Antibiotic Combination Therapy: A Strategy to Overcome Bacterial Resistance to Aminoglycoside Antibiotics

Streptomycin was the first discovered aminoglycoside antibiotic (AGA) to be used for tuberculosis treatment in the mid-1940s ( Schatz et al., 2005 ). Thereafter, a series of aminoglycoside antibiotics (AGAs) were discovered, including neomycin (1949), gentamicin (1963), tobramycin (1967), sisomycin (1970), amikacin (1972), and plazomicin (2006) ( Figure 1 ), and all were found to have good antibacterial activities not only for gram-negative bacteria but also for some gram-positive bacteria ( Becker and Cooper, 2013 ; Clark and Burgess, 2020 ). AGAs are composed of amino sugars and aminocyclic alcohols. 2-deoxystreptamine (2-DOS) is the central unit, which is connected with 2–3 sugar units through glycosidic bonds ( Figure 2 ) ( Stead, 2000 ). According to the structural differences, AGAs are divided into two categories: one is with a 2-deoxystreptamine (2-DOS) core site and another is without a 2-DOS core site (e.g., streptomycin). In turn, according to the substituent linkage position the core site is divided into 4,5-disubstituted 2-DOS and 4,6-disubstituted 2-DOS. The chemical structure of 2-deoxystreptamine is as follows ( Figure 2 ). The structural feature of 4,5 disubstituted 2-DOS AGAs is that the hydroxyl groups at the C-4 and C-5 positions of the 2-DOS ring are substituted and connected to the sugar ring by a glycosidic bond; in 4,6-disubstituted 2-DOS AGAs, the hydroxyl groups at the C-4 and C-6 positions of the 2-DOS ring are substituted and connected to the sugar ring by a glycosidic bond. These sugar units generally have multiple hydroxyl and amino groups, so they have the advantages of good water solubility, good functionality and wide antibacterial spectrum ( Johnston et al., 2002 ). FIGURE 1 Timeline of aminoglycoside antibiotics development. FIGURE 2 Chemical structure of 2-deoxystreptamine (2-DOS). However, because of the widespread use of these antibiotics, side effects such as nephrotoxicity, ototoxicity and neuromuscular blockade have been found, and bacterial resistance has become more and more frequent. Therefore, the third-generation AGAs such as amikacin (1972), arbekacin (1973), and netilmicin (1976) developed ( Kudo and Eguchi, 2016 ). AGAs development can be divided into three stages, also known as three generations, based on chemical structure, antibacterial spectrum and resistance to bacterial modification enzymes ( Lin, 2001 ). The first generation of AGAs is represented by kanamycin, which is characterized by the binding of fully hydroxylated amino sugars and cyclic alcohols and no antimicrobial activity against pseudomonas aeruginosa ( P. aeruginosa ). The second generation is represented by gentamicin, which contains deoxyamino sugars in the structure and has antibacterial activity against P. aeruginosa . The third generation is represented by amikacin, a semi-synthetic derivative of amino cyclic alcohol substituted at the nitrogen position. The third generation of AGAs is characterized by preserving their antibacterial activity while reducing their side effects after modification of their mother nucleus, and has the potential for clinical application due to its characteristics of low drug resistance potential and high plasma concentration ( Ristuccia and Cunha, 1982 ). However, it was subsequently discovered that these AGAs had limited antibacterial activities against aminoglycoside-modifying enzyme (AME)-producing bacteria. With the development and use of broad-spectrum antibiotics with fewer side effects such as β-lactam antibiotics and fluoroquinolones, the market shares of AGAs decreased and the search for new AGAs has been scaled back. Plazomicin (formerly ACHN-490) is a new type of parenteral drug targeting multidrug-resistant Enterobacteriaceae , including AME-producing bacteria, extended-spectrum beta-lactamase (ESBL) and carbapenemase of microorganisms ( Haedersdal et al., 2016 ; Castanheira et al., 2018 ), which was approved by the Food and Drug Administration (FAD) in June 2018 for the treatment of complicated urinary tract infections (cUTI) and pyelonephritis caused by microorganisms ( Saravolatz and Stein, 2020 ). It became the first FDA-approved AGAs after amikacin was approved in 1981, marking the return of AGAs to the market ( Figure 1 ). In 2013, the Center for Disease Control (CDC) released the first threat report on antibiotic-resistant (AR) bacteria in the United States. The CDC’s US Antibiotic Resistance Threat 2019 (AR Threat Report 2019) includes up-to-date estimates of the number of deaths and bacterial infections nationwide, re-emphasizing the ongoing threat of antibiotic resistance in the United States. More than 2.8 million infections resulting from antibiotic resistance occur in the United States each year, and more than 35,000 people die from infections. New CDC data show that despite the growing threat of antibiotic resistance in the United States, for example, erythromycin-resistant invasive group A streptococcus increased by 351%, drug-resis

3.1 Enzymatic Modification of Aminoglycoside Antibiotics Among various bacterial resistance mechanisms to AGAs, AMEs is the most common mechanism ( Wachino et al., 2020 ). AMEs are divided into three aminoglycoside modifying enzyme families: phosphotransferases (APHs), acetyltransferases (AACs), and adenyl transferases (ANTs) ( Sparo et al., 2018 ). These AMEs use cofactors, acetyl-coenzyme A or ATP to modify NH 3 or OH groups of AGAs, thereby inactivating them. The genes encoding AMEs are usually carried in plasmids, which promotes the bacterial-bacterial spread of aminoglycoside resistance ( Bassenden et al., 2016 ). Clinically isolated aminoglycoside resistant strains often harbor multiple AME genes, and it is reported that the AME gene is encoded on the same plasmid as the 16S-ribosomal RNA methyltransferases (RMTase) gene ( Garneau-Tsodikova and Labby, 2016 ; López Díaz et al., 2017 ). The three types of AMEs comprise many subtypes, each with a certain specificity for resistance to AGAs ( Garneau-Tsodikova and Labby, 2016 ). 3.1.1 Acetyltransferases Acetyltransferases (AACs) acetylate the amino group (-NH 2 ) on aminoglycoside molecules, and are members of the N-acetyltransferase (GNAT) superfamily, which are thought to be related to general control non-depressible 5 (GCN5) ( Ramirez et al., 2013 ). The enzyme is divided into four subtypes according to the location of catalytic acetylation: AAC (1), (2′), (3), and (6′) ( Ramirez and Tolmasky, 2010 ). AAC (6′) enzymes are widespread in gram-negative as well as gram-positive bacteria and are by far the most common, and their genes have been found in plasmids and chromosomes, and are part of mobile genetic elements ( Tolmasky, 2000 ; Centrón and Roy, 2002 ; Soler Bistué et al., 2008 ). A group of gram-negative and gram-positive bacteria caused by nosocomial infections called as ESKAPE ( E. faecium , staphylococcus aureus ( S. aureus ), klebsiella pneumoniae ( K. pneumoniae ), acinetobacter baumannii , P. aeruginosa , and enterobacter ) are highly resistant to antibiotics including AGAs ( Rice, 2008 ). The most common gram-negative member of the AME genes they carry is aac (6′) -Ib ( Ramirez and Tolmasky, 2010 ; Shaul et al., 2011 ; Herzog et al., 2012 ). N-acetyltransferase AAC (3)-I was established as a selectable marker for gentamicin-based oomycete transformation, and it was found that N-acetyltransferase AAC (3)-I was responsible for gentamicin resistance in phytophthora palmivora and phytophthora infestans ( Evangelisti et al., 2019 ).

Adenyl transferase (ANT) enzymes transfer the adenosine monophosphate (AMP) group in ATP to the hydroxyl group (-OH) of aminoglycoside molecules. There are five classes of this of enzyme: ANT (2″), (3″), (4′), (6), and (9). ANT (2″)-Ia is an enzyme normally encoded by plasmids and transposons, widely distributed as gene cassettes in class 1 and class 2 integrons ( Vakulenko and Mobashery, 2003 ; Ramírez et al., 2005 ). This enzyme mediates resistance to gentamicin, tobramycin, dibecacin, sisomicin and kanamycin of Enterobacteriaceae and non-fermentative gram-negative bacilli ( Ramirez and Tolmasky, 2010 ). Compared with AACs and APHs, ANTs are found less frequently in enterobacteriaceae and P. aeruginosa , but still act as an important role of bacterial resistance to AGAs ( Almaghrabi et al., 2014 ; Holbrook and Garneau-Tsodikova, 2018 ).

3.2.2.1 Mutations of Genes Encoding Porins To take up nutrients, bacteria have evolved many absorption mechanisms. One of which is the formation of water-filled pores extending through the membrane to promote the absorption of hydrophilic compounds. The proteins that form these channels are called porins. Drug resistance caused by porins mutations include: decreased expression, no expression, and structural mutations. Antibiotic resistance caused by changes in porin expression and their mutations might be a limited resistance mechanism because they can also lead to reduced nutrient intake by bacteria ( Garneau-Tsodikova and Labby, 2016 ). AGAs have a hydrophilic structure and a positive charge; therefore, they are believed to penetrate bacterial cell walls through porin channels instead of directly diffusing through the phospholipid bilayer ( Nikaido and Pagès, 2012 ). Beta-lactams, fluoroquinolones, tetracycline antibiotics, and other antibiotics also enter the bacterial membrane through porins, and the loss of porins on the membrane will lead to resistance to these antibiotics. It was found that mutants of E. coli lacking porin OmpF are resistant to AGAs ( Bafna et al., 2020 ). However, the survival rate of the OmpF mutant of E. coli is lower than that of the wild-type; thus it might not be widespread in clinically isolated strains ( Zhao et al., 2014 ).

The ability of bacteria to form biofilm enhances their ability to infect the host, which is usually associated with chronic infections by invasive bacteria ( Yarlagadda and Wright, 2019 ). A biofilm is a collection of microorganisms attached to the surface of living organisms or non-living organisms, and is embedded in the matrix of extracellular polymeric substances (EPSs), including proteins, extracellular polysaccharides, and extracellular DNA (eDNA) ( Das et al., 2013 ). The eDNA produced during cell lysis is an important part of the biofilm. The eDNA in P. aeruginosa can acidify the environment and induce the function of the PhoPQ and PmrAB two-component regulatory system to regulate gene expression, thereby enhancing aminoglycoside resistance ( Wilton et al., 2016 ). The complex structure of a biofilm can protect the bacteria from the host’s immune system and antibacterial drugs ( Lebeaux et al., 2014 ). The general mechanisms by which biofilm-mediated resistance protects bacteria from antibiotic attack include preventing antibiotic penetration, altering the microenvironment to induce slow growth of biofilm cells, induction of adaptive stress responses, and sustained cell differentiation ( Stewart, 2002 ). Biofilm-mediated drug resistance is an adaptive drug resistance, when bacteria lose biofilm protection, antibiotic susceptibility can be rapid ( Walters et al., 2003 ). Chronic infections caused by P. aeruginosa have been found to be usually accompanied by biofilm formation ( Hall and Mah, 2017 ). While the adaptive resistance mechanism of P. aeruginosa infection and recurrence is related to biofilm-mediated resistance ( Pang et al., 2019 ).

Aminoglycoside resistance is usually not caused by bacterial target mutations, because most bacteria have more than one copy of the rRNA coding gene. To induce aminoglycoside resistance through this mechanism requires mutations in every gene copy ( Serio et al., 2018 ). AGAs affect ribosome mutation to produce drug resistance via the rrs and tlyA genes. The rrs gene, encoding 16S rRNA ( Honoré et al., 1995 ), and the tlyA gene, encoding a 2′-O-methyltransferase, an modifies nucleotide C1409 in Helix 44 of 16S rRNA and nucleotide C1920 in Helix 69 of 23S rRNA, respectively ( Johansen et al., 2006 ). It was found that the most common mutations related to AGA resistance in clinically resistant mycobacterium tuberculosis were A1401G, C1402T, and G1484T in the rrs gene ( Georghiou et al., 2012 ).

The carbon source and cellular respiration play important roles in bacterial pathogenicity and antibiotic sensitivity ( Vitko et al., 2015 ; Vilchèze et al., 2017 ). Crc, Hfq and a small RNA CrcZ act as central regulators in carbon metabolism of P. aeruginosa . The catabolite repression control protein (Crc) forms a complex with the RNA chaperone Hfq, and directly inhibits the translation of catabolic genes via binding to the target mRNA ( Sonnleitner et al., 2018 ). Hfq is considered critical for biofilm formation ( Pusic et al., 2016 ), and also contributes to bacterial resistance to antibiotics such as gentamicin ( Pusic et al., 2018 ). The triphosphate isomerase, TpiA, can reversibly convert glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, which is a key step linking glucose metabolism to glycerol and phospholipid metabolism. Mutations in the TpiA gene have been found to enhance carbon metabolism, respiration, and oxidative phosphorylation in bacteria, thereby increasing the membrane potential and promoting AGA uptake. Studies have found that the level and stability of CrcZ in bacteria with TpiA mutations are increased. CrcZ mutations can restore the expression of type III secretion system (T3SS) genes and enhance the resistance of bacteria to AGAs ( Xia et al., 2020 ).

In addition to bacterial resistance, side effects of AGAs are another reason that hinders the clinical application of AGAs. The represent toxic side effects include ototoxicity caused by AGAs, cochlear nerve injury caused by ototoxicity and nephrotoxicity caused by proximal tubular epithelial cell injury ( Kros and Steyger, 2019 ; Jospe-Kaufman et al., 2020 ; Rosenberg et al., 2020 ). However, the antibacterial effect of AGAs in combination with different compounds is better than that when the antibiotics are used alone. The investigation found that the combination cannot only exert a synergistic antibacterial effect but also reduce the dosage of the AGAs, leading to the reduced chance of occurrence and degree of side effects. Herein, synergistic antibacterial effects in combination with AGAs reported in recent years are summarized below. 4.2.1 Combination With Compounds With Antibacterial Activities 4.2.1.1 Combination With Other Antibacterial Agents 4.2.1.1.1 Beta-Lactam Antibiotics Combinations of AGAs such as gentamicin can expand the scope of clinical treatment, accelerate bacterial clearance, and improve antibiotic resistance, especially the because of the synergistic antibacterial effects with β-lactam antibiotics ( Le et al., 2011 ; Rhodes et al., 2017 ). β-lactam antibiotics cause non-fatal damage to the bacterial cell wall, thus promoting AGA entry inro bacteria and enhancing their killing ability ( Davis, 1982 ). AGAs in combination with β-lactam antibiotics are commonly used in severe hospital-acquired infections by multidrug resistant species, such as acquired pneumonia, ventilator-associated pneumonia, and sepsis ( Tamma et al., 2012 ; Sick et al., 2014 ; May 2016 ).

Polymyxins are one of the most commonly used drugs against gram-negative bacteria, especially against P. aeruginosa . The combination of a polymyxin and amikacin presented a synergistic antibacterial effect on polymyxin-sensitive and resistant P. aeruginosa . Polymyxin-sensitive P. aeruginosa (FADDI-PA111) and polymyxin-resistant P. aeruginosa (LESB58) were selected for the combined antibacterial treatment with polymyxin B and amikacin. After their co-administration, a metabonomic analysis showed that the necessary bacterial membrane lipid levels involved in the biosynthesis of phospholipids and lipopolysaccharide (LPS) were reduced significantly, demonstrating that polymyxin B combined with amikacin could inhibit the intermediates in LPS synthesis significantly ( Hussein et al., 2019 ). The pentose phosphate pathway (PPP) is the key source for the synthesis of LPS precursors, and the antibacterial effect might result from early inhibition of the PPP. The study also found that polymyxin B combined with amikacin interfered with the tricarboxylic acid (TCA) and glycolysis pathways in FADDI-PA111, but this effect was not found in LESB58. Bacterial central carbohydrate metabolism, represented by glycolysis and TCA cycle, has been an important target in research into new antibiotics in recent years. The inhibitory effect of polymyxin B combined with amikacin on the pyridine nucleotide cycle (PNC) might also be one of the mechanisms of its synergistic antibacterial effect. The combined treatment resulted in a significant reduction of D-ribose-5-phosphate, a key initial intermediate for purine and pyrimidine metabolism in FADDI-PA111, indicative of nucleotide degradation. The combination of the two drugs had a significant effect on the metabolism of arginine and proline in FADDI-PA111, leading to the destruction of amino acid pathways ( Hussein et al., 2019 ). The disruption of amino acid pathways, represented by arginine metabolism, is considered to be a new way to deal with bacterial infection and destroy their mechanism of action ( Xiong et al., 2016 ).

Plant essential oil has antibacterial, synergistic antibacterial and other pharmacological effects; they are one of the important sources of natural medicine extracts ( Bakkali et al., 2008 ). Therefore, during the development of higher antibacterial activity against a variety of gram-negative bacteria and gram-positive bacterial infections with antibacterial drugs combined with plant essential oils, it was found that gentamicin combined with the essential oils extracted from Pelargonium graveolens and Aniba rosaeodora had synergistic effects. The combination can reduce the minimum effective dose of gentamicin. Especially in the essential oil of Acinetobacter baumannii , gentamicin combined with this had a significant synergistic antibacterial effect. The chemical composition of essential oils was studied using gas chromatographic analysis, which showed that the essential oils extracted from P elargonium graveolens and Aniba rosaeodora contained a high percentage of terpene alcohols ( Rosato et al., 2010 ). The antibacterial mechanism of monoterpenes was found to be due to the destruction of the microbial plasma membrane, resulting in changes in membrane permeability and intracellular substance efflux ( Trombetta et al., 2005 ), such that a high content of terpene alcohols benefitted the antibacterial effect of gentamicin, mainly due to the blocking protein synthesis by binding to the 30S subunit of the bacterial ribosome. Camellia sinensis (green tea) has been proved to have a wide range of antibacterial activity. A study found a high content catechin especially epigallocatechingallate (EGCG) in green tea ( Song and Seong, 2007 ). Studies have found that EGCG combined with gentamicin has a strong synergistic antibacterial effect against multi-drug resistant E.coli and S. aureus , because EGCG in green tea can destroy the bacterial cell membrane and eventually lead to bacterial rupture ( Parvez et al., 2019 ). Triphala, comprising the dried pericarp of the fruit of three plant from India, Myrobalans Terminalia chebula Retz . (Haritaki, Family: Combretaceae), Terminalia bellirica Roxb. (Bibhitaki, Family: Combretaceae), and Phyllanthus emblica Linn. or Emblica officinalis Gaertn. (Amalaki or the Indian gooseberry, Family: Euphorbiaceae), is an important medicine in the Indian traditional medicine system ( Baliga, 2010 ; Baliga et al., 2012 ). Triphala and its single drug extracts have certain antibacterial activity against gram-negative and gram-positive bacteria ( Srikumar et al., 2007 ; Tambekar and Dahikar, 2011 ; Parveen et al., 2018 ). Its phenolic content is closely related to its antibacterial activity ( Bag et al., 2013 ). A study showed that gentamicin and triphala have a synergistic antibacterial effect on some multidrug resistant gram-negative bacilli. Phenolic compounds can destroy the cell membrane of the bacteria, thus, in the synergistic antibacterial effect of gentamicin and triphala. The mechanism may be similar to the synergistic effect of gentamicin and β-lactam antibiotics ( Manoraj et al., 2019 ). Brazilian red propolis contains a large number of phenolic compounds, of which the largest proportion is flavonoids, which are believed to be related to the antibacterial effect of red propolis (F. B. Carvalho et al., 2016 ; da Cruz Almeida et al., 2017 ). Previous studies have found that combinations of antibiotics with phenolic compounds cause damage to the bacterial cell wall or cell membrane and enhance antibiotic activity ( Mun et al., 2013 ; Guo et al., 2015 ), making it easier for antibiotics to enter the interior of the bacterial cell. This might be the main mechanism of action by which propolis compounds enhance antibiotic activity ( Orsi et al., 2012 ). Studies have reported the synergistic effect of gentamicin or imipenem combined with Brazilian red propolis on S. aureus and P. aeruginosa , and discovered the effect of seasonal humidity changes on resin plants: The levels of active ingredients in propolis in specimens collected in dry season period were obviously higher than those in specimens collected in the rainy season. Therefore, the study found that the combination of propolis collected in the drier period and gentamicin or imipenem has a synergistic effect ( Regueira et al., 2017 ). 5-hydroxy-3,7,4-trimethoxyflavone (VG.EF.CLII) was isolated from Vitex gardneriana leaves. Nuclear magnetic resonance analysis found that the isolated 5-hydroxy-3,7,4 - trimethoxyflavone had the chemical formula C 18 H 16 O 6 . To study its antibacterial activity against E. coli and S. aureus , the minimum inhibitory concentration (MIC) was used to evaluate the antibacterial effect of the component and its ability to regulate the activity of antibiotics. VG.EF.CLII showed antibacterial activity against multidrug resistant bacterial strains. When combined with the antibiotics norfloxacin and gentamicin, respectively, and tested against 27 strains of E. coli and 358 strains of S. aureus isolated from

As mentioned above, plant essential oils have antibacterial, synergistic antibacterial and other pharmacological effects. Although the essential oil of Mikania cordifolia (EOMc) and its major constituent, limonene, has no direct antibacterial activity, it can reverse antibiotic resistance by regulating the action of antibiotics. For P. aeruginosa , EOMc combined with gentamicin and norfloxacin displayed a synergistic antibacterial effect; and for S. aureus , EOMc reduