Mechanism of action, resistance, synergism, and clinical implications of azithromycin

Abstract Background Azithromycin (AZM), sold under the name Zithromax, is classified as a macrolide. It has many benefits due to its immunomodulatory, anti‐inflammatory, and antibacterial effects. This review aims to study different clinical and biochemisterial aspects and properties of this drug which has a priority based on literature published worldwide. Methods Several databases including Web of Science, Google Scholar, PubMed, and Scopus were searched to obtain the relevant studies. Results AZM mechanism of action including the inhibition of bacterial protein synthesis, inhibition of proinflammatory cytokine production, inhibition of neutrophil infestation, and macrophage polarization alteration, gives it the ability to act against a wide range of microorganisms. Resistant organisms are spreading and being developed because of the irrational use of the drug in the case of dose and duration. AZM shows synergistic effects with other drugs against a variety of organisms. This macrolide is considered a valuable antimicrobial agent because of its use as a treatment for a vast range of diseases such as asthma, bronchiolitis, COPD, cystic fibrosis, enteric infections, STIs, and periodontal infections. Conclusions Our study shows an increasing global prevalence of AZM resistance. Thus, synergistic combinations are recommended to treat different pathogens. Moreover, continuous monitoring of AZM resistance by registry centers and the development of more rapid diagnostic assays are urgently needed.

and body fluids. 4 Due to the reversible cutting of the 50S bacterial ribosomal subunit, AZM inhibits protein synthesis and hinders the growth of bacteria. 5,6 Moreover, it can penetrate into bacterial extracellular vesicles, a kind of secretory defense system. 5 2 | PHARMACOLOGY

| Pharmacodynamic of AZM
Azithromycin is classified as a macrolide antibiotic because of its unique ability. 5 In virtue of its dual-base structure, AZM is actively absorbed by a variety of cells, including fibroblasts and white blood cells. 7 This antibiotic agent works in vitro against many pyogenic bacteria (e.g., Neisseria gonorrhoeae [N. gonorrhoeae] and Moraxella catarrhalis [M. catarrhalis]) and beta-lactam-resistant bacteria (e.g., Legionella and Chlamydia spp.). 8 AZM has immunomodulatory, anti-inflammatory, and antibacterial modulatory effects; thus, it is beneficial for patients with varying inflammatory diseases of the respiratory tract. 9 AZM is also effective in patients with COVID-19 and has been used in clinical trials for the prevention of bacterial infection in these patients. It has been reported that AZM in combination with hydroxychloroquine (HCQ) can mitigate the viral load of SARS-CoV-2. 10 Moreover, AZM can modulate the features of the immune system, that is, reducing cytokine production, maintaining epithelial cell integrity, and preventing lung fibrosis. 11 Treatment with AZM involves a short period of time. The method of its administration in adults is 1500 mg immediate-release (IR) AZM, that is, 500 mg once daily for 3 days or 500 mg on the first day and 250 mg on Day two up to Day five. 12 The highest oral dose approved for the treatment of gonococcal urethritis is 2.0 g of IR AZM. 12 2.2 | Structure of drug AZM (9-deoxo-9a-methyl-9a-aza-9a-homo erythromycin A) with the chemical formula C 38 H 72 N 2 O 12 is produced by replacing carbonyl (9a) in the aglycone ring with methyl nitrogen. Unlike erythromycin (ERY), AZM improves the durability and strength, blocks the internal reaction for hemiketal formation, and leaves the acid hydrolysis of the ether bond to the neutral sugar of L-cladinosis, as the main decomposition pathway (Figure 1). 13

| Mechanism of action
Similar to other macrolide antibiotics, the main objective of AZM is inhibiting bacterial protein synthesis by targeting the 50S subunit of the sensitive bacterial ribosome (Figure 2). The reduction in protein synthesis is correlated with the increase in macrolide concentration. 14 The unionized form of AZM membrane passage rate is higher, and this could be the reason behind the increased antimicrobial activity of AZM at alkaline pH. 15 AZM binds at a site near peptidyl transferase center on 23S rRNA called nascent peptide exit tunnel (which is approximately 100 Å long and 10-20 Å wide) and partially occludes it. 16,17 The binding process of AZM is almost similar to erythromycin. Resting of erythromycin on a surface formed by three bases (U2611, A2058, and A2059), utilizing three axial methyl groups belonging to the lactone ring of the drug is the key to this process based on research on H. marismortui. There is also a hydrogen bond between the 2′ OH group of the desosamine sugar of erythromycin and the N1 atom of A2058, which stabilizes erythromycin in its position. These interactions result in base movement and nascent peptide exit tunnel occlusion due to the placement of bases within van der Waals contact of the amino group of P-site tRNA. 18 Novel findings show that the context of the nascent peptide has an important role in changing the possibility of being allowed to pass from the peptide exit tunnel, namely AZM does not completely occlude the passage (although the nascent peptide exit tunnel has various responsibilities rather than being a normal passage to the cytoplasm such as modulating the ribosome functions in response to sequences of the novel peptide and environment). 16 These events result in faster penetration of the outer membranes; hence, it has effects on the entrance into the bacteria and increases the activity against Gram-negative bacteria. 17 AZM also showed anti-inflammatory effects on various studies; for instance, Cigana et al. demonstrated that AZM reduces TNFα mRNA expression, TNFα protein levels, and NF-κB DNA-binding activity in human cystic fibrosis (CF) cell lines subsequent to the confirmation of a higher rate of TNFα mRNA expression, TNFα protein levels, and NF-κB DNA-binding activity in CF cell lines compared with isogenic non-CF cell lines. 19 The reduction in NF-κB DNA-binding activity is associated with the inhibition of the degradation of IκBα, the protein that prohibits the translocation of NF-κB active subunits into the nucleus. 20 Inflammatory cell F I G U R E 1 Chemical structure of azithromycin (https://go.drugb ank.com/drugs/ DB00207, accessed on December 18, 2021) signaling is affected by AZM, and these impacts include a decrease in NF-κB (and subsequent IL-6 and IL-8 production), which is mentioned above, inhibition of LPS-induced expression of PLA2, which is involved in cytokine and chemokine production in macrophages, neutrophils, and endothelial cells and cell signaling pathways, which result in arachidonic acid and eicosanoids production, and inhibition of AP-1 signaling in neutrophils isolated from the lungs of mice induced by LPS administration, which consequently reduce IL-1b concentrations. 21 AZM affects neutrophils directly and indirectly. 21 The anti-inflammatory properties of AZM are the reason behind the indirect effects of AZM on neutrophils. Direct effects include reduction in IL-8 release and neutrophil airway infiltration, degranulation and degradation of extracellular myeloperoxidase, reduction in neutrophil oxidative burst, 22,23 and decrease in the production of leukotriene B4 (LTB4; a potent neutrophil chemoattractant that stimulates neutrophil IL-8 release). 24 AZM also helps macrophages shift from M1 type to M2 alternative-like phenotype in vitro by inhibiting pro-inflammatory cytokine expression (including IL-12 and IL-6) and shifting surface receptor expression. 25

| Pharmacokinetic parameters
Demethylation is the major route of metabolism, and the metabolites are not considered to have any significant antimicrobial activity. 26 As a result of oral administration, the bioavailability of AZM reached 37%. AZM absorption may be dropped by up to 50% when administered with a large meal. 27 AZM coadministration with aluminum-and magnesium-containing antacids may reduce peak plasma concentrations by 24%, but the overall extent of absorption is not altered. 28 The mean plasma clearance of AZM following a single 500 mg oral and intravenous dose is 630 ml/min. The primary route of AZM elimination, particularly as an unchanged drug, is through biliary excretion, and the feces are a prominent route of elimination. 26 Moreover, over a period of 1 week, approximately 6% of the administered dose is discharged as an unchanged drug in urine; therefore, urinary excretion of AZM appears to be a minor elimination route. 26  The long-term studies have demonstrated AZM has no carcinogenic and mutagenic potential in standard laboratory animals and tests. 28 The main possible adverse effects related to AZM include gastrointestinal upset, headache, dizziness, hearing loss, and cardiovascular arrhythmias. In rare cases, hepatotoxicity has been reported. In patients with a prolonged QT interval, disturbed hepatic function, and renal GFR <10 ml/min, caution should be taken when administrating AZM. 28,30

| New formulation of AZM
A new formulation of AZM, designed as a microsphere with longterm release (ER) to delay the release of AZM, is released slowly through bypassing the upper gastrointestinal tract after reaching its lower part. In this method, by alkalizing the formulation, elevation in the pH of the suspension minimizes the release of the drug from the microspheres in the mouth and stomach and the microsphere matrix. AZM is soluble, and this feature helps control the drug release. It spreads through the pores formed at the site of the microspheres. This ER formulation does not significantly compromise the oral bioavailability of AZM, although it bypasses a small portion of the uptake site in the upper gastrointestinal system. It achieved approximately 83% bioavailability over the IR formulation, the released microsphere formulation of AZM, allowing patients to well tolerate a full course of AZM at a dose of 2.0 g. This formulation should be taken on an empty stomach together with antacids. 31 A new oral-free release microsphere formulation of AZM is the first antibacterial drug approved in the USA for adult patients with mild-to-moderate acute bacterial sinusitis or community-acquired pneumonia. 32 The mentioned formulation of AZM is an oral powder that should be reconstituted with water and given in a single dose of 2.0 g. Continuous release of the drug is achieved through F I G U R E 2 Schematic view of AZM mechanism inhibiting translation of mRNA diffusion from the microspheres; the time to reach a peak serum concentration is 5 h. AZM is well absorbed by free release. The mean maximum serum concentration is 0.82 μg/ml, and AUC24 is about 8.62 μg/ml. Free-release AZM should be taken on an empty stomach to ensure slower absorption. AZM is mainly excreted unchanged in feces. The final half-life of AZM secretion is 59 h. 33 Drug delivery to the site of infection by phagocytes and fibroblasts is characterized by tissue-directed AZM, which provides 5-day once-daily diets for most infections that respond to oral therapy and 7-10 days for more serious intravenous infections. Metabolism occurs through hepatic pathways other than cytochrome P450, thus minimizing the risk of drug interactions. 8

| Activity in biofilms
The potential role of AZM as an antibiofilm has been studied and shown to have a planktonic state when used in aerobic conditions. It has been observed that AZM can significantly inhibit the formation and motility of biofilm in Pseudomonas aeruginosa (P. aeruginosa). 34 Inhibition of biofilm mass in Porphyromonas gingivalis has also been reported among the AZM-treated isolates. 35 AZM in combination with Dapsone can decline the glycosaminoglycan and durability of biofilms produced by Borrelia burgdorferi isolates. 36 Additionally, when combined with ciprofloxacin (CIP) or rifampin, AZM is able to completely kill the biofilm of Bartonella henselae within 6 days. 37 The antibiofilm activity of the AZM pattern has also been studied among Stenotrophomonas maltophilia isolates and demonstrated that AZM/ tigecycline combination can hamper the formation of biofilms. 38

| Mechanisms of resistance
Like other drugs, the suboptimal use of AZM has been assumed the most important cause of the development of resistant bacteria. The administration of an improper dose or duration of treatment results in the emergence and spread of resistant organisms (Table 1). 39 Two strategies have been generally involved in gonococcal resistance against AZM: The mutations in the mtrR coding region resulted in overexpression of the MtrCDE efflux pump. Moreover, the affinity of N. gonorrhoeae to AZM decreases due to mutations in genes encoding the 23S rRNA subunit. 39 The modification of the drug target is associated with methylation of the 23S ribosomal subunit (related to the presence of erm genes) or by mutations in rrl alleles of the 23S rRNA gene, which blocks macrolide binding to this subunit. 40 The molecular basis of the AZM resistance mechanism in P. aeruginosa showed that the overexpression of efflux pumps particularly mexAB-oprM and mexCD-oprJ 41 and mutations in the ribosomal target of drugs in the 23S rRNA gene can cause the development of resistant strains in the biofilm community of cystic fibrosis patients. 42 Although the better permeability and higher intracellular uptake of AZM resulted in the better activity of this antibiotic, the majority of macrolides are ineffective against Enterobacteriaceae due to intrinsic low macrolide permeability. In Enterobacteriaceae, the relevance of 23S rRNA alterations as being responsible for macrolide resistance is low since E. coli, Salmonella spp., Shigella spp., and Klebsiella spp. possess up to one or more rrn loci. 43 The methylation of 23S rRNA mediated by methylases encoded in erm genes is the most relevant mechanism of macrolide resistance.
These genes have been located in mobile elements such as plasmids carrying more than one erm gene. 43,44 Another type of modification related to macrolide resistance is pseudouridylation of 23S rRNA. Studies showed that mutations of the rplD gene contributed to less sensitive C. trachomatis serovar L2 isolates to AZM and ERY.
It has been reported that the mutations in L4 protein conclude in the conformational modification of the 23S rRNA in domains II, III, and V resulting in disorder in the translational activity of ribosomes.
Moreover, mutations in the peptidyl transferase region of 23S rRNA genes and the non-conserved region of the protein L22 have been seen in clinical isolates resistant to C. trachomatis. 45 The dramatic increase in macrolide-resistant Treponema pallidum (T. pallidum) spp.
pallidum has been reported since 2000. The emergence of macrolide resistance isolates evolves by a two-step process including either A2058G or A2059G mutation in one copy of the 23S rRNA that subsequently results in gene conversion of both rRNA genes. 46 The resistance mechanism of S. pneumoniae is associated with horizontal gene transfer of efflux pump Mef (E) genes. Moreover, streptococcal methylase ErmB can develop high-level crossresistance to macrolides through methylation of A2058 nucleotide of 23S rRNA. Other mechanisms, including mutations in domain V of 23S rRNA and in ribosomal proteins L4 or L22, can also appear more rarely in macrolide resistance isolates of S. pneumoniae. 47 The genetic mechanism of macrolide resistance of S. aureus strains isolated from cystic fibrosis patients has been well documented by mutations in genes of 23S rRNA domain II, V (rrl), and ribosomal protein L4 (rplD) and L22 (rplV). In addition, acquired resistance genes such as erm (encoding a ribosomal methylase) and msr(A) (encoding an efflux protein) can lead to macrolide resistance in S. aureus strains. 48 Although Salmonella isolates have intrinsic resistance to ERY which is associated with active efflux of drugs, these strains are naturally susceptible to AZM. Resistance to macrolides is related to mutations in nucleotides A2058 and A2059 of 23S rRNA domain V.
Additionally, the modification of the 50S ribosomal subunit proteins L4 and L22 may contribute to macrolide resistance. 49 Haemophiles influenzae strains are intrinsically resistant to macrolide due to the presence of a homologous efflux pump to the acrAB efflux mechanism in E. coli or other efflux pumps. In a few strains, higher MICs related to mutations in 23S rRNA and L4 and L22 ribosomal proteins. 50 In Legionella pneumophila strains, mutations in the upstream sequence of lpeAB (lpp2879-lpp2880) operon result in the overexpression of protein products. Lpp2879-Lpp2880 together with TolC forms a tripartite efflux pump of the resistance-nodulation-division (RND) family. Moreover, in AZM-resistant isolates, mutations in 23S rRNA genes and L4/L22 ribosomal proteins have been identified. 51 In Campylobacter spp., the most common mechanism for high-level resistance to macrolides is substitutions in the domain V of the 23S rRNA gene (A2075G, A2074C/G). 52 The substitutions and insertions in ribosomal proteins are another resistance mechanism in the absence of mutations in 23S rRNA genes. Moreover, CmeABC efflux pumps (a member of the RND transporter family) have an important role in resistance to macrolides. 52 These three mechanisms synergistically contribute to high-level macrolide resistance. 53 Another mechanism of macrolide resistance in Campylobacter spp. is antibiotic exclusion through the major outer membrane porin (MOMP). Campylobacter spp. can alter membrane permeability mediated by overexpression of MOMP, chromosomally encoded by porA. 53 A novel mechanism for resistance in E. coli isolates associated with erm (B) transferred by multidrug resistance

Mechanisms of resistance References
Neisseria gonorrhoeae (1) Over expression of an efflux pump (due to mutations at mtrR coding region) (2) Decreased antimicrobial affinity (due to mutations in genes encoding the 23S ribosomal subunit) 39,40 Pseudomonas aeruginosa (1) Efflux pump of P. aeruginosa confers resistance to AZM during biofilm formation (2) Mutations in the 23S rRNA gene 41,42 Enterobacteriaceae (1)

| South America and Caribbean
Most published studies from America have examined the rate of AZM resistance and related mechanisms of Shigella spp. isolates.
The resistance rate of AZM has been reported at 23.5-100% among Shigella spp. [54][55][56] (Table 2). Although mostly mphA plasmid-encoded genes were reported as determinants of reduced susceptibility to AZM in these isolates, ermB is identified in Shigella spp. isolated from men who have sex with men in Canada. 56 The gonococcal AZM susceptibility in South America and the

| Asia
Several studies on the resistance of Neisseria isolates to AZM have been reported from East Asia. These studies examined the susceptibility of N. gonorrhoeae isolates between 2009 and 2016, and the calculated AZM resistance ranged between a high of 28.6% and 3.6% of isolates tested in China. [83][84][85][86] Moreover, two studies from India and Taiwan presented the overall percentage of AZM resistance in N. gonorrhoeae isolates-5% and 14.6%, respectively. 75,79 The resistance mechanism mentioned in relation to these resistant isolates was mutations in 23S rRNA, penA, and mtrA genes. 83

| Africa
The assessment of antibiotic susceptibility of diarrhoeagenic bacteria collected from children in Morocco showed the prevalence of AZM resistance of Shigella spp., E. coli, and Salmonella spp. was 11.1%, 15.5%, and 20% respectively; however, the mechanisms involved in the antibiotic resistance of these isolates have not been identified. 69  and has a successful history in malaria treatment. 97 Ohrt et al.
demonstrated that CQ/AZM combination is efficacious against CQ-resistant Plasmodium falciparum (P. falciparum) and AZM has an additive to synergistic activity on CQ in vitro and this therapy should be evaluated for malaria prophylaxis. 98 In an Indian study, it was indicated that CQ/AZM combination is much more effective than AZM or CQ alone as single-drug therapy in P. falciparum treatment. 99 To discover the reason behind this synergy, Cook et al. performed a study, which revealed that synergism is not due to a systemic drugdrug interaction or the following factors: (1) the enhancement of exposure to one or both drugs because of improved bioavailability; (2) a decrease in clearance. 100  Quinine is also a quinoline-containing drug that is effective against P. falciparum-induced malaria. 97 It is claimed that Quinine/ AZM drug therapy is the best way to counteract MDR-resistant P. falciparum in vitro. 101 A randomized, dose-ranging study in Thailand indicated that the combination of Quinine/AZM (quinine: 30 mg salt/kg divided three times a day and AZM: ≥1 g/ day for 3 days) was effective against MDR-resistant P. falciparum malaria. 106 In a case of uncomplicated P. falciparum malaria, a randomized, phase 2 clinical trial was conducted in Thailand, which showed high cure rates for Quinine/AZM combination plus quinine, for a total dose of 4.5 g of AZM plus 60 mg/kg quinine or 3 days of AZM plus quinine. This study also demonstrated that AZM, which is a slow-acting drug, should be combined with a fast-acting drug to reach a quicker initial parasite clearance. 107

| Synergism against Pythium insidiosum
Pythiosis is a zoonosis disease caused by a fungus-like pathogen, named Pythium insidiosum (P. insidiosum), which presents many clinical manifestations based on the type of infection. 110 Jesus et al. demonstrated that AZM has synergistic effects with some antifungal agents such as terbinafine, amphotericin B, itraconazole, voriconazole, micafungin, caspofungin, and anidulafungin against P. insidiosum in vitro. 111 Furthermore, in another study, AZM showed a synergistic effect with Carvacrol and Thymol against P. insidiosum in vitro. 112 A lack of antagonism between AZM and topical drugs such as benzalkonium, cetrimide, cetylpyridinium, mupirocin, and triclosan in vitro and a lack of topical therapeutics against P. insidiosum suggests that these combinations may provide a potential therapy for pythiosis treatment. 113 In vivo studies showed that AZM could be a remarkable anti-P. insidiosum therapy in combination with minocycline or alone. 114

| Synergism against Naegleria fowleri
Naegleria fowleri is an ameba that causes a rapidly fatal infection called primary amebic meningoencephalitis (PAM) in humans.
Amphotericin B, a broad-spectrum drug, acts against most human fungal pathogens and is used to treat PAM. 115 Soltow et al. mentioned that AZM has synergistic effects with amphotericin B against Naegleria fowleri; each of these drugs had less than 50% efficacy while administrated alone; however, when they were utilized with each other, the combination had 100% efficacy in vitro; therefore, it might be an acceptable regimen to treat PAM. 116

| Synergism against Pseudomonas aeruginosa
Due to increasing CIP-resistant P. aeruginosa isolates, new approaches should be further investigated to treat the caused infections. 117 Combination therapy might be the key to this subject. After the synergism of CIP and AZM was confirmed in vitro, on the peak infection day, the use of CIP/AZM combination improved clearance from the kidney and bladder and exhibited anti-inflammatory and immunomodulatory effects in P. aeruginosa biofilm induced acute pyelonephritis. 118 Saini et al. showed that this combination can also be used as a material to construct a special catheter that prevents catheter-associated urinary tract infections in vitro. 119 The efficacy of a novel CIP/AZM sinus stent (CASS) was evaluated in a subsequent study, and results showed that CASS delivers a sustainable amount of CIP and AZM which causes antibiofilm activity against P. aeruginosa in vitro. 120

| Synergism against Escherichia coli
Colistin, also called polymyxin E, is a molecule that is often used as a last-line therapy to treat MDR-resistant Gram-negative bacteria and can be administered either intravenously or in oral form. 123 Li The absence of antagonism is the reason for the continuation of this therapy. 128

| Asthma
Azithromycin accumulates in the lysosomes of phagocytic cells. In the lungs, the concentration of macrolides in neutrophils and macrophages is much higher than that measured in extracellular compartments. This information represents important cellular sites of immunomodulatory function in asthma. 129

| Bronchiolitis
Azithromycin is mostly used to treat lung infection and viral bronchiolitis. 132 In a secondary analysis of a randomized double-blinded

| Chronic obstructive pulmonary disease (COPD)
Azithromycin has been shown to have the greatest effect on subjects with COPD. 135 In a retrospective observational study, Naderi et al. randomized patients to receive AZM (250 mg, at least three times weekly for at least 6 months (n = 126) or neither (n = 69)). In AZM-treated patients, the rate of exacerbations per patient in a year before the treatment period was 3.2, but during the following year on therapy, the rate was 2.3. In the control group, the exacerbation rates were 1.7 and 2.5 during the first and second follow-up year, respectively. Therefore, long-term AZM reduced the rate of exacerbation in severe COPD patients. 136 Han et al. carried out a secondary cohort analysis study to demonstrate the effect of AZM in reducing exacerbation in COPD patients. They randomly grouped 1113 COPD patients, of which 557 and 556 subjects were received AZM and placebo, respectively.
For a year, an AZM dose of 250 mg or placebo was prescribed daily.
AZM was more effective than placebo in reducing COPD, although antibiotic and steroid therapy was required. The data also uncovered that AZM is more effective in older patients and patients with mild illness. 137 In a randomized double-blinded placebo-controlled trial performed by Uzun

| Cystic fibrosis
Azithromycin has displayed great effects on cystic fibrosis patients.

| Sexually transmitted infections
Azithromycin has been shown to be highly efficient in bacterial STIs caused by C. trachomatis, N. gonorrhoeae, and T. pallidum. 144  Overall, the data show that the global prevalence of AZM resistance is increasing among bacteria. Resistance to AZM is developing similar to many other drugs; therefore, synergistic combinations are prescribed and being studied to confront different pathogens.
Therefore, it is necessary to discover the AZM mechanism of action and the underlying mechanism behind the synergism with different drugs that effectively act against different organisms. A great variety of combinations could be studied in various outlooks including synergism and effects on the human body and different AZM combinations are no exception. Therefore, continuous monitoring of AZM resistance by AST methods, the establishment of an antibiotic resistance registry center, using electronically reporting systems, and the development of more rapid diagnostic assays are recommended.

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest in this work.