance to vancomycin is mediated by the acquisition of the VanA phenotype from Enterococcus sp. strains through the Tn1546 transposon carrying the vanA gene [20].

Community-associated MRSA (CA-MRSA) is caused by strains that are genetically distinguished from hospital-associated MRSA (HA-MRSA). CA-MRSAs are more virulent than typical HA-MRSA probably due to the fact that many of them have been found to carry genes for Panton-Valentine Leukocidin (PVL) a virulence factor associated with MRSA infections [1, 21-23]. In CA-MRSA, mecA is carried within SCCmec type IV, which is dis-tinct from the SCCmec types I, II, and III that are typically found in HA-MRSA [24-26]. In fact, CA-MRSA strains have now been found to display enhanced virulence causing severe infections and they appear to spread faster than HA-MRSA [27].

A plethora of disease states are caused by MRSA and most of them are quite challenging to treat. MRSA is found to be among the most frequently identified pathogens causing pneumonia, and is associated with increased morbidity and mortality rates [28, 29], accounting for 20%–40% of all hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) [30-32]. Skin and soft tissue infections (SSTIs), such as diabetic MRSA wound infections, lead to increased costs, prolonged healing time and poor prognosis [33, 34]. MRSA infections may also be associated with persistent or recurrent bacteremia especially in long-term hemo-dialysis patients with renal disease [35-38]. Persistent MRSA bac-teremia is associated with infective endocarditis eventually leading to heart failure and even death [39-42]. Bone infections constitute another difficult-to-treat clinical entity, with diabetes and peripheral vascular disease predisposing patients to MRSA osteomyelitis [43-45]. Last but not the least, toxic shock syndrome, a life-threatening condition gathered the public attention because of its association with the use of tampons colonized with S. aureus during use by the female population [46].

2. CLINICALLY AVAILABLE TREATMENTS

The use of penicillin was rapidly associated with the spread of resistant S. aureus strains in both the hospital and community set-tings soon after its introduction in the 1940s [1]. Methicillin was introduced in 1961 to treat penicillin-resistant S. aureus isolates, but methicillin-resistant strains soon made their appearance through the acquisition and expression of a chromosomally encoded gene for PBP2A. Hyper-virulent staphylococcal strains have evolved resis-tance against the most renowned antibiotic classes commonly used in clinical practice. The diversity in the different mechanisms of resistance that have evolved results from the interplay between microbial physiology, the nature of the genetic elements, as well as the landscape in which those resistance mechanisms occur (Table

1). Especially, mutations and genetic rearrangement carried out by mobile genetic elements are found among the most common resis-

tance mechanisms of Staphylococcus. The US Food and Drug Ad-ministration (FDA) (Table 2) has approved a number of antibacte-rial agents to treat MRSA infections.

Vancomycin has been the gold standard for the treatment of MRSA infections for decades and still remains the first-line option for the clinical treatment of multiple diseases caused by MRSA. Failure rates of vancomycin treatment of staphylococcal bactere-mia, endocarditis and pneumonia are now emerging, raising ques-tions about the supremacy of vancomycin in the management of MRSA infections [45, 54-56]. MRSA with reduced vancomycin susceptibility often leads to VISA or heterogeneous VISA (hVISA) with documented worse clinical outcomes [56]. Teicoplanin is a semi-synthetic anti-MRSA drug which has been known since the 1980s. It has similar effects to vancomycin, yet is more cost-effective and less toxic. Initially approved in Europe, teicoplanin was subsequently approved in many other countries, but is not yet approved by the FDA due to its unusual pharmacokinetic properties [57].

Linezolid is a bacteriostatic synthetic drug against MRSA that also exhibits in vitro efficacy against VRSA and VISA [58]. Pa-tients treated with Linezolid are more likely to have better out-comes than those treated with vancomycin for MRSA pneumonia and especially for MRSA VAP; according to a variety of clinical trials this is most likely due to its enhanced ability to penetrate tis-sues [59]. Despite the fact that linezolid is a well-established antibi-otic for the treatment of serious MRSA infections, the reported cases of linezolid-resistant S. aureus worldwide raise concerns about its possible overuse in clinical practice [60].

