Natural Products for the Treatment of Chlamydiaceae Infections

Due to the global prevalence of Chlamydiae, exploring studies of diverse antichlamydial compounds is important in the development of effective treatment strategies and global infectious disease management. Chlamydiaceae is the most widely known bacterial family of the Chlamydiae order. Among the species in the family Chlamydiaceae, Chlamydia trachomatis and Chlamydia pneumoniae cause common human diseases, while Chlamydia abortus, Chlamydia psittaci, and Chlamydia suis represent zoonotic threats or are endemic in human food sources. Although chlamydial infections are currently manageable in human populations, chlamydial infections in livestock are endemic and there is significant difficulty achieving effective treatment. To combat the spread of Chlamydiaceae in humans and other hosts, improved methods for treatment and prevention of infection are needed. There exist various studies exploring the potential of natural products for developing new antichlamydial treatment modalities. Polyphenolic compounds can inhibit chlamydial growth by membrane disruption, reestablishment of host cell apoptosis, or improving host immune system detection. Fatty acids, monoglycerides, and lipids can disrupt the cell membranes of infective chlamydial elementary bodies (EBs). Peptides can disrupt the cell membranes of chlamydial EBs, and transferrins can inhibit chlamydial EBs from attachment to and permeation through the membranes of host cells. Cellular metabolites and probiotic bacteria can inhibit chlamydial infection by modulating host immune responses and directly inhibiting chlamydial growth. Finally, early stage clinical trials indicate that polyherbal formulations can be effective in treating chlamydial infections. Herein, we review an important body of literature in the field of antichlamydial research.


Introduction
Effective management of infectious diseases is one of most important endeavors of modern times. Infectious diseases impact human, animal, and environmental health, and beyond the direct cost of human disease management they influence the productivity of a wide range of agricultural practices. Chlamydiae are obligate intracellular bacteria that are known to be responsible for a wide range of serious global health-care challenges. From the Chlamydiaceae family within the Chlamydiae order, Chlamydia trachomatis and Chlamydia pneumoniae cause common human diseases, while Chlamydia abortus, Chlamydia psittaci, and Chlamydia suis represent zoonotic threats or are endemic in human food sources [1]. C. trachomatis is the leading cause of trachoma, which can lead to blindness  Compounds are injected into the host cell to initiate internalization and establish an antiapoptotic state. The EB is incorporated into an endosomal membrane to form an inclusion, which is freed from the cell wall and passes into the host cell cytoplasm. Through bacterial protein synthesis the EB converts to reticulate bodies (RBs), which redirect host cell nutrients and divide by binary fission. The RBs direct host cell function and continue to replicate exponentially. If the RBs are excessively stressed they enter a dormant persistent state to promote survival, and reactivate upon removal of stress. Within 30-48 h the RBs differentiate back to EBs, which then exit the host cell through lysis or extrusion. Adapted with permission from [28]. Copyright 2016 Nature Publishing Group. Compounds are injected into the host cell to initiate internalization and establish an antiapoptotic state. The EB is incorporated into an endosomal membrane to form an inclusion, which is freed from the cell wall and passes into the host cell cytoplasm. Through bacterial protein synthesis the EB converts to reticulate bodies (RBs), which redirect host cell nutrients and divide by binary fission. The RBs direct host cell function and continue to replicate exponentially. If the RBs are excessively stressed they enter a dormant persistent state to promote survival, and reactivate upon removal of stress. Within 30-48 h the RBs differentiate back to EBs, which then exit the host cell through lysis or extrusion. Adapted with permission from [28]. Copyright 2016 Nature Publishing Group.
Frontline antibiotics for chlamydial infections include tetracyclines (TET) or macrolides (MAC), with doxycycline and azithromycin being the most common [5]. C. trachomatis within the human population remains manageable [7,8], although the potential for resistant forms has been reported [13]. Conversely, C. suis, which primarily infects swine, has shown to resist clearance at a herd level with a diverse range of antibiotics [12], and is the first of the Chlamydiaceae to develop stable TET-resistant forms in vivo [30]. C. psittaci responds well to TET or MAC treatment in humans and birds at an individual level [9,31], however, no reliable treatment is known for infections in cattle [6,32]. C. pneumoniae within the human population also remains manageable [5]. If detected and treated early, C. abortus in humans responds well to treatment with TET and erythromycin (MAC) [33], however, it is endemic in the livestock industry, and although oxytetracycline (TET) will reduce the number of abortions and bacterial shedding, antibiotics are ineffective in clearing the infection [9]. C pecorum is also endemic in the livestock industry [6], and in particular, no reliable treatment is known for infections in cattle [32], although, within Koala populations there has been some treatment success using chloramphenicol or enrofloxacin [34,35]. Other chlamydial species, such as C. muridarum, C. felis, and C. caviae, are generally manageable with TET and/or MAC treatment regimes [36][37][38]. C. avium and C. gallinacea have only recently been distinguished as distinct from C. psittaci, and for now it may be assumed that they have similar treatment and infection characteristics. Table 1. Standard antibiotic treatments, potential resistance, and infection characteristics.

Chlamydial Species Standard Treatments
In Vitro and/or Natural Antibiotic Resistance [13] Asymptomatic Persistence in Host [27] Infected Tissues [27] C. trachomatis

C. abortus
Humans [33] Responds well to early treatment with tetracyclines (TET) and erythromycin (MAC). Livestock (Ruminants) [9,10] Oxytetracycline (TET) will reduce the number of abortions and bacterial shedding. Antibiotics are ineffective in clearing the infection.

C. felis
Cats [37] Doxycycline (TET) and topical tetracycline (TET Livestock infections from C. suis, C. psittaci, C. abortus, and C pecorum all exhibit significant difficulty to clear, especially at a herd level [6,12,32]. Treatment failure, persistence, and reinfection are all common in a range of animal hosts, with antibiotics typically providing only a reduction in infection severity for the duration of the treatment period [6,10]. It is proposed that livestock often exhibit infection by multiple chlamydial species at a time [12,32], and chlamydial species found in livestock are commonly present in the digestive tract (Table 1), which may complicate clearance and allow for reinfection [39]. Recent research points to asymptomatic chlamydial infections being responsible for huge reductions in livestock productivity and profits [6]. There is a strong need for new treatment options to avoid widespread prophylactic antibiotic treatment in the industry as a means to effectively manage potential financial losses.

Biomedical Phytochemical Groups and Anti-Infective Action
Approximately 80% of the world relies on plant-based medicine for treatment of all ailments and 70% of pharmaceutical therapeutics used today are models of natural products [40]. Herbal medicines may be useful as a starting point for the development of new antichlamydial drugs [41]. Natural products comprise a diverse range of biochemical compounds. Plant secondary metabolites comprise the largest group of compounds used in plant-based therapeutics with polyphenols being the most commonly studied secondary metabolite group with respect to the treatment of Chlamydiaceae infections. Additionally, lipidic compounds and proteinaceous compounds are other commonly derived natural materials which exhibit antichlamydial activity. There have also been studies exploring the efficacy of cellular metabolites and probiotics to inhibit chlamydial infections. As examples highlighting the potential of natural product-based therapeutics, three broad-spectrum antimicrobial polyherbal formulations, which exhibit potent antichlamydial activity, have been developed and studied up to Stage II clinical trials.

Polyphenolic Compounds
Polyphenols are organic chemicals found in plants and have been shown to help prevent degenerative diseases and exhibit antimicrobial properties against a wide range of bacteria, including Chlamydiaceae [42]. Table 2 provides a summary of existing studies exploring the antichlamydial properties of polyphenols. The mechanism by which polyphenols have antichlamydial activity is not entirely known, however, based on these studies it appears that the antichlamydial activity of polyphenols is due to various modes of action. Results from a comprehensive study by Alvesalo et al. suggested that compound structural variations play a key role in the antichlamydial effect of polyphenolic compounds. This study evaluated the in vitro antichlamydial activity of 57 natural flavonoids and other natural polyphenols and structurally similar synthetic compounds against C. pneumoniae in human cells. Thirty-seven percent (21/57) of the studied compounds, all of which were non-toxic to the host cells at tested concentrations, were highly active against C. pneumoniae. From the remaining compounds, 28% (16/57) were considered active, 11% (6/57) moderately active, and 24% (14/57) inactive ( Figure 3). Even when present only before infection, some compounds had the ability to accumulate inside cells or in cell membranes, causing inhibition of C. pneumoniae [43]. From this data, there are several observations of the relationship between molecular structure and observed antichlamydial activity. Compound structural variations, either free from sugar moieties, or with greater hydrophobicity, were found to be more active.
Early studies explored a group of polyphenols found in tea, known as catechins, which have been observed to exhibit broad-spectrum antimicrobial properties [44]. It has been proposed that catechins cause cytoplasmic membrane damage by damaging [45] or disrupting the permeability [46] of lipid bilayers. Yamazaki et al., performed an in vitro study on the effect of Polyphenon 70S, a tea polyphenol extract from a green tea high in catechins, on C. pneumoniaeand C. trachomatis-infected human cells.
Complete inhibition of C. pneumoniae occurred at 1.6 mg/mL Polyphenon 70S for the strain AC-43 and at 0.8 mg/mL for the Ar-39 strain, and complete inhibition of C. trachomatis at 1.6 mg/mL for serovar D and at 0.4 mg/mL for the L2 strain. Polyphenon 70S comprises: epigallocatechin, epicatechin, epigallocatechin gallate, epicatechin gallate, and gallocatechin gallate. Epigallocatechin gallate is the dominant constituent and is attributed to be a major contributor to the observed antimicrobial effects. Although Polyphenon 70S was toxic to human cells at 0.25 mg/mL for treatment post-inoculation with the bacteria, it was non-toxic when treatment began pre-inoculation [47]. A further study by Yamazaki et al. showed that five biosynthesized tea polyphenols were active against C. trachomatis and analyzed the varying toxicity of the catechins. All five catechins, of which (−)-epicatechin (EC) was the least toxic, had an inhibitory effect on the proliferation of C. trachomatis in vitro. Because the concentration of tea polyphenols required for complete inhibition of C. trachomatis is high compared to antibiotics, tea polyphenols are not currently suitable for treating systemic infections. Modification of the catechin structure to reduce the required dose and toxicity may help circumvent the toxicity problem for use of catechins in a topical microbicide [48].
Other antichlamydial mechanisms have also been observed with several other polyphenols. Tormakangas et al., evaluated the effects of treatment of acute C. pneumoniae infection with the flavonoids quercetin and luteolin and an alkyl gallate, octyl gallate, in a mouse model. The lowest presence of C. pneumoniae in lung tissue was detected in mice treated with luteolin. The response to quercetin treatment was not favorable, which contradicts other studies performed with the flavonoid [43]. C. pneumoniae is reported to inhibit apoptosis of the infected host cell and it has been proposed that luteolin negates the antiapoptotic effect of chlamydia by inducing cellular apoptosis via interference with the mitochondrial pathway [49,50] and thereby allowing the chlamydial infection to become vulnerable to the hosts natural immune responses. The polyphenolic flavonoid, baicalin, also results in a more effective immune response to clear chlamydial infections. Baicalin is an antimicrobial, anti-inflammatory flavonoid isolated from Scutellariae baicalensis, or Scutellariae radix, a plant used in traditional oriental medicine and is a potential agent for therapy of C. trachomatis infections. Hao  24 days after inoculation with C. trachomatis. Some antichlamydial activity was observed with baicalin concentrations between 0.12 mg/mL and 0.48 mg/mL. At 48 mg/mL, baicalin strongly inhibited C. trachomatis and blocked further infection almost completely. When the RFX5 and chlamydial protease-like activity factor (CPAF) genes in chlamydia-infected cells were examined, it was found that RFX5 was upregulated and CPAF was downregulated by baicalin, with CPAF as the primary target [51]. It has been suggested that the CPAF degradation of RFX5 may play a role in chlamydia escaping efficient host immune detection, and therefore the down-regulation of CPAF may allow the hosts immune system to more effectively detect the chlamydial infection [41]. The lupine-class triterpene, betulin, is extensively distributed in nature and has been shown to be highly biologically active in the treatment of intracellular pathogens. Salin et al. extracted betulin from birch bark and evaluated thirty-two betulin derivatives for potential use against C. pneumoniae in vitro. Five promising compounds were identified. The betulin derivative, betulin dioxime, had a minimum inhibitory concentration (MIC) of 1 mM against the CWL-029 C. pneumoniae strain, with 50% inhibition achieved at 290 nM. A clinical isolate confirmed the antichlamydial activity, with an MIC of 2.2 mM. The mechanism by which inhibition occurs is unknown [52].
Studies on extracts from two different mint species showed positive antichlamydial effects. Corn mint (Mentha arvensis) contains various phenolic compounds, with rosmarinic acid, linarin, and acacetin compounds being most prevalent. Salin et al. examined the efficacy of corn mint extracts and several pure versions of compounds present in the corn mint extracts in treating C. pneumoniae infections both in vitro and in vivo. Both the mint extracts and pure compounds exhibited high levels of inhibitory activity in vitro, with low host cell toxicity. In a mouse model, intraperitoneally administered corn mint extracts delivered at nutritionally relevant concentrations resulted in reduced inflammatory symptoms but were not as effective as antibiotics in clearing the infection [53]. Kapp et al., performed an in vitro study on the effect of seven peppermint (Mentha × piperita L.) tea extracts on C. pneumoniae-infected human cells. While all seven tea extracts were active against C. pneumoniae, at an extract concentration of 250 µg/mL growth inhibition varied between 20.7% and 69.5%. Peppermint teas contain secondary metabolites, predominantly catechins and glycosides of flavanones and flavones, including luteolin and apigenin glycoside, which, along with rosmarinic acid, were related to higher antichlamydial activity [54].
Biochanin A is the main isoflavone component of red clover, and was compared with formononetin, genistein, daidzein, genistin, and daidzin for their antichlamydial effect against both C. trachomatis and C. pneumoniae in vitro. IC50 values ranged from 12 to >100 µM for all compounds, except formononetin and daidzein, which exhibited no inhibition of C. trachomatis. Overall, C. pneumoniae was more susceptible to inhibition. Hanski et al., determined that biochanin A exhibited the greatest antichlamydial activity overall, and significantly suppressed inclusion counts and decreased the mean bacterial inclusion size in C. trachomatis-infected cell cultures. Biochanin A prevented the formation of C. pneumoniae inclusions at concentrations of 25 µM or higher and prevented the formation of new infectious progeny. The greater efficacy of biochanin A was attributed to the presence of a methylated hydroxyl group. This study went on to improve bioavailability and the resulting efficacy of biochanin A with the development of oromucosal buccal dosage forms to improve dissolution and achieve permeation of buccal tissue so as to overcome digestive demethylation and conversion to genistein [55].
prevented the formation of new infectious progeny. The greater efficacy of biochanin A was attributed to the presence of a methylated hydroxyl group. This study went on to improve bioavailability and the resulting efficacy of biochanin A with the development of oromucosal buccal dosage forms to improve dissolution and achieve permeation of buccal tissue so as to overcome digestive demethylation and conversion to genistein [55]. pneumoniae at 50 μM concentration (n = 4 or more). Activity is determined in comparison to controls: highly active (black bar) = 85%-100% inhibition; active (striped bar) = 50%-84%; moderately active (black dotted bar) = 30%-49%; inactive (white bar) = <30%. Adapted with permission from [43]. Copyright 2005 Elsevier Inc. . Activity is determined in comparison to controls: highly active (black bar) = 85%-100% inhibition; active (striped bar) = 50%-84%; moderately active (black dotted bar) = 30%-49%; inactive (white bar) = <30%. Adapted with permission from [43]. Copyright 2005 Elsevier Inc.  Polyphenolic synergistic effects with antibiotics or calcium modulators were explored and shown to result in improvements in antichlamydial efficacy. In an attempt to increase the efficacy of both polyphenols and doxycycline against C. pneumoniae in vitro, Salin et al. combined the polyphenols luteolin, quercetin, rhamnetin, and octyl gallate with either doxycycline or one of three calcium modulators-verapamil, isradipine, or thapsigargin. The polyphenol-doxycycline combinations did not increase the efficacy of treatment and some combinations had antagonistic effects. However, combining calcium modulators with polyphenols had some synergistic effect, although calcium modulators alone are not active against C. pneumoniae. While isradipine was synergistic with high concentrations of luteolin and quercetin, verapamil was synergistic with low concentrations of the same polyphenols. Thapsigargin had the greatest synergistic effect, significantly increasing the chlamydial growth inhibitory effect of polyphenols [56]. However, although this provides interesting insights, it should be highlighted that the synergistic use of calcium modulators in humans may lead to significant side effects. Rizzo et al. studied the synergistic effects of polyphenolic pretreatment followed by subsequent antibiotic treatment on C. pneumoniae. Either of the phenolic compounds resveratrol or quercetin was administered, followed by either clarithromycin or ofloxacin. With resveratrol concentrations of 40 µM and quercetin concentrations of 20 µM, both phenolic compounds exhibited significant inhibitory effects when combined with clarithromycin or ofloxacin, in comparison to controls. Chlamydial inhibition was linked to the immunomodulatory effects of decreased IL-17 and IL-23 production in a time-dependent manner in C. pneumoniae-infected cells [57]. Overall, the existing studies indicate that some polyphenolic compounds directly inhibit chlamydial activity by various mechanisms and may also work synergistically with other compounds to achieve increased efficacy.

Lipidic Compounds
It has long been known that lipidic compounds, such as fatty acids, monoglycerides, and terpenoids, exhibit broad-spectrum antimicrobial effects [58]. Recent evidence supports that different kinds of lipidic compounds such as fatty acids and monoglycerides induce different kinds of morphological responses in lipid membranes, and hence are attractive to explore for therapeutic applications [59]. Table 3 provides a summary of existing studies that have explored the efficacy of lipidic compounds on chlamydial infections. Bergsson et al. evaluated the antichlamydial effects of 12 lipidic compounds on C. trachomatis. From these, monocaprin, lauric acid, and capric acid were shown to have the greatest antichlamydial effects. At a 5 mM concentration, the most active, monocaprin, caused a greater than 100,000-fold inhibition of C. trachomatis when exposed for five minutes. Capric acid was the least active of the three lipids, losing most of its activity when diluted. Monocaprin at 30 µg/mL was 50% effective when incubated with C. trachomatis for two hours. Chlamydial EBs were exposed to the lipid and then removed and inoculated into cell cultures. Observations of EBs exposed to monocaprin for 1, 5, and 10 min, indicated that after 10 min exposure, the EBs were irreversibly deactivated and were observed to rupture and disintegrate ( Figure 4A-D) [60]. These observations support the proposal that lipidic compounds primarily inactivate the bacteria by affecting and disrupting the outer membrane.
Further studies went on to explore the potential of synthetic lipids for use in developing topical microbicides. Lampe et al. determined that 2-O-octyl-sn-glycerol, a synthetic lipid developed from naturally occurring human breast milk lipids was an effective inhibitory agent against C. trachomatis. Complete growth inhibition occurred after two hours of contact with a 7.5 mM concentration of 2-O-octyl-sn-glycerol. Four other lipids were studied, but were significantly less effective. When tested in conditions similar to the human vagina-10% human blood and with pH alterations between 4.0 and 8.0-lipid activity was not affected. When EBs were exposed to lipids for 90 min, the EBs appeared to be hollow shells with ruptured cell walls ( Figure 4E,F). All of these lipids have also been shown to exhibit broad-spectrum antimicrobial properties [61], which supports cell wall disruption being the primary mechanism of action. However, it should be noted that these synthetic lipids were determined to exhibit no cellular toxicity, and preliminary observations indicate no vaginal irritation in rabbit models from lipid concentrations as high as 120 mM [62]. As a continuation of the work done in 1998 by Lampe et al., Skinner et al. explored the development of a topical microbicide with the synthetic lipid, 3-O-octyl-sn-glycerol , and an engineered antimicrobial peptide, WLBU2, as the active compounds. After in vitro activity and toxicity analysis of the components, concentrations found to be toxic were omitted from further tests. While both WLBU2 and 3-OG were effective in vitro against C. trachomatis, the two components combined showed synergy, with significantly increased inhibitory activity. Although simulated fluids reduced activity, the combination shows potential for the development of a topical microbicide [63].  , and an engineered antimicrobial peptide, WLBU2, as the active compounds. After in vitro activity and toxicity analysis of the components, concentrations found to be toxic were omitted from further tests. While both WLBU2 and 3-OG were effective in vitro against C. trachomatis, the two components combined showed synergy, with significantly increased inhibitory activity. Although simulated fluids reduced activity, the combination shows potential for the development of a topical microbicide [63]. Another lipidic compound shown to have potential for topical application formulations is a tropolone-related compound, hinokitiol, which is found in the heartwood of trees in the Cupresseceae family. Hinokitiol has previously been shown to have antimicrobial activity against several bacterial species, including Staphylococcus aureus and Schistosoma mansoni. Yamano et al. studied the inhibitory effects of hinokitiol on C. trachomatis. Although, hinokitiol was found to be active against C. trachomatis, with an MIC of 32 μg/mL in vitro, the MIC was significantly greater than for hinokitiol against S. aureus or the MICs of antibiotics. Because high concentrations of hinokitiol are cytotoxic, topical application would be recommended for treatment of C. trachomatis infections [64]. While there are only a few studies exploring the antichlamydial efficacy of lipidic compounds, the results support the widely held opinion that lipidic compounds typically exhibit broad-spectrum antimicrobial properties, which is primarily due to disrupting the cellular membrane of pathogenic Another lipidic compound shown to have potential for topical application formulations is a tropolone-related compound, hinokitiol, which is found in the heartwood of trees in the Cupresseceae family. Hinokitiol has previously been shown to have antimicrobial activity against several bacterial species, including Staphylococcus aureus and Schistosoma mansoni. Yamano et al. studied the inhibitory effects of hinokitiol on C. trachomatis. Although, hinokitiol was found to be active against C. trachomatis, with an MIC of 32 µg/mL in vitro, the MIC was significantly greater than for hinokitiol against S. aureus or the MICs of antibiotics. Because high concentrations of hinokitiol are cytotoxic, topical application would be recommended for treatment of C. trachomatis infections [64]. While there are only a few studies exploring the antichlamydial efficacy of lipidic compounds, the results support the widely held opinion that lipidic compounds typically exhibit broad-spectrum antimicrobial properties, which is primarily due to disrupting the cellular membrane of pathogenic microbes. This supports the potential antichlamydial activity of a wide range of unexplored lipidic compounds, and highlights the need for ongoing research in this field. [60] In vitro Pre-treatment: incubated with EBs for 0, 30, 60, 90, or 120 min prior to inoculation.
2-O-octyl-sn-glycerol, at 7.5 mM, completely prevented growth of C. trachomatis after 120 min of contact with the organism. The lipids were shown to have disrupted the chlamydial inner membrane, allowing leakage of the cytoplasmic contents from the cell. [62] Lipidic compound: In vitro Pre-treatment: incubated with EBs for 5 or 120 min prior to inoculation.

Proteinaceous Compounds
There is a wide range of proteinaceous compounds known to exhibit antimicrobial effects. Table 4 summarizes existing studies reporting protein-based antichlamydial compounds. An early reference to the use of desert truffle (Terfezia claveryi) aqueous extracts in treating trachoma, a common eye disease resulting from a C. trachomatis infection, highlights the importance of exploring ethnobotanical therapeutics as leads for new antimicrobial compounds. Desert truffles are a mycorrhizal fungus, or dark brown truffle, native to the Arabian Peninsula, and are a traditional ethnobotanical medicine in Bahrain. A protein extracted from T. claveryi has displayed antimicrobial activity against a wide spectrum of bacteria and against C. trachomatis in particular. In a pilot clinical study conducted by Al-Marzooky in 1981, which used sterilized aqueous extracts of T. claveryi to treat patients with trachoma, the truffle extracts were found to be effective, although slower acting than conventional antibiotic treatment. The treatment that most effectively inhibited C. trachomatis was with partially purified proteins extracted from an aqueous extract of T. claveryi. Effective in treating a broad range of bacterial infections as well as other diseases, T. claveryi and other species of desert truffles have many potential uses in medicine in addition to treating C. trachomatis [65].
Peptides comprise short chains of amino acids and are a common form of antimicrobial proteinaceous compounds. There is significant research indicating the broad-spectrum antimicrobial properties of various peptide classes. During studies in the mid-1990s, Yasin et al. explored the antichlamydial properties of human defensin HNP-2 and porcine protegrin PG-1 against C. trachomatis in vitro. Both HNP-2 and PG-1 inhibited chlamydial infection, but HNP-2 was the most potent. Examination of PG-1 treated EBs revealed morphological changes, membrane damage, and loss of cytoplasmic contents [66]. Later, melittin, as a principle active component of bee venom, was explored by Lazarev et al. to develop a potential treatment for C. trachomatis infections. Chlamydial inhibition was achieved in vitro with the introduction and activation of recombinant plasmid vectors expressing the melittin gene. Melittin is known to be cytotoxic and it is believed that the main antichlamydial mechanism is its direct cytotoxic effect, however, a secondary mechanism may be due to lowering the transmembrane potential of a transfected cell, which disturbs chlamydial adhesion to the cell [67]. During in vivo mouse studies, half of infected mice were free from infection 3-4 weeks after exposure [68].
Cecropin is a peptide found in cecropia moths (Hyalophora cecropia) that has been shown to have broad-spectrum antimicrobial activity. As part of the development of a cecropin-based topical microbicide, Ballweber et al. determined the minimum bactericidal concentration (MBC) of gel formulations of cecropin peptides D2A21 and D4E1 for in vitro activity against C. trachomatis. The gel formulations were equally effective against two C. trachomatis strains and the addition of 10% human blood did not alter the results significantly. Although pH values above and below 7 reduced D2A21 activity, the 2% D2A2 gel formulation remained effective with experimental pH variation. After D2A21 exposure for 90 min, ultrastructural observations showed that C. trachomatis EB membranes had been disrupted, causing the leaking of cytoplasm ( Figure 5A,B) [69]. Previous studies have also suggested that this class of peptides inhibits microbial growth due to the creation of pores or channels through the bacterial membrane [70,71]. However, other studies suggest that similar peptides exhibit broad-spectrum microbial inhibitory activity due to the release of mitochondrial respiratory control, the inhibition of protein import, and the inhibition of bacterial respiration [72]. In an extension of the work done by Ballweber et al., as mentioned above, Skinner et al. explored the development of a topical microbicide with a synthetic lipid, 3-OG, and an engineered antimicrobial peptide, WLBU2. Both WLBU2 and 3-OG were effective in vitro against C. trachomatis, however, when combined, the two components together showed significantly increased inhibitory effect [63]. C. trachomatis in vitro. Both HNP-2 and PG-1 inhibited chlamydial infection, but HNP-2 was the most potent. Examination of PG-1 treated EBs revealed morphological changes, membrane damage, and loss of cytoplasmic contents [66]. Later, melittin, as a principle active component of bee venom, was explored by Lazarev et al. to develop a potential treatment for C. trachomatis infections. Chlamydial inhibition was achieved in vitro with the introduction and activation of recombinant plasmid vectors expressing the melittin gene. Melittin is known to be cytotoxic and it is believed that the main antichlamydial mechanism is its direct cytotoxic effect, however, a secondary mechanism may be due to lowering the transmembrane potential of a transfected cell, which disturbs chlamydial adhesion to the cell [67]. During in vivo mouse studies, half of infected mice were free from infection 3-4 weeks after exposure [68].
Cecropin is a peptide found in cecropia moths (Hyalophora cecropia) that has been shown to have broad-spectrum antimicrobial activity. As part of the development of a cecropin-based topical microbicide, Ballweber et al. determined the minimum bactericidal concentration (MBC) of gel formulations of cecropin peptides D2A21 and D4E1 for in vitro activity against C. trachomatis. The gel formulations were equally effective against two C. trachomatis strains and the addition of 10% human blood did not alter the results significantly. Although pH values above and below 7 reduced D2A21 activity, the 2% D2A2 gel formulation remained effective with experimental pH variation. After D2A21 exposure for 90 min, ultrastructural observations showed that C. trachomatis EB membranes had been disrupted, causing the leaking of cytoplasm ( Figure 5A,B) [69]. Previous studies have also suggested that this class of peptides inhibits microbial growth due to the creation of pores or channels through the bacterial membrane [70,71]. However, other studies suggest that similar peptides exhibit broad-spectrum microbial inhibitory activity due to the release of mitochondrial respiratory control, the inhibition of protein import, and the inhibition of bacterial respiration [72]. In an extension of the work done by Ballweber et al., as mentioned above, Skinner et al. explored the development of a topical microbicide with a synthetic lipid, 3-OG, and an engineered antimicrobial peptide, WLBU2. Both WLBU2 and 3-OG were effective in vitro against C. trachomatis, however, when combined, the two components together showed significantly increased inhibitory effect [63]. Granulocyte-and epithelium-derived antimicrobial peptides, protegrin-1, RTD-1, cryptdin-4, and indolicidin, were studied by Chong-Cerrillo et al. in vitro. Protegrin-1 was found to have the strongest antichlamydial activity, inhibiting infectivity by 50% at a concentration of 6 μg/mL. Several chlamydial serovars were examined and results suggested that specific peptide/bacteria interactions are complex and remarkably specific. Overall, it was observed that protegrins may have a broader antimicrobial activity than defensins [73]. Then, in a highly comprehensive study by Yasin et al., 48 structurally diverse antimicrobial peptides were examined against C. trachomatis serovar L2. By Granulocyte-and epithelium-derived antimicrobial peptides, protegrin-1, RTD-1, cryptdin-4, and indolicidin, were studied by Chong-Cerrillo et al. in vitro. Protegrin-1 was found to have the strongest antichlamydial activity, inhibiting infectivity by 50% at a concentration of 6 µg/mL. Several chlamydial serovars were examined and results suggested that specific peptide/bacteria interactions are complex and remarkably specific. Overall, it was observed that protegrins may have a broader antimicrobial activity than defensins [73]. Then, in a highly comprehensive study by Yasin et al., 48 structurally diverse antimicrobial peptides were examined against C. trachomatis serovar L2. By examining both natural and synthetic peptides from five major peptide groups: full-length β-sheet (×13), truncated protegrins (×7), PG-1 disulfide variants (×7), α-helical peptides (×12), and circular peptides (×6), it was possible to gain insight into some general properties regarding the antichlamydial properties of peptides. From this, it was proposed that moderate-sized cationic peptides may be useful in microbicide preparations designed to prevent chlamydial infection [74].
Cathelicidin peptides are found in lysosomes of macrophages and polymorphonuclear leukocytes (PMNs), and keratinocytes. In an important series of studies, Donati et al. explored the antichlamydial characteristics of cathelicidin peptides from various sources upon a wide range of chlamydial species. Initially, the cathelicidin peptides: SMAP-29 (sheep), LL-37 (humans), BMAP-27 (cattle), BMAP-28 (cattle), BAC-7 (cattle), and PG-1 (pigs), were tested against a total of 25 strains from the chlamydial species: C. trachomatis, C. pneumoniae, C. felis, C. abortus, C. psittaci, and C. pecorum. It was observed that: (1) SMAP-29 was most active against C. trachomatis, and was also active against C. pneumoniae and C. felis; (2) C. pneumoniae strains were less susceptible to peptides than C. trachomatis; (3) most animal Chlamydiae were not sensitive to cathelicidins at concentrations of around 10 to 80 µg/mL; and (4) PG-1 resulted in an increase in the number of inclusions in some animal chlamydial species at a concentration of 80 µg/mL [75]. In a follow-up study, the same group of peptides was examined against nine C. suis isolates obtained from pigs with conjunctivitis. Again, SMAP-29 was most active, followed by BAC-7 and BMAP-27, with LL-37 and PG-1 showing no activity at 80 µg/mL [76]. Next, to further explore the earlier observation that PG-1 resulted in increased activity in some animal chlamydial species, Donati et al. more carefully explored the effect of PG-1 on C. abortus infectivity.

From this it was identified that infection of PG-1-pretreated cells resulted in an eight-fold increase in the number of inclusions and that PG-1 treatment after chlamydial infection had no increase in infectivity.
Additional experiments demonstrated that PG-1 pretreatment facilitates the entry of C. abortus into host cells [77].
Dermaseptins are a family of peptides isolated from skin of the Phyllomedusa genus of frogs. Bergaoui et al. performed an in vitro evaluation of antichlamydial and cytotoxic properties of dermaseptin S 4 and derivatives: D 4 D 20 S 4 , K 4 K 20 S 4 , S 4 (5-28), and S 4 (1-12). S 4 provided 81% inhibition after 48 h at a concentration of 5 µg/mL, whereas K 4 K 20 S 4 provided 96% inhibition after 48 h at 5 µg/mL. The 50% cytotoxic concentration (CC50) was determined to be higher than 25 µg/mL for each peptide, except for S 4 , which appeared to be more toxic than the other peptides. However, increasing the number of peptide positive charges reduced cytotoxicity [78]. Overall, there is a wealth of information regarding peptide/Chlamydiae interactions that could be used for the further development of peptide-based antichlamydial therapeutics.
Transferrins are a family of iron-binding glyco-proteins found in milk, tears, saliva, and vaginal secretions that have been shown to be both bactericidal and bacteriostatic [79]. A series of studies was performed to explore the efficacy of various transferrins on inhibiting poultry-related C. psittaci infections. Initially, Beeckman et al. tested the effect of ovotransferrin (ovoTF), human lactoferrin (hLF), and bovine lactoferrin (bLF) against C. psittaci in vitro. While all three transferrins exhibited inhibitory activity, ovoTF was more effective in inhibiting irreversible attachment and cell entry by C. psittaci, though transferrins had no effect on bacterial activity within eukaryotic cells. The antichlamydial activity of ovoTF is believed to stem from ovoTF incorporation into the bacterial membrane, followed by subsequent binding of bacterial lipopolysaccharides leading to the bacterial membrane becoming more rigid. It is proposed that increased membrane rigidity results in interference with the actin host cell recruitment pathway by disrupting the stability of bacterial T3SS translocon proteins, or by disrupting secretion of Type III secretion effector proteins. Additionally, interference with the actin host cell recruitment pathway may occur due to proteolytic degradation of the Type III secretion effector proteins. Such disruption of the actin host cell recruitment pathway is believed to inhibit actin host cell recruitment at the bacterial entry site, which is a key step in host cell internalization of the bacteria [80]. In a follow-up study, Van Droogenbroeck et al. investigated using ovoTF to prevent C. psittaci infections by treating turkeys with aerosolized ovoTF, and then inoculating the turkeys with a virulent strain of C. psittaci. While the ovoTF did not prevent infection, the severity of infection was significantly diminished. It is proposed that iron sequestration is involved in the in vivo antibacterial activity of ovoTFs and may also be the underlying mechanism which results in ovoTFs activating both innate and adaptive immune responses [81]. In a later study, newborn turkeys infected with C. psittaci, avian metapneumovirus, or Ornithobacterium rhinotracheale were treated with ovoTF. For the first nine weeks, the turkeys remained healthy, after which symptoms of respiratory disease appeared. OvoTF treatment reduced the shedding of C. psittaci into the air, reducing the risk of zoonotic transmission, and respiratory disease was delayed for the first half of the brood period, resulting in a 46% reduction in mortality. Although infection was not prevented, the symptoms of infection were less severe and the cost of antibiotics required for treatment lowered. As the C. psittaci infection was not cured, ovoTF is currently recommended for use in conjunction with antibiotics unless further studies increase the effectiveness of ovoTF [82]. These studies highlight the potential diversity in antimicrobial efficacy and mechanism of action for proteinaceous compounds. While transferrins can disrupt the biological pathways necessary for host cell infection, peptides have been shown to rupture the membrane of infective EBs thereby leading to bacterial lysis and death. Sterilized aqueous T. claveryi extracts of were found to be effective, although slower acting than conventional antibiotic treatment. Partially purified proteins extracted from the aqueous T. claveryi extract were more effective. [65] Peptides: Human defensin HNP-2, Porcine protegrin PG-1 In vitro Pre-treatment: incubated with EBs for 2 h prior to inoculation.
Both HNP-2 and PG-1 inhibited chlamydial infection, but HNP-2 was the most potent. PG-1-treated EBs exhibited morphological changes, membrane damage, and loss of cytoplasmic contents. [66] Peptide: Melittin In vitro Pre-treatment: incubated with EBs for 24 h prior to inoculation.
C. trachomatis inhibition after the introduction of recombinant plasmid vectors expressing the melittin gene. Main mechanism is its direct cytotoxic effect. Secondary mechanism is lowering the transmembrane potential of a transfected cell, which disturbs chlamydial adhesion to the cell. [67] Peptide: Melittin In vivo Pre-& Post-inoculation: administered 1 day prior to inoculation; administered at 14 days p.i.

Vaginal administration and induction of melittin gene transcription with doxycycline inhibited subsequent infection in mice.
Half of the mice were free from infection within 3-4 weeks. [68] Peptides: Cecropin D2A21, Cecropin D4E1 In vitro Pre-treatment: incubated with EBs for 5 or 120 min prior to inoculation.
WLBU2, at 50 µM, was 89% inhibitory after 5 min of exposure, and 100% after 120 min. Coadministration with 3-OG produces significantly increased activity. WLBU2 could be used at up to 60 µM without causing toxicity.
β-Sheet protegrins and α-helical peptides were equally active. Enantiomers were as active as native structures.
Moderate-sized circular mini-defensins were less effective against C. trachomatis.
Moderate-sized cationic peptides may be useful in microbicide preparations designed to prevent chlamydial infection. In vitro Pre-treatment: incubated with EBs for 2 h prior to inoculation.
SMAP-29 was most active, C. trachomatis inhibition by >50% at 10 µg/mL, with BMAP-27, BMAP-28, and BAC-7, >50% at 80 µg/mL. SMAP-29 also active against C. pneumoniae and C. felis. C. pneumoniae strains were less susceptible to peptides than C. trachomatis. Most animal chlamydiae were not sensitive to cathelicidins at concentrations of around 10-80 µg/mL. PG-1 at 80 µg/mL resulted in an increase in the number of inclusions in some animal chlamydial species. In vitro Pre-treatment: incubated with EBs for 2 h prior to inoculation.
Ovotransferrin was more effective than human and bovine lactoferrin in inhibiting bacterial irreversible attachment and cell entry. [80] Transferrin: Ovotransferrin In vivo Pre-inoculation: one dose administered pre-inoculation. Pre-& Post-inoculation: one dose administered pre-inoculation; administered daily for 12 days p.i. (turkeys) A single pre-infection dose of 10 mg or a daily dose of 5 mg did not prevent turkeys from becoming infected with C. psittaci. Treatment significantly reduced the severity of infection. [81] Transferrin: Ovotransferrin Respiratory disease occurred at 9 weeks although, overall treatment was associated with 46% reduction of mortality. [82]

Cellular Metabolites & Probiotics
Mechanisms of immune response in controlling microbial infections and the complex interplay between symbiotic host microbiota and pathogenic microbes are important considerations in the development of new treatment strategies. Table 5 summarizes results of investigations into the effect of cellular metabolites and probiotic microbes with regards to chlamydial infections. Carratelli et al. explored the role of the cellular metabolite, nitric oxide (NO), in inhibiting C. pneumoniae from infecting macrophage J774 cells, as well as the ability of NO to directly damage isolated C. pneumoniae cells. Infected cells were exposed to recombinant murine gamma interferon (MurIFN-γ) so as to activate inducible nitric oxide synthase (iNOS). This resulted in increased production of NO and reduced viability of the infected cells. In addition, 2-(N,N-diethylamino)-diazenolase-2-oxide was added to cells before infection or during chlamydial cultivation. 2-(N,N-diethylamino)-diazenolase-2-oxide is a complex of diethylamine with NO, which can be used to generate a controlled release of NO in solution. The increase in NO concentration, before cell infection or during chlamydial cultivation, resulted in C. pneumoniae inhibition in a dose-dependent manner. These results suggest that the host immune response to chlamydial infection triggers cellular pathways which activate NO release and subsequently lead to the inhibition of chlamydial growth [83]. Cellular NO production also has implications for the role of symbiotic microbes in the control of pathogenic microbial infections, as a wide range of host symbiotic microbiota are known to produce NO as a cellular metabolite [84].
A growing trend in research is the role of symbiotic microbes in human health and results indicate that the host microbiome plays a significant role in the inhibition of pathogenic microbial infections. Pollmann et al. explored the efficacy of probiotic feed supplements to reduce rates of chlamydial congenital infections in swine. However, C. suis isolate, S-45, is identical to C. trachomatis serovar D, and consequently is a concern for zoonotic infection, such that limiting the spread of C. suis infection in swine is important for human safety. The large-scale antibiotic treatment of swine is undesirable, which has led to much interest in the development of alternative treatments. Enterococcus faecium is a bacterium that has previously shown beneficial effects as a probiotic feed supplement. When Pollmann et al. used E. faecium as a probiotic in the feed of pregnant C. suis-positive sows for thirteen weeks and then for eight weeks after giving birth, the rate of piglets born infected was reduced from 85% to 60% and the appearance of infection was delayed. The Pollman et al. study was the first to test the effect of probiotics on an obligate intracellular bacteria. It was proposed that E. faecium may function as an antibiotic by reducing the proliferation of bacteria, inhibiting host cell infection, or facilitating a more rapid clearance of the infection. Additionally, as a probiotic, E. faecium is expected to balance the native microbial population of the swine digestive tract, which is beneficial for maintaining the hosts innate defense system [85].
To gain greater insight into the mechanism by which probiotic bacteria may exhibit antichlamydial activity, there are several studies which explore the antichlamydial effect of various lactobacilli on C. trachomatis. Mastromarino et al. explored the antichlamydial effects of the vaginal lactobacilli, Lactobacillus brevis and Lactobacillus salivarius, on C. trachomatis. Both lactobacilli had an adverse effect on chlamydial EBs, on chlamydial adsorption to epithelial cells, and on intracellular phases of chlamydial replication, although L. brevis was significantly more effective than L. salivarius. Significantly, L. brevis inhibited the development of persistent forms of C. trachomatis induced by coinfection with herpes simplex virus type 2 (HSV-2) [86]. Gong et al. went on to explore the mechanism of lactobacilli antichlamydial properties on C. trachomatis by preparing and testing lactobacillus-conditioned media (LCM) from Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. The LCM from each of the lactobacillus strains exhibited similar inhibitory activity. Through pH analysis and modification of the LCM, chlamydial inhibition was shown to be due to acidic pH conditions arising from lactic acid production. As another cellular metabolite of interest, H 2 O 2 , was shown to not inhibit chlamydial activity at levels present in the LCM [87]. Rizzo et al., explored the ability of L. crispatus to influence the infectivity of C. trachomatis. HeLa and J774 cells were infected with C. trachomatis and exposed to L. crispatus and its supernatant. It was determined that L. crispatus and its supernatant had no cytotoxic effect on the epithelial cells or macrophages. Importantly, L. crispatus and its supernatant inhibited the adhesion of C. trachomatis cells to human epithelial cells or macrophages, and inhibited C. trachomatis infectivity. The immunomodulatory effect of L. crispatus was evaluated by variations in the expression of inflammatory cytokines, IL-6, IL-8, TNF-α, and IL-10. It was observed that L. crispatus and its supernatant reduced the production of the pro-inflammatory cytokines, IL-6, IL-8, and TNF-α. In contrast, L. crispatus and its supernatant significantly increased the production of the anti-inflammatory cytokine, IL-10. L. crispatus commonly resides in the urogenital microbiome of healthy women and these results suggest that increasing the presence of such microbes can play an important role in protecting the genitourinary tract against pathological conditions [88]. Nardini et al. performed a comprehensive study of eight strains of L. crispatus, six strains of L. gasseri, three strains of Lactobacillus vaginalis, and lactic acid as a lactobacilli cellular metabolite, on the infectivity of C. trachomatis. All lactobacilli exerted a strong inhibitory effect, although, L. crispatus exhibited the highest efficacy. Greater antichlamydial activity was correlated to increased cellular metabolites resulting in a lower pH, and the acidic conditions produced by lactic acid production were shown to be necessary for chlamydial inhibition. However, lactobacilli supernatants exhibited greater inhibition than only lactic acid, suggesting synergism with other lactobacilli metabolites. Interestingly, both shorter EB/lactobacilli supernatant incubation times, as well as increased lactobacilli consumption of glucose by the most active strains, were related to higher inhibitory activity [89]. Overall, these studies indicate the potential of a range of probiotics in inhibiting and managing chlamydial infections. Swine consuming E. faecium for 13 weeks before and 8 weeks after giving birth, reduced the rate of infected piglets from 85% to 60%. The appearance of infection was also delayed. [85] effects, such as skin irritation or sensitization. Neem seeds contain water-soluble polysaccharides that stimulate antitumor and antiviral cytokines, including γ-interferon, which cause cell-mediated immune responses [90]. Praneem is more useful for preventing the spread of infections and treating some symptoms, as the polyherbal cream treats local, not systemic, infections [91,92]. In Phase I clinical trials, daily topical application of 5 mL of the cream for eight days, resulted in C. trachomatis being cleared from the cervicovaginal region of every subject studied [90]. The polyherbal formulation, CH-005, was also developed by Talwar et al. and studied alongside Praneem for its efficacy as a broad-spectrum antimicrobial against reproductive tract infections and sexually transmitted pathogens. The polyherbal formulation, CH-005, comprises purified saponins from S. mukorossi, Mentha citrata oil, and a natural polycationic polymer. In a mouse model study, topical application of either CH-005 or Praneem was effective in blocking the vaginal transmission of C. trachomatis, with CH-005 resulting in a 4.17% transmission rate and Praneem resulting in a 13.9% transmission rate ( Figure 6A). It is claimed that additional studies, at John Hopkins University, to inhibit the vaginal transmission of C. trachomatis with a range of potential microbicides resulted in polyherbal formulations providing the best results [91].
Microorganisms 2016, 4, 39 24 of 31 purified extract from neem (Azadirachta indica) seeds as the main active ingredient. Toxicity studies indicate a lack of side effects, such as skin irritation or sensitization. Neem seeds contain water-soluble polysaccharides that stimulate antitumor and antiviral cytokines, including γ-interferon, which cause cell-mediated immune responses [90]. Praneem is more useful for preventing the spread of infections and treating some symptoms, as the polyherbal cream treats local, not systemic, infections [91,92]. In Phase I clinical trials, daily topical application of 5 mL of the cream for eight days, resulted in C. trachomatis being cleared from the cervicovaginal region of every subject studied [90]. The polyherbal formulation, CH-005, was also developed by Talwar et al. and studied alongside Praneem for its efficacy as a broad-spectrum antimicrobial against reproductive tract infections and sexually transmitted pathogens. The polyherbal formulation, CH-005, comprises purified saponins from S. mukorossi, Mentha citrata oil, and a natural polycationic polymer. In a mouse model study, topical application of either CH-005 or Praneem was effective in blocking the vaginal transmission of C. trachomatis, with CH-005 resulting in a 4.17% transmission rate and Praneem resulting in a 13.9% transmission rate ( Figure 6A). It is claimed that additional studies, at John Hopkins University, to inhibit the vaginal transmission of C. trachomatis with a range of potential microbicides resulted in polyherbal formulations providing the best results [91]. BASANT is a polyherbal formulation developed by Bhengraj et al. that can be used as a cream or tablet as a vaginal microbicide, and the cream has gone through Phase II clinical trials in India. The incubation of cells with BASANT, both before inoculation with doxycycline-resistant C. trachomatis and after, showed antimicrobial activity against sexually transmitted serovars of C. BASANT is a polyherbal formulation developed by Bhengraj et al. that can be used as a cream or tablet as a vaginal microbicide, and the cream has gone through Phase II clinical trials in India. The incubation of cells with BASANT, both before inoculation with doxycycline-resistant C. trachomatis and after, showed antimicrobial activity against sexually transmitted serovars of C. trachomatis. BASANT is comprised of Aloe vera, curcumin, saponins from S. mukerossi, and amla (Phyllanthus emblica).
It is proposed that the components have a synergistic effect that enhances the properties of each ingredient and broadens the spectrum of treatable infections. A. vera has wound healing properties and has been shown to inhibit HIV and HPV. Curcumin has antiseptic, anti-inflammatory, and antitumor properties. Amla has antioxidant, anti-inflammatory, and antimutagenic properties. In pre-infection in vitro studies, HeLa cells were infected with C. trachomatis serovar D and exposed to BASANT solutions of varying concentration. Based on pre-incubation exposure, complete inhibition was achieved with 15 min incubation at a concentration of 65 µg/mL, 30 min incubation at 35 µg/mL, and 60 min incubation at 15 µg/mL ( Figure 6B). Based on in vitro post-incubation exposure, the minimum inhibitory concentration (MIC) was determined to be~9 µg/mL ( Figure 6C,D). With C. trachomatis isolates from a doxycycline treatment failure patient, the MIC was 30 µg/mL. There are no known side effects of BASANT, which is equally effective as a cream or tablet [93,94]. In vitro pre-incubation exposure, 100% inhibition was achieved in 15 min at 65 µg/mL, 30 min at 35 µg/mL, and 60 min at 15 µg/mL. In vitro post-incubation exposure, the MIC was determined to bẽ 9 µg/mL. There are no known side effects of BASANT, which is equally effective as a cream or tablet. [93,94]

Conclusions
Through the utilization of various compound classes identified within these studies it seems likely that ongoing research in this field could lead to the development of effective antichlamydial products and treatment strategies. A wide range of natural compounds have been studied which exhibit varying degrees of antichlamydial activity. Polyphenolic compounds have been explored from commonly available sources, such as tea, mint, bark, clover, and traditional herbal therapeutics. Lipidic compounds have been explored in the form of purified fatty acids, breast milk lipids, and tree heartwood terpenoids. Proteinaceous compounds have been explored in the form of aqueous protein extracts from desert truffles, various peptides extracted from humans, pigs, sheep, cattle, moths, bee venom, and frog skin, and iron-binding glyco-proteins from humans, pigs, and chickens. Cellular metabolites & probiotics have been explored in the form of nitric oxide (NO), lactic acid, E. faecium, and six species of lactobacilli. Perhaps most importantly, three polyherbal formulations, utilizing various extracts from reetha tree fruit, cinchona bark, neem seeds, lemon mint, Aloe vera, turmeric, and Indian gooseberry, have demonstrated significant antichlamydial activity and highlight the potential for utilizing existing natural products to develop effective antichlamydial therapeutics.
To ensure long-term effective management of all Chlamydiaceae-related infections we should both prepare for the further development of clinical antibiotic resistance, as well as develop new options for providing better livestock management with the aim of preventing the development of antibiotic resistance. The exploration for new naturally derived or synthetic compounds is important, however, many modern antibiotics are already derivatives of natural products [95], and the further development of antibiotic resistance is always a possibility [96]. Although working with natural products is often challenging due to the biochemical complexity typically present, it is likely that this biochemical complexity offers a more reliable solution to infectious disease management.
Antibiotic resistance is the result of the natural evolutionary struggle between pathogens and hosts, and is often due to mutations in pathogenic bacteria, which eventually result in evolutionary solutions to improve on compounds with single mechanisms of action [96]. A standard approach to treating antibiotic resistant pathogens is multidrug therapy wherein two or more antibiotics are administered in an attempt to circumvent a pathogen's ability to quickly adapt to a single stressor [97]. A multi-drug treatment may be more effective in treating chlamydial infections than a single drug approach, and it is important to devote greater effort into exploring synergistic antichlamydial compound combinations [98], as well as pursuing greater standardization of herbal therapeutics. Growing application of nanomedicine formulations for natural products in the context of antimicrobial applications of various kinds also deserves attention and could lead to improved formulations [99]. Overall, natural products show significant potential in treating chlamydial infections and the development of these products into novel drugs may help in the global management of Chlamydiae-related infections.