Targeting the hard to reach: challenges and novel strategies in the treatment of intracellular bacterial infections

Abstract

Infectious diseases continue to threaten human and animal health and welfare globally, impacting millions of lives and causing substantial economic loss. The use of antibacterials has been only partially successful in reducing disease impact. Bacterial cells are inherently resilient, and the therapy challenge is increased by the development of antibacterial resistance, the formation of biofilms and the ability of certain clinically important pathogens to invade and localize within host cells. Invasion into host cells provides protection from both antibacterials and the host immune system. Poor delivery of antibacterials into host cells causes inadequate bacterial clearance, resulting in chronic and unresolved infections. In this review, we discuss the challenges associated with existing antibacterial therapies with a focus on intracellular pathogens. We consider the requirements for successful treatment of intracellular infections and novel platforms currently under development. Finally, we discuss novel strategies to improve drug penetration into host cells. As an example, we discuss our recent demonstration that the cell penetrating cationic polymer polyhexamethylene biguanide has antibacterial activity against intracellular Staphylococcus aureus.

Linked Articles

This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro‐organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc

Abbreviations

AMP

antimicrobial peptides

AS‐ODN

antisense oligonucleotides

CPP

cell penetrating peptides

MRSA

methicillin‐resistant Staphylococcus aureus

PHMB

polyhexamethylene biguanide

Pip

piperazine

PMO

phosphorodiamidate morpholino oligomer

PNA

peptide nucleic acid

Tables of Links

TARGETS
P‐glycoprotein (ABCB1)

LIGANDS
Azithromycin	Gentamicin	Penicillin G
Cathelicidin LL‐37	Lipopolysaccharide (LPS)	Peptidoglycan
Doxycycline	Nitric oxide (NO)	Rifampicin

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016), and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (Alexander et al., 2015).

Antibacterial resistance and the challenge of infectious disease

Infectious disease remains a major threat to both human and animal populations. Approximately 15 million human deaths were due to infectious diseases during 2010, and the World Health Organization forecasts that this figure will fall only marginally by 2050 (Dye, 2014). In animals, infectious diseases continue to affect the health and welfare of livestock, resulting in threats to food security. Infectious diseases in animals not only causes huge economic losses but also increase the risk of possible transmission of zoonotic disease to the human population (Tomley and Shirley, 2009).

The discovery of penicillin as an antibacterial in the early 20th century revolutionized treatment for infectious diseases caused by bacteria (Fleming, 1929). Soon after, chloramphenicol, streptomycin and several other antibiotics provided further therapy options (Aminov, 2010). While the discovery and development of antibacterials improved infectious disease control, the triumph of antibacterial therapy has been short‐lived. Increasing consumption of antibacterials to treat illnesses, the use of antibacterials as growth promoters in livestock and their largely un‐controlled release into nature, has resulted in the development of resistance (Tomley and Shirley, 2009). The World Economic Forum recently stated that antibacterial resistance is the greatest risk to human health (Howell, 2013). Many infections are now difficult to treat, resulting in high dose administration of antibacterials, in‐tolerable toxicity and delays in effective treatment (WHO, 2012). It has been estimated that infections by antibacterial resistant pathogens claim a total of 700 000 lives every year globally, with 10 million projected deaths in the year 2050 (O'Neill, 2014).

The impact of antibacterial resistance is also important within animal health. In livestock, a high prevalence of β‐lactam resistant Staphylococcus aureus (S. aureus), one of the pathogens responsible for bovine mastitis, has made existing therapies less effective, prolonging the disease and increasing the costs of treatment (Barkema et al., 2006). Also, in companion animals, the emergence of methicillin‐resistant Staphylococcus pseudointermedius, the causative agent of skin, ear and wound infections is a new challenge for veterinary medicine (van Duijkeren et al., 2011). Animals are recognized as an important source of pathogen transmission to humans, and there are recent examples of antimicrobial resistance trait transmission from animal to human populations (Woolhouse et al., 2015).