Daptomycin exhibits bactericidal activity and reduced toxicity and is often used to treat multi-resistant pathogens [61]. Daptomy-cin was first approved by the US FDA in 2003 for the treatment of MRSA SSTIs and in 2006 for the treatment of bacteremia and right-sided endocarditis. It is not recommended for the treatment of respi-ratory infections due to its inactivation by pulmonary surfactant [58]. Daptomycin-resistant S. aureus strains have been reported due to failures in treatment. These failures may be either attributed to unsuccessful therapy with daptomycin or to inappropriate surgical intervention [62].

Topically administered mupirocin is a naturally occurring anti-biotic, introduced into clinical practice in 1985, but only two years after its introduction there was the first report of the isolation of mupirocin-resistant S. aureus strains [63, 64].

Fusidic acid is a bacteriostatic drug that has been approved for topical use worldwide to treat bacterial skin infections since the 1960s, but is not yet approved by the US FDA. Fusidic acid still remains a valuable treatment option for skin infections with a high therapeutic potential and low resistance rates [61, 65].

Table 1. S. aureus resistance mechanisms to antibiotics according to their genetic base.

Resistance Mechanism Genetic Basis Target-Phenotype References

�-lactamase production Plasmid-encoded bla genes penicillins [47]

Acquired low-affinity PBP2A mecA gene in SCCmec cassettes �-lactams (MRSA) [48]

Modified peptidoglycan target vanA and vanB genes on transposons glycopeptides (VRSA) [49]

Altered cell wall physiology Chromosomal mutations glycopeptides [50]

Ribosomal target modification G2576T mutation linezolid [51]

Ribosomal methylation at A2503 residue Plasmid-encoded cfr genes linezolid [52]

Amino acid substitutions Chromosomal mutations in the genes gyrA, grlA and gyrB fluoroquinolones [53]

2060 Current Pharmaceutical Design, 2015, Vol. 21, No. 16 Magana et al.

Table 2. Current therapeutic options for MRSA infections.

Drug Structure Route Activity

Range

Reg.

Status Class Target

Medical

conditions

treated

Daptomycin PO Gram-

positive

FDA

(2003) Lipopeptide

cell

wall

SSTIs

bacteremia

endocardi-

tis

Vancomycin IV/PO broad

spectrum

FDA

(1964) Glycopeptide

cell

wall

bacteremia

endocardi-

tis osteo-

myelitis

septic

arthritis

meningitis

Teicoplanin IV/IM Gram-

positive - Glycopeptide

cell

wall SSTIs

Retapamulin TD Gram-

positive

FDA

(2007) Pleuromutilin

protein

synthe-

sis

SSTIs

Telavancin IV Gram-

positive

FDA

(2009)

Lipoglycopept

ide/vancomy-

cin derivative

cell

wall

SSTIs

HAP

New Treatments for Resistant Staphylococci Current Pharmaceutical Design, 2015, Vol. 21, No. 16 2061

(Table 2) Contd….

Drug Structure Route Activity

Range

Reg.

Status Class Target

Medical

conditions

treated

Telithromycin PO broad

spectrum

FDA

(2004) Macrolide

protein

synthe-

sis

CAP

Oritavancin IV Gram-

positive

FDA

(2014) Glycopeptide

cell

wall SSTIs

Dalbavancin IV broad

spectrum

FDA

(2014) Glycopeptide

cell

wall SSTIs

Tedizolid

phosphate IV/PO

Gram-

positive

FDA

(2014)

Oxazolidi-

none

protein

synthe-

sis

SSTIs

2062 Current Pharmaceutical Design, 2015, Vol. 21, No. 16 Magana et al.

(Table 2) Contd….

Drug Structure Route Activity

Range

Reg.

Status Class Target

Medical

conditions

treated

Linezolid IV/PO broad

spectrum

FDA

(2000)

Oxazolidi-

none

protein

synthe-

sis

pneumo-

nia

meningitis

SSTIs

Clindamycin IV/PO/

TD

broad

spectrum

FDA

(1970) Lincosamide

protein

synthe-

sis

SSTIs

Sepsis

Necrotiz-

ing infec-

tions

Gentamicin TD broad

spectrum

FDA

(1964)

Aminoglyco-

side

protein

synthe-

sis

SSTIs

Bacitracin TD/IM broad

spectrum

FDA

(1973) Polypeptide

cell

wall SSTIs

Fusidic acid TD Gram-

positive -

Natural prod-

uct

protein

synthe-

sis

SSTIs

Mupirocin TD broad

spectrum

FDA

(1987)

Natural prod-

uct

protein

synthe-

sis

SSTIs

Ceftaroline

fosamil IV

broad

spectrum

FDA

(2010) Cephalosporin

cell

wall

SSTIs

CAP

New Treatments for Resistant Staphylococci Current Pharmaceutical Design, 2015, Vol. 21, No. 16 2063

(Table 2) Contd….

Drug Structure Route Activity

Range

Reg.

Status Class Target

Medical

conditions

treated

Doxycycline PO broad

spectrum

FDA

(1967) Tetracycline

protein

synthe-

sis

SSTIs

Minocycline PO broad

spectrum

FDA

(1971) Tetracycline

protein

synthe-

sis

SSTIs

Quinopristin/

Dalfopristin IV

Gram-

positive

FDA

(1999) Streptogramin

protein

synthe-

sis

SSTIs

Tigecycline IV broad

spectrum

FDA

(2005) Tetracycline

protein

synthe-

sis

SSTIs

CAP

Compli-

cated

intra-

abdominal

infections

Nadifloxacin TD broad

spectrum

FDA

(2009)

Fluoroquinolo

ne

DNA

synthe-

sis

SSTIs

(IV: intravenous, PO: per os, TD: transdermal, IM: intramuscular, SSTIs: skin and soft tissue infections, HAP: hospital-acquired pneumonia, CAP: community-acquired pneumonia)

Bacitracin is a complex of cyclic peptide antibiotics produced by the Tracy-I strain of Bacillus subtilis. The zinc salt of bacitracin is combined with other topical antibiotics (polymyxin B and neo-mycin) to form an ointment, that is used for topical treatment of a variety of localized skin and eye infections [66].

Fifth-generation cephalosporins (ceftobiprole and ceftaroline fosamil) have demonstrated extensive activity against Gram-positive bacteria. Ceftaroline fosamil was approved by the FDA in 2010 and by the European Medical Agency (EMA) in 2012 for the treatment of MRSA SSTIs and community-acquired pneumonia (CAP), and is the only cephalosporin active against hVISA and VRSA [65, 67]. On the other hand, ceftobiprole was rejected by the

FDA in 2008 and by EMA in 2010 due to invalid data provided by the clinical studies [67]. Especially, the FDA and EMA Good Clinical Practice (GCP) inspections discovered that there was a scarcity in monitoring the studies and in the study conduct [68].

Clindamycin has a primarily bacteriostatic effect and is useful for the treatment of a number of infections [58]. Inducible macrol-ide-lincosamide-streptogramin resistance caused by erythromycin limits its broad use for the treatment of empiric MRSA infection [69, 70]. Doxycycline and minocycline are broad-spectrum tetracy-clines with efficient oral bioavailability and tissue penetration. They present an efficient alternative for the treatment of non-invasive MRSA infections that do not require intravenous (IV) therapy [71-

2064 Current Pharmaceutical Design, 2015, Vol. 21, No. 16 Magana et al.

73]. Minocycline does not exhibit a good safety profile and for this reason it was added to the FDA Adverse Event Reporting System (AERS) in 2009 [74]. Tigecycline exhibits bacteriostatic efficacy against MRSA. It is structurally similar to the tetracyclines but not efficient when used for the treatment of HAP or VAP since it has demonstrated low therapeutic effects [75].

Quinupristin-dalfopristin is used for the treatment of life-threatening infections but it is rarely applied in clinical practice due to its poor safety profile and the availability of better treatment options [58].

Dalbavancin and oritavancin are semi-synthetic compounds with high efficacy against bacterial skin infections. Dalbavancin belongs to the first-line antibiotics for the treatment of diabetic foot infections compared to vancomycin, daptomycin and linezolid [76]. Oritavancin is a newly approved lipoglycopeptide with antibacterial activity [77]. Oritavancin exhibits bactericidal activity against an array of antibiotic resistant isolates of S. aureus including methicil-lin-susceptible S. aureus (MSSA), VRSA, VISA, daptomycin non-susceptible S. aureus and MRSA in planktonic and biofilm species [77]. Both dalbavancin and oritavancin are active against MRSA and have a long half-life thus requiring a shorter administration regimen which allows them to be a promising outpatient option [78]. Telavancin is another recently-approved antibiotic that exhib-its concentration-dependent bactericidal activity against MRSA. It is as active as vancomycin and other antistaphylococcal penicillins in the treatment of SSTIs and hospital-acquired pneumonia [79].

Nadifloxacin is a topical fluoroquinolone that acts by inhibiting DNA gyrase and it has been shown to be effective against a broad-spectrum of bacteria including MRSA and coagulase-negative staphylococci. It is approved for use in the treatment of acne vul-garis and other skin infections in Japan where it has been used for decades representing good safety and efficacy profiles [80].

3. THE PRECLINICAL PIPELINE

The increased mortality rates as well as the variety of estab-lished resistance mechanisms constitute a therapeutic challenge, and underline the urgent need for more effective alternatives for MRSA treatment. There is a wealth of preclinical leads against MRSA stemming from validated targets and a rapidly growing pipeline of molecules arising from preliminary investigations based on the re-positioning of underexplored or newly discovered targets and approaches. Table 3 summarizes lead molecules that represent advances in the preclinical pipeline for staphylococcal infections. Antimicrobial peptides (AMPs) and natural derivatives exhibit strong antistaphylococcal activity with unique and rather unex-plored characteristics that could make them potential therapeutic agents [81, 82].

Two lipopeptide molecules have a unique mode of action com-pared with any other approved lipopeptides. MX-2401 is a semi-synthetic analog of the naturally occurring lipopeptide, amphomy-cin [83]. MX-2401, unlike daptomycin, is not affected in vitro by the presence of lung surfactant and has shown to be active in vivo in a bronchial-alveolar pneumonia mouse model [84]. Tripropeptin

C (TPPC) exhibits high efficacy against antibiotic-resistant strains such as MRSA and VRE [85]. Drugs targeting small molecular substrates usually delay the emergence of resistant bacteria. TPPC inhibits enzymatic reactions through binding to substrates repre-senting a promising novel class of antibiotics [86].

Although the utility of tetracyclines has been reduced since the onset of bacterial resistance, two newly introduced agents of this class, eravacycline and omadacycline exhibit good oral bioavail-ability and activity which is not impaired by the lung surfactant [67]. Plazomicin is a next-generation aminoglycoside that has demonstrated in vitro synergistic activity with daptomycin or cefto-biprole against MSRA, VISA, hVISA and VRSA [87]. Fluoroqui-nolones are of particular interest as many antibiotics from this class

are currently in clinical trials. Delafloxacin eradicates quinolone-resistant or susceptible MRSA as it becomes highly potent at re-duced pH of tissue following inflammation [88]. JNJ-Q2 appears to be among the most promising new members of this class with low potential resistance and increased potency compared to other marketed fluoroquinoles [89].