Bacteria acquire resistance to antibiotics through spontaneous mutation and the acquisition of resistance traits. A classic example of this is the acquisition of β‐lactamases, which are hydrolytic enzymes that break down β‐lactam antibiotics rendering them ineffective. However, the acquisition of resistance traits is only one of the contributing factors in treatment failure. For an antibacterial to be effective in the clinic, it should be able to reach both the bacteria and its molecular targets at effective concentrations that are not toxic to the host. It has been recognized for some time that infections with Gram‐negative bacteria can be difficult to treat, because the outer membrane provides a barrier against the diffusion of antibacterials. At the population level, the ability of bacterial communities to form biofilms also provides barriers to drug penetration. These three dimensional multicellular aggregates are inherently resistant to antibacterials. The formation of biofilms by S. aureus on medical devices, such as artificial joints or catheters, and Pseudomonas aeruginosa (P. aeruginosa) on the surfaces of infected sites can bring additional hurdles to existing therapies (McConoughey et al., 2014; Winstanley et al., 2016). For example, in the case of bovine mastitis, biofilm formation by S. aureus on the mammary gland reduces the effectiveness of therapies, creating persistent infections (Melchior, 2011). The importance of antimicrobial resistance and biofilm structures has been extensively reviewed previously (Abee et al., 2011). In this review, we focus on another significant challenge to successful therapy: the problem of antibacterials gaining access to bacteria residing within host cells.

Intracellular bacteria represent hard to reach targets

Certain species of bacteria are able to localize inside host cells, followed by multiplication and modulation of the host cell biology. In this way, these bacteria create a niche, from which they can continue the infection cycle (Silva and Silva, 2013). This group of bacteria, known as intracellular bacteria, can also manipulate the host immune system to permit dissemination to different sites of the body. The classical examples of intracellular bacteria are Mycobacterium tuberculosis (M. tuberculosis), Salmonella enterica (S. enterica), Chlamydia trachomatis and Listeria monocytogenes (Armstrong and Hart, 1971; Gaillard et al., 1987; Kumar et al., 2006; Ibarra and Steele‐Mortimer, 2009). Additionally, evidence suggests that some classical extracellular bacteria, such as S. aureus, Escherichia coli (E. coli) and P. aeruginosa also have the ability to invade and localize inside host cells (Angus et al., 2008; Garzoni and Kelley, 2009; Dikshit et al., 2015). Table 1 provides a list of intracellular bacteria and their associated disease. Below, we discuss the mechanisms of invasion used by three clinically important pathogens, S. enterica, M. tuberculosis and S. aureus.

Table 1.

Summary of diseases associated with intracellular bacteria

Bacteria	Associated disease	Host cells	Localization inside host cells	Reference
Salmonella enterica	Typhoid and paratyphoid	Macrophages	Modified phagosomea	Gorvel and Méresse, 2001, Brumell et al., 2002
Mycobacterium tuberculosis	Tuberculosis	Macrophages	Phagosome, cytosol	Armstrong and Hart, 1971; Rohde et al., 2012, Watson et al., 2012
Chlamydia species	Ocular and genital infections	Conjunctiva and genital epithelial cells	Vacuoleb	Kumar et al., 2006
Listeria monocytogenes	Listeriosis	Epithelial cells	Cytosol	Gaillard et al., 1987
Staphyloccocus aureus	Skin infections, mastitis, osteomyelitis	Keratinocytes, bovine mammary epithelial cells, osteoblast	Endosome, cytosol	Brouillette et al., 2003 Fraunholz and Sinha, 2012
Escherichia coli	Urinary tract infections, mastitis.	Bladder epithelial cells, mammary epithelial cells	Vacuole	Dikshit et al., 2015

^a Modified phagosome also known as Salmonella containing vacuole.

^b Vacuole also known as inclusion.

Salmonella enterica

Host infections start when S. enterica is ingested. On reaching the gastrointestinal tract, S. enterica can induce its own uptake into specialized epithelial cells, M cells, that cover Peyer's patches of the intestine (Jensen et al., 1998). The bacteria injects effector proteins into the host cell, triggering membrane ruffling and actin rearrangement from inside the cells, leading to bacterial internalization (Patel and Galán, 2005). Internalization into M cells allows the bacteria to cross the intestinal barrier. The bacteria are then engulfed by macrophages and reside in a phagosome called the Salmonella containing vacuole. While inside vacuoles, S. enterica secrete effector proteins that can prevent fusion of the phagosome with a lysosome, therefore avoiding lysosomal activities within macrophages (Gorvel and Méresse, 2001). Other evidence suggests that S. enterica can also escape into the cytosol (Brumell et al., 2002). Migration of infected macrophages can further disseminate bacteria into other organs, such as the liver and spleen (Monack et al., 2004).

Mycobacterium tuberculosis

Transmission of M. tuberculosis occurs via inhalation of droplets containing the bacilli. Once the pathogen reaches the lung airways, bacteria are phagocytosed by alveolar macrophages. Numerous studies show that M. tuberculosis can evade killing processes in macrophages by arresting phagosome fusion with the lysosome, thereby establishing a survival niche within macrophages where replication occurs (Armstrong and Hart, 1971; Rohde et al., 2012). However, more recent studies demonstrate that certain M. tuberculosis strains can escape into the cytosol, by permeabilizing the phagosome membrane (Watson et al., 2012; Peng et al., 2016). Although there is growing evidence that suggests M. tuberculosis has a cytosolic phase, localization of the bacteria inside the vacuole is still thought to be critical for bacterial survival (Russell, 2011). Strategies that target both niches are thought to be equally important in for optimal therapy, and both are difficult to achieve in practice.

Staphylococcus aureus

S. aureus is a Gram‐positive pathogen that can cause various disease conditions including complicated skin infections (Dryden, 2010) and bloodstream infections in hospitalized patients (hospital acquired infections) (Burton et al., 2009). In animals, S. aureus is one of the main pathogens that causes mastitis, a disease manifested by inflammation of the udder (Jamali et al., 2014). S. aureus was historically known as an extracellular bacterium. However, accumulating evidence suggests that S. aureus can invade and survive in various host cells including keratinocytes, endothelial cells, epithelial cells, fibroblast, osteoblasts and bovine mammary epithelial cells (Hébert et al., 2000; Mempel et al., 2002; Sinha and Hermann, 2005; Garzoni et al., 2007; Reott et al., 2008; Hanses et al., 2011).

S. aureus invades host cells through a zipper uptake mechanism involving adhesion to the host cell surface (Fraunholz and Sinha, 2012). Attachment leads to signal transmission that results in cytoskeletal rearrangement, allowing movement of S. aureus into host cells (Sinha et al., 1999; Ahmed et al., 2001; Edwards et al., 2011). Once inside host cells, S. aureus can either survive and replicate within the acidic phagolysosome (Brouillette et al., 2003) or escape from the phagosome into the cytosol (Fraunholz and Sinha, 2012). S. aureus invasion can induce cell death, allowing the bacteria to escape and start a new cycle of infection, subsequently entering the blood stream to cause septicaemia (Soong et al., 2012).

Challenges in the treatment of intracellular bacterial infections

The problem of antibacterial delivery to bacteria within host cells is of paramount importance. Some of these bacteria are responsible for our most devastating diseases. For example, M. tuberculosis, the causative agent of tuberculosis, causes approximately 1.5 million deaths a year and is the second leading cause of death due to a single infectious agent (Lewandowski and co‐investigator, 2015). Species belonging to the Salmonella group are important foodborne pathogens responsible for enteric diseases and cause over one billion infections annually (Buckle et al., 2012). Moreover, methicillin‐resistant S. aureus (MRSA) is responsible for 20% of mortality due to the bloodstream infections in the hospital‐acquired setting (Thomer et al., 2016). Therefore, the effective delivery of antibacterials into host cells containing these pathogens is a critical goal of novel antibacterial therapies.

The delivery of antibacterials into desired locations in the body is one of the main challenges for successful therapeutics. Depending on the routes of uptake and the location of the infections, antibacterials may need to cross the epithelial cells of the gastrointestinal tract to reach the bloodstream (oral antibacterial), the thick stratum corneum for skin infections (topical antibacterial) or the mucosa for respiratory tract infections (pulmonary antibacterial). When bacteria reside intracellularly, antibacterials face another challenge; they need to cross the host cell membrane(s) either through diffusion or endocytosis. Localization of bacteria inside host cells provides protection. Although there are multiple antibacterial options available for treatment, more than two thirds are ineffective against intracellular pathogens (Abed and Couvreur, 2014).

The plasma membrane of mammalian cells is composed of a lipid bilayer embedded with peripheral and integral proteins. The membrane is impermeable to most polar or charged solutes. Small (< 700 Da in size) lipophilic antibacterials such as β lactams, macrolides and quinolones enter mammalian cells via diffusion across the lipid bilayer (Tulkens, 1991). Uptake via endocytosis may occur when a compound is large or does not readily diffuse across the membrane. Endocytosis involves internalization of molecules bound within vesicles from the membrane, followed by invagination of the vesicles into host cells. Once taken up by the host cells, the vesicles are directed to the endosomal route, where acidification takes place. Certain compounds such as cationic amphiphilic peptides can trigger endosome rupture and release of vesicle contents into the cytosol (Varkouhi et al. 2011).

For certain antibiotics, the host cell entry route has been well characterized. For example, aminoglycoside antibiotics are known to enter host cells via endocytosis. They bind to megalin, the endocytic receptor abundantly expressed in the renal proximal tubule that promotes uptake into the host cells (Nagai and Takano, 2004). Because of the specificity of aminoglycoside towards megalin, accumulation in the kidney can cause nephrotoxicity in patients (Nagai and Takano, 2004).

Once inside host cells, to be effective, antibacterials must be retained and accumulate at sufficient concentrations for a sufficient period. Although macrolides and quinolones enter the host cells via diffusion, they are then depleted by host P‐glycoprotein efflux pumps (Seral et al., 2003). For compounds entering host cell via endocytosis, if the compound remains in the endosome, it may be exported out from the host cell via the exocytosis route. Therefore, drugs that enter host cells may subsequently be removed by efflux or exocytosis, and thus unable to reach the pathogen.

In addition to penetration and retention inside host cells, antibacterials must also reach the sub‐cellular compartment that contains target bacteria. As discussed previously, intracellular bacteria can reside within intracellular compartments or the cytosol. Therefore, antibacterials must penetrate the specific compartment where bacteria reside. The specific intracellular location (vesicle or cytosol) may bring additional challenges to treatment. Certain bacteria, such as Salmonella, localize and replicate in acidified phagosomes where the pH is in between 4.0 and 5.0 (Rathman and Sjaastad, 1996). In such cases, antibacterials must also resist pH insult (Lemaire et al., 2011).

To survive the stress of the host cell environment, intracellular bacteria may transform to a non‐replicating or slowly replicating state (Grant and Hung, 2014). For example, S. enterica can change into a state of non‐replicating persistence inside macrophages (Helaine et al., 2014), and S. aureus can change into small colony variants inside epithelial cells (Vesga et al., 1996). M. tuberculosis enters a non‐replicating state within the host to cause latent infections that are resistant to conventional treatment (Wayne and Sohaskey, 2001). Such physiological changes reduce susceptibility to antibacterials (Nguyen et al., 2009). Therefore, to be able to clear intracellular infections by non‐replicating bacteria, antibacterials must be effective against both replicating and non‐replicating states. Figure 1 illustrates bacterial localization inside the host cells and cellular barriers to antibacterial access intracellular targets.

Figure 1.

Illustration of bacterial localization inside the host cells and cellular barriers to antibacterial access intracellular targets. Following host cell entry, bacteria are typically enclosed inside endosomes. Bacteria can continue to survive and replicate inside endosomes as they mature into phagosomes or phagolysosomes. Certain bacteria can also induce endosome rupture and enter the cytosol. Red arrows depict three major barriers that limit antibacterial access to intracellular bacteria: (1) the plasma membrane of host cells, (2) the phagosomal/phagolysosomal membrane and (3) the bacterial cell wall. For a more detailed description of the composition of the mammalian and bacterial plasma membranes, see Silhavy et al. (2010), Marquardt et al. (2015) and Simons and Sampaio (2016).

The potency of existing therapies against intracellular pathogens

Quinolones are often considered to be the best choice for treatment of intracellular infections. They have potent effects against a range of Gram‐positive and Gram‐negative bacteria and Mycobacteria (Dalhoff, 1999; Jacobs, 1999; Van Bambeke et al., 2005). They enter and accumulate in mammalian cells (Tulkens, 1991) and diffuse across subcellular compartments (Carlier et al., 1990). Although quinolones have been shown to be more effective against intracellular bacteria, compared with other classes of antibacterials (Carryn et al., 2003), their potency against bacteria that are located intracellularly is still much lower relative to their potency against extracellular bacteria (Seral et al., 2005).

Derivatives of tetracycline, such as tigecycline, have also shown efficacy against intracellular bacteria. This antibiotic has potent activities against a range of Gram‐positive and Gram‐ negative bacteria (Peterson, 2008). Tang et al. (2011) demonstrated bactericidal activities of tigecycline at 0.5 mgL⁻¹ against intracellular S. Typhimurium in peripheral blood mononuclear cells. In contrast, another study found that tigecycline at 1 mgL⁻¹ only displayed bacteriostatic activities against intracellular S. aureus in polymorphonuclear neutrophils (Ong et al., 2005). These observations provide examples of how intracellular localization influences the activity of antibacterials.

Existing antibiotics can be improved by increasing their ability to penetrate host cells. Barcia‐Macay et al. (2006) made a comparison between vancomycin and televancin (a hydrophobic derivative of vancomycin), against intracellular S. aureus in macrophages. Televancin displayed bactericidal activities against intracellular S. aureus within 6 h of treatment, while vancomycin required 24 h to demonstrate the same efficacy (Barcia‐Macay et al., 2006). The reduced efficacy of existing antibiotics has driven the need to improve existing therapies. Four examples of platforms that can potentially improve the outcome and provide a better solution for intracellular infections are discussed below.

Novel promising therapies in the treatment of intracellular infections

Antimicrobial peptides

Antimicrobial peptides (AMP) are chains of amino acids produced by living organisms as part of the host's innate immunity (Zasloff, 2002). They are expressed on the primary barriers of organisms such as the skin or the mucosal epithelial cells (Guan‐Guerra et al., 2010). AMPs display potent antimicrobial activities against bacteria, viruses and fungi. The antibacterial activities of AMPs often involve potent membrane disruption and pore formation, causing leakage of the cellular contents. Additionally, AMPs may enter and interact with intracellular molecules within bacteria, thereby inhibiting DNA, RNA and protein synthesis (Peters et al., 2010).

Certain AMPs are amphiphilic (contain hydrophilic and hydrophobic regions). This property facilitates AMPs penetration into mammalian cells. A number of studies have shown their promise as a potential therapy for intracellular infections. One example of an AMP that can enter mammalian cells is cathelicidin LL‐37, which is naturally expressed by the human skin. Noore et al. (2013) demonstrated that LL‐37 was effective against intracellular S. aureus in osteoblasts. Temporin, an AMP isolated from frog skin was found to be bactericidal against intracellular methicillin‐sensitive S. aureus and MRSA in keratinocytes, and promoted wound healing by stimulating keratinocyte migration (Di Grazia et al., 2014). Another study found that the equine α‐helical antimicrobial peptide eCATH1 killed Rhodococcus equi in macrophages (Schlusselhuber et al., 2013). Brinch et al. (2010) demonstrated that plectasin, an AMP derived from the pezizalean fungus Pseudoplactenia nigrella was effective against intracellular S. aureus in human and mouse monocytes.

Antisense oligonucleotide based technologies

Antisense oligonucleotide (AS‐ODN)‐based technology is a strategy designed to control gene expression at the RNA level. AS‐ODNs are short oligomers of nucleic acids or nucleic acid mimics; consisting of typically 10–30 residues that are complimentary to the target mRNA of interest. Hybridization of AS‐ODN to the target mRNA can inhibit translation, resulting in repression of gene expression (Sahu et al., 2007). Phosphorothioate, peptide nucleic acid (PNA), locked nucleic acid and phosphorodiamidate morpholino oligomer (PMO), are among the most studied AS‐ODNs (Chan et al., 2006). To improve delivery into bacterial or mammalian cells, AS‐ODNs are often attached to cell penetrating peptides (CPP) (Nekhotiaeva et al., 2003).

For antibacterial purposes, AS‐ODNs are designed to target genes essential for the survival of the bacteria (Good, 2002). In this way, antisense technology serves to silence the expression of targeted genes. Antisense PNAs and PMOs have been shown to effectively inhibit bacterial growth in vitro and in vivo. Good et al. (2001) demonstrated bactericidal activity of a peptide‐PNA conjugate targeted to the acyl‐carrier protein (acp), an essential gene involved in fatty acid biosynthesis in E. coli. Similarly, Tilley et al. (2007) showed that CPP conjugated PMO targeted to the acp gene reduced bacteraemia and promoted survival of mice infected with E. coli.

For the potential of AS‐ODNs to be realized in the clinic, the challenge of delivery across both bacterial and mammalian membranes must be overcome. Ma et al. (2014) showed that electroporation improved the delivery of a peptide‐PNA targeting bacterial RNA polymerase and killed intracellular S. Typhimurium. Also, Mitev et al. (2009) introduced piperazine (Pip) linkages between bases of PMO, to introduce cationic charges to the peptide‐PMO, to further enhance its delivery into mammalian cells. The Pip‐peptide‐PMO showed potent efficacy against intracellular S. Typhimurium and killed >99% of the bacteria inside macrophages (Mitev et al., 2009). These studies suggest that antisense technology, with further improvements in delivery technologies, will represent a promising strategy against intracellular bacteria.

Nanoparticles

Nanoparticles are nano‐scale materials derived from metallic, metal oxide, semiconductors, polymers or carbon‐based materials (Hajipour et al., 2012). Nanoparticles have long been applied in the material sciences field. Also, large number of FDA approved nanomedicines demonstrates their utility in a range of therapeutic areas (Bawa, 2011). Certain nanoparticles display potent antibacterial activities and may help to potentiate small molecule antibiotics. For example, Azam et al. (2012) demonstrated the antibacterial activities of zinc oxide, cuprum oxide and ferum oxide nanoparticles against Gram‐positive and Gram‐negative pathogens.

Nanoparticles display antibacterial activities through various mechanisms. For example, the cationic charges of titanium and aluminium oxide nanoparticles promote their adsorption onto bacterial surfaces, resulting in destabilization of the membrane, leading to cellular leakage (Ruparelia et al., 2008; Pal et al., 2015). Silver nanoparticles can produce free radicals that can cause lipid peroxidation of the membrane, resulting in loss of respiratory activities (Allahverdiyev et al., 2011). Zinc nanoparticles internalized by bacteria can induce production of ROS, resulting in ROS‐mediated cell damage (Zhao and Drlica, 2014; Patra et al., 2015).

Although nanoparticles are very large structures relative to drug molecules, they are able to improve cell entry properties. This effect is being exploited in several therapeutic areas, aiming to improve the intracellular delivery and cell type targeting of biomolecules or drugs. Nanoparticles are thought to enter mammalian cells through phagocytosis or the pinocytosis pathways (Oh and Ji‐Ho, 2014). This activity makes nanoparticles useful weapons in the fight against intracellular bacteria. Pati et al. (2014) demonstrated zinc oxide uptake by macrophages. Zinc oxide induced ROS and nitric oxide production in the cells and subsequently killed intracellular Mycobacterium smegmatis.

Certain nanoparticles such as liposomes, polymeric nanoparticles, solid lipid nanoparticles and dendrimers can be tailored to display desired charge or composition for combination with other biomolecules; for example, drugs, antibodies, proteins and oligonucleotides. The nanoparticle surfaces can also be decorated with material that is responsive to certain stimuli (e.g pH or temperature) allowing for controllable drug release in a specific place, for example in the acidified endosome (Xu et al., 2006). Therefore, together with the ability to enter mammalian cells, the nanoparticle platform can be utilized to improve the delivery of existing antibiotics into host cells (Zhang et al., 2010). A number of studies have investigated the ability of nanoparticles to potentiate antibiotic activities against intracellular bacteria, and these are listed in Table 2. The efficacies of penicillin, gentamicin and tetracycline against intracellular S. aureus (Ranjan et al., 2009; Di Meo et al., 2012; Maya et al., 2012), rifampicin and isoniazid against intracellular M. tuberculosis (Clemens et al., 2012), streptomycin and doxycycline against intracellular of Brucella melitensis (Seleem et al., 2009) and rifampicin and azithromycin against intracellular Chlamydia trachomatis (Toti et al., 2011), have all been markedly improved over the free drug through nanoparticle‐mediated delivery.

Table 2.

Summary of studies showing the promise of nanoparticles in the improvement of therapies against intracellular infections

Host cell/ organs	Bacteria	Nanoparticles platform	Antibiotic	Outcome	Reference
Macrophages	Staphylococcus aureus	Squanelene	Penicillin G (PenG)	Nanoparticle (NP)‐Pen G killed 87%, and free PenG killed 56%	Semiramoth et al. 2012
THP‐1 and HEK‐ 293 cells	Staphylococcus aureus	Chitosan	Tetracycline (Tet)	NP‐Tet killed ~97% in THP‐1 and ~95% in HEK 293 Free Tet killed ~83% in THP‐1 and ~85% in HEK 293	Maya et al., 2012
Macrophages	Mycobacterium tuberculosis	Polyethylenimine coating mesoporous siica	Rifampicin (Rif)	NP‐Rif reduced 3.3 log, and free Rif reduced 1.6 log	Clemens et al., 2012
Lung epithelial Hep2 cells	Chlamydia trachomatis	Poly d‐L‐lactide‐co‐glycolide polymer	Rifampicin (Rif) or azithromycin (Azi)	NP‐ Rif reduced 40%, and free Rif reduced 20% NP‐Azi reduced 40%, and free Azi showed no reduction	Toti et al., 2011
Murine Spleen and liver	Brucella melitensis	Poly ethylene oxide‐b‐sodium acrylate (PEO‐b‐PAA‐+Na) and poly sodium acrylate (PAA‐+Na) co polymers	Streptomycin (Strep) and doxycycline (Dox)	NP‐Strep‐Dox reduced 0.72 log in spleen and 0.79 in liver Free Strep‐Dox reduced 0.51 log in spleen and 0.42 log in liver	Seleem et al. 2009
Murine Spleen and liver	S. Typhimurium	PAA‐ +Na‐b‐PEO‐b‐PPO‐b‐PEO‐b‐PAA‐+Na block copolymers	Gentamicin (Gent)	NP‐Gent reduced 0.29 log in spleen and 1.07 log in liver Gent only reduced 0.23 log in liver but not in spleen (0.34 increase)	Ranjan et al., 2009

Recent advances with a polymeric biocide

Polyhexamethylene biguanide (PHMB) is a cationic polymer composed of repeating biguanide groups linked by hexamethylene chains (Figure 2). PHMB alone is a potent topical antimicrobial against Gram‐positive and Gram‐negative bacteria (Gilbert and Moore, 2005), fungi (Messick et al., 1999; Hiti, 2002), parasites and viruses (Romanowski et al., 2013). It has been widely used in the clinic, food industries and for domestic applications (Gilbert and Moore, 2005). PHMB applications include as an antiseptic, impregnation of wound dressings (Moore and Gray, 2007), water treatment (Kusnetsov et al., 1997), mouthwash and disinfection in contact lenses (Hiti, 2002). Although PHMB has been used for over 40 years, there are no reports of acquired bacterial resistance towards this compound (Gilbert and Moore, 2005).

Figure 2.

The structure of PHMB. PHMB is a cationic polymer of repeating hexamethylene biguanide groups, with n average = 10–12 (n is the number of structural unit repeats) and MW 3025 g·mol⁻¹. Reprinted from Kamaruzzaman et al., 2016.

PHMB's antibacterial activities involve the interaction of biguanide groups with the cytoplasmic membrane, lipopolysaccharide and peptidoglycan of the bacterial cell wall. This binding is believed to displace the divalent cation Ca^{2 +}, causing membrane destabilization and cellular leakages (Gilbert and Moore, 2005). Also, the hexamethylene segment can interact with phospholipids on the membrane, causing a phase separation that disturbs random distribution of lipids, further destabilizing the membrane structure (Broxton et al., 1984). Recent findings in our laboratory demonstrated that PHMB enter bacteria cells, and this leads to chromosome condensation (Chindera et al., 2016). Therefore, PHMB appears to have at least two mechanisms of action, and this may help to explain why acquired antibacterial resistance to PHMB has not yet been reported.

PHMB is able to enter a range of cell types, including many mammalian cells (Firdessa et al., 2015, Chindera et al., 2016). Moreover, following mammalian cell entry, PHMB co‐localizes with intracellular MRSA inside the host cells (Kamaruzzaman et al., 2016). Following treatment of infected cells, PHMB causes a marked reduction in survival of intracellular pathogens inside the host cells (Figure 3). These findings show that PHMB has potential value to be further developed as a novel therapy for intracellular infections.

Figure 3.

Intracellular localization and bactericidal activities of nadifloxacin and PHMB against intracellular MRSA. (A) Colocalization of PHMB‐FITC with epidemic methicillin‐resistant S. aureus (EMRSA)‐15 in keratinocytes. Keratinocytes were infected with EMRSA‐15 followed by treatment with PHMB‐FITC (green). Keratinocytes were labelled with DAPI (blue) for keratinocytes and EMRSA‐15 nuclei staining and wheat germ agglutination (WGA) (red) for keratinocyte membrane stain. Upper panels are images of infected cells and merged images. Lower panels are enlarged images that clearly show colocalization between PHMB‐FITC (green) and EMRSA‐15 (blue). White scale bar is 25 μm. (B) Survival of EMRSA‐15 within keratinocytes after treatment with nadifloxacin and PHMB. Keratinocytes infected with strains of EMRSA‐15 were either untreated or treated with increasing concentrations of nadifloxacin or PHMB. Untreated cultures were used to establish colony forming unit values corresponding to 100% survival. Reprinted from Kamaruzzaman et al., 2016.

Summary and outlook

The current problem of antibacterial resistance threatens our ability to treat and control infections. Intracellular bacteria, which are generally harder to reach than extracellular bacteria, may not be resistant to antibacterials in the conventional sense, yet they represent a population of bacteria that are particularly difficult to treat, resulting in frequent treatment failures and limited treatment options. The difficulty of reaching intracellular bacteria was recognized over 50 years ago (Holmes et al., 1966), and in this review, we have discussed the challenges of antibacterial therapy for intracellular infections.

While intracellular bacteria will remain a serious challenge to infection control, as we better understand intracellular infections, it should be possible to develop better‐targeted therapies. There are a number of new technologies that offer promise over conventional treatments. AMPs and AS‐ONDs have the potential to increase the choice of treatments and offer the advantage that acquired resistance to these compounds may be infrequent (Guilhelmelli et al., 2013). However, such technologies still face challenges in gaining entry into host cells. Perhaps the most promising advances will be found in the nanoparticle arena and the application of cationic polymer, where several studies have shown that both platforms can improve antibacterial efficacy against bacteria residing within host cells.

Author contributions

N.F.K., S.K. and L.G. contributed to the writing and editing of this article.

Conflict of interest

The authors declare no conflicts of interest.

Kamaruzzaman, N. F. , Kendall, S. , and Good, L. (2017) Targeting the hard to reach: challenges and novel strategies in the treatment of intracellular bacterial infections. British Journal of Pharmacology, 174: 2225–2236. doi: 10.1111/bph.13664.