Iclaprim is a broad-spectrum drug that is in Phase III clinical trials and was found to be effective against MRSA exhibiting high cure rates along with a well tolerated toxicity profile [90]. The EMA accepted for review a marketing authorization application for iclaprim in 2008 but, the Anti-infective Drugs Advisory Committee of the FDA requested additional clinical efficacy data [91].

4. EMERGING NON-ANTIBIOTIC APPROACHES

A number of preclinical studies have been conducted utilizing natural viral predators of bacteria, bacteriophages. The use of bacte-riophage (phage) therapy has been tested for the elimination of multidrug resistant infections. Special emphasis has been placed on a class of highly evolved molecules, the bacteriophage lytic en-zymes (lysins) that digest the bacterial cell wall. Small quantities of purified recombinant lysin were found to cause rapid and many log-fold lyses of specific Gram-positive bacteria [92]. After replicating within the bacterial host, the bacteriophage must exit the bacterial cells to disseminate. The lytic enzymes have been refined by evolu-tion to serve this purpose [93, 94]. Lysins target one of the four major peptidoglycan bonds, thus weakening the cell wall. They can function either as an endo-beta-N-acetylglucosaminidase or an N-acetylmuramidase with activities targeted against the sugar moiety of the bacterial wall, as an endopeptidase acting on the peptide moiety, or as an N-acetylmuramoyl-L-alanine amidase that hydro-lyzes the amide bond connecting the glycan strand and peptide moieties [93]. Although lysins have been well known since 1940s, the rapid industrial advancement of antibiotics shifted the focus away from them.

Emerging infections have triggered a thorough reexamination of phage therapy in Russia [95]. Phage-based therapies were devel-oped in the 1930s in Eastern Europe (Russia and Poland) to treat a vast array of pathogenic microbes [96]. The first FDA approval of phage therapy was granted in 2006, for a cocktail of six individu-ally purified phages to serve as a therapeutic alternative for meat and poultry products contaminated with Listeria monocytogenes. This was the first time FDA approved the use of a phage prepara-tion as a food additive [92]. Moreover, whole phage derived lysins have been evaluated for their potential therapeutic effect. Phage lysins have been used to control a wide range of pathogens in vitro such as Group A Streptococci, Streptococcus pneumoniae, Bacillus anthracis, Enterococcus faecalis and S. aureus [97-101]. Lysin PlyC is potent in killing logarithmically grown Streptococcus pyo-genes at nanomolar concentrations [102].

The bacteriolytic activity of lysins has been effective in control-ling multidrug resistant Gram-positive bacteria in animal studies, with a selective effect on mucosal surfaces and in blood, infections often without a demonstrated immune response [103] and most importantly with no evidence of lysin inactivation by blood con-stituents. Lysin Cpl-1 (a muramidase that binds to choline) for ex-ample, exhibited in vivo activity in a penicillin-resistant pneumo-coccal bacteremia mouse model [93, 104].

The key advantages of phages over antibiotics have been sum-marized including specificity for the pathogen without documented effects on the normal flora nor the microbiome, low possibilities for resistance development, and the unique ability to eradicate coloniz-ing pathogens on mucosal surfaces.

Quorum sensing, secretion systems, as well as cell adhesion pathways have been extensively validated as potential targets but with very few preclinical studies available [104-106]. Bacterial cell

New Treatments for Resistant Staphylococci Current Pharmaceutical Design, 2015, Vol. 21, No. 16 2065

Table 3. Promising compounds currently in clinical trials.

Name Structures Class Target/ MOA Reg. Status

Brilacidin

(PMX-30063)

1224095-98-0

Drug belonging to

the ‘defensin-

mimetics’ class of

antibiotics

cell membrane Phase II com-

pleted

TD-1792

(Theravance)

1393900-12-3

Chimeric molecule

from the linking of

vancomycin to a

cephalosporin

cell membrane Phase II

XF-73

718638-68-7 Porphyrin cell membrane Phase II

Radezolid

(RX1741)

869884-78-6

Oxazolidinone protein synthesis Phase II

Omadacycline

(PTK-0796)

389139-89-3

Aminomethylci-

cline protein synthesis Phase III

Eravacycline

(TP-434)

1207283-85-9

Aminomethylci-

cline protein synthesis Phase III

Plazomicin

(ACHN-490)

1154757-24-0

Aminoglycoside protein synthesis Phase III

GSK1322322

1152107-25-9

Peptide deformy-

lase inhibitor protein synthesis Phase II

2066 Current Pharmaceutical Design, 2015, Vol. 21, No. 16 Magana et al.

(Table 3) Contd….

Name Structures Class Target/ MOA Reg. Status

BB-83698

325795-25-3 Oxadiazole

Peptide de-

formylase in-

hibitors

Phase I

Delafloxacin

(RX3341)

189279-58-1

Fluoroquinolone DNA gyrase

inhibitor Phase III

Avarofloxacin

JNJ-Q2

878592-87-1

Fluoroquinolone DNA gyrase

inhibitor

Phase III/Fast

track

Iclaprim

192314-93-5 Diaminopyrimidine DHFR inhibitor withdrawn

surfaces are of utmost importance for bacterial integrity and viabil-ity. The pathways and the enzymes that catalyze wall teichoic acid (TarO, TarA) and lipoteichoic acid (LtaA, LtaS) synthesis have been identified and exploited as plausible discovery targets [107].

Advances in the field of host-directed therapeutic strategies could eventually lead to the elimination of infections caused by MRSA. The innate immune system constitutes a complex defense mechanism against various microbial threats and from this perspec-tive it has become a promising target for the development of thera-peutics to control such infections [108]. Antistaphylococcal vac-

cines and immunoglobulins have been reported to be effective in vitro; although, both active and passive immunizations have failed to show efficient results in human studies [105]. The complex pathogenic mechanisms of S. aureus represent the major challenge leading to the failure of passive immunization strategies [109]. Monovalent vaccines failed to express sufficient efficacy in humans due to the partial protection they offered. To date, the vaccines that have been so far tested in humans targeted only single antigens, and ultimately led to inefficient antibody and cytokine production. Ide-ally, an antistaphylococcal vaccine should protect against the wide range of S. aureus toxins and immune evasion factors. Antibody-mediated protection is a significant factor that is clearly affected by the complex staphylococcal evasion factors that hamper the im-mune response of the host. Components of the host’s immune sys-tem may play a significant role in the battle against resistance. Pep-tides, complement fragments as well as cells of humoral immunity

and their derivatives could very well be in practice in near future always considering the complexity inherent to host-pathogen inter-actions [108, 110]. Peptides and synthetic analogs may be useful adjuvants for an increase in antibody titers and their efficacy lead-ing to the activation of both cell-mediated (opsonophagocytosis) and also humoral immunity. The design of proper adjuvant formu-lations through a more efficient combination of different antigens, the elucidation of various staphylococcal factors that inhibit the host immune response and the identification of the particular vul-nerabilities of the pathogen could ultimately lead to the develop-ment of improved multivalent antistaphylococcal vaccines [111].

5. NEW APPROACHES

Staphyloccocal drug efflux systems are potentially attractive targets, but they have never been systematically explored, partially due to the fact that their role in the physiology of the pathogen is far more complicated than just a central resistance mechanism to an-timicrobials. Efflux systems are membrane transporter proteins with classes identified as effluxing tetracyclines and fluoroquinolones in S. aureus [112, 113]. Multi-antibiotic resistance is attributed to the chromosomally encoded Major Facilitator Superfamily (MFS) in-cluding NorA, NorB, NorC, MdeA, the mepRAB, Multidrug And Toxic compound Extrusion (MATE) family, and the SepA of the SMR (small multidrug resistance) Family [114, 115]. Plasmid en-coded systems such as the QacA, QacB and Tet(K) that function as tetracycline-divalent metal complex/H

+ antiporters are contributing

New Treatments for Resistant Staphylococci Current Pharmaceutical Design, 2015, Vol. 21, No. 16 2067

in antibiotic efflux [116, 117]. These systems have a broad and overlapping substrate specificity including quinolones, tetracy-clines, monovalent and divalent antimicrobial cations, as well as plant secondary metabolites [118]. Efflux inhibition is considered a viable strategy for enhancing antibiotic efficacy. The discovery of small molecules that block efflux systems is a rapidly expanding field. The efforts to discover these small molecules have yielded a number of natural and synthetic staphylococcal efflux modulators [119, 120]. Although the concept of enhancing the utility of antibi-otics by employing efflux inhibitors appears appealing, it remains challenging in terms of clinical implementation [121, 122].

Photodynamic therapy (PDT) is a light-based technology platform, which uses harmless visible light in combination with non-toxic photosensitizers (PS) to control infections. Historically, PDT has had a prominent role in the cure of many serious diseases like cancer, producing reactive oxygen species (ROS) that non-specifically kill cells. This mechanism is the reason PDT is cur-rently being investigated as an alternative therapeutic approach for localized infections as resistance to ROS is unlikely to develop [123, 124]. Most often, the molecules tested as antimicrobial PSs are organic, aromatic dyes with a high degree of electron delocali-zation (porphyrins, halogenated xanthenes, chlorins, bacteriochlo-rins, phthalocyanines, perylenequinones, cationic fullerenes and psoralens) [125, 126]. The molecules should be designed to have affinity to bacterial cells and not to host cells, and methods to de-liver light to all parts of the body via fiberoptics have now been designed. Phenothiazinium dyes (methylene blue (MB), toluidine blue (TBO)) are the only clinically approved antimicrobial PS [127]. Phenothiazenium PSs are physicochemically similar to the natural product, the antibacterial alkaloid berberine, a well-characterized substrate of the NorA efflux system in S. aureus and other Gram-positive bacteria [118, 128]. It has been demonstrated that phenothiazinium PSs are substrates of microbial efflux systems and small molecule efflux pump inhibitors can enhance their photo-toxic effect in a range of pathogens including S. aureus [129]. This synergistic discovery platform along with the therapeutic efficacy of visible light, without the addition of exogenous PS, are under investigation for preclinical development in localized staphylococ-cal infections [126].

With the advent of the nanotechnology revolution, antistaphylo-coccal nanotechnology-based therapeutic applications are being intensively explored with an emphasis on the discovery of novel antimicrobial nano-structures or employing nanoparticles for deliv-ery purposes [130-132]. Nanotechnology by definition refers to the design, development and application of materials in the nano-scale range (< 100nm). The physicochemical properties of nanoparticles (small size and high volume-to-surface ratio) allow them to gain access to a variety of biologic structures and systems. Nanosized materials are quite amenable to manipulation of their size, shape, and chemical characteristics. This feature offers multiple opportuni-ties to engineer nanoparticles as vehicles for therapeutic or diagnos-tic agents in medical applications, including targeted drug delivery, gene therapy and cell labeling [133-136]. Nanoparticles have been deployed in a variety of localized staphylococcal infections and are under investigation for delivering antibiotics, new experimental therapeutics with promising in vitro MRSA efficacy profiles [137-141].

6. BIOINFORMATICS AS A TOOL FOR DRUG DISCOV-ERY IN S. AUREUS

Microbiology research in the post-genomic era has inevitably involved bioinformatics (genomics and proteomics) as a tool to investigate the physiology of microbes, micro and macro-evolution, dynamics of mutations and adaptation within different host popula-tions. As sequencing techniques (ST) mature, fuller microbial ge-nomes will become available and the need for the development of powerful bioinformatics tools for the analysis of the growing data is

imperative. As of November 2014, a total of 4,193 entries (full or draft genomes) regarding S. aureus were available at the GenBank database [142]. Applying MLST (Multi Locus Sequence Typing), a total of 2,832 ST sequences have already been identified for S. aureus which are grouped in multiple clonal complexes (CC), un-derlying the extreme genetic diversity for this pathogen [143]. The study of MRSA genomics in silico offers valuable insights into the development of novel therapeutic interventions.

One research direction involves the study of transmission and molecular tracing of various strains –molecular epidemiology of MRSA strains- within human or animal populations or between human and animals. Transmission of such strains from animals to humans may be associated with novel virulence genes and homo-logues [144, 145]. Therefore, the study of MRSA molecular epide-miology has a direct impact on the design of novel drugs since it offers an insight into the diversity of existing therapeutic targets. Studies pertaining to the genetic sequence of specific MRSA strains (particularly USA300) have given insight into the origin, the trans-mission route and networks of this clone in the US delineating its diversity and spread [146-148]. Other studies have pointed out the existence of MRSA strains within animals whereas extensive re-search has been conducted using other STs and CC in different geographic areas [149-162]. Shepheard et al. analyzing strains from human and animals showed a total of 15 historical switching events and at least two S. aureus CC (CC25 and 29) which have arisen from animal-associated ancestors [163].

Another intervention concerns the identification of novel viru-lence genes in MRSA. Phylogenetics have been applied in the iden-tification of novel virulence genes and toxins as well the exchange of genetic material between different bacterial species leading to the development of novel phenotypes in MRSA [145, 164, 165]. Study-ing the diversity of known virulence factors as well as the identifi-cation of conserved regions may provide a pipeline of potential novel drug targets [166-172]. Likewise, the study of the interaction between MRSA and hosts (animal or human) could help in pharma-ceutical design [173-175]. A study designed to identify novel tar-gets for the development of drugs specific to MRSA, employed a genome-wide scan for balancing selection in S. aureus strains iden-tifying ninety nine candidate genes at sixty two different genomic loci [176]. An alternative approach for identification of novel therapeutic targets involves subtractive genome analysis. A recent study on MRSA ST398 and MRSA 252 revealed six and twenty-one possible therapeutic targets, respectively, for future drug devel-opment [177].

Overall, the employment of bioinformatics may give insight into the evolution and molecular epidemiology of MRSA, as well as the identification of novel therapeutic targets for drug discovery.

7. SYNOPSIS AND PERSPECTIVES

The treatment of resistant MRSA strains is complicated both in systemic and Acute Bacterial Skin and Skin Structure Infections (ABSSSI). The clinical need for alternative therapeutic interven-tions targeting multi-antibiotic resistant pathogens remains high as: (a) the evolutionary adaptability of staphylococcal strains to antibi-otic pressure has been proven to be a successful survival strategy for the pathogen [178]; (b) the potent current major systemic anti-biotics (linezolid, daptomycin, vancomycin) and topical mupirocin have all been found to have at least some (higher or lower) inci-dence of resistance or non-susceptibility [178]; (c) the use of con-ventional broad-spectrum antibiotics is a risk factor for elevated resistance in healthcare facilities [179]; (d) the recent FDA ap-proval of oxazolidinones for ABSSSI [180] as well as the com-pounds in the preclinical development pipeline are promising but may provide only a temporary answer; (e) the economic burden of treatment persists [181].

The empirical use of antibiotics in patients with S. aureus infec-tions should be limited in clinical settings. Monitoring of patient

2068 Current Pharmaceutical Design, 2015, Vol. 21, No. 16 Magana et al.

compliance and susceptibility testing of the isolates to antibiotics used in current therapeutics should be taken into consideration in order to guide treatment.

CONFLICT OF INTEREST

This article content has no conflict of interest.

ACKNOWLEDGEMENTS

George P Tegos was supported by the US NIH grant 5U54MH084690-02 (CDP1) and by the US DTRA contract HDTRA1-13-C-0005 and Michael R Hamblin was supported by US NIH grant R01AI050875.

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Received: January 21, 2015 Accepted: March 5, 2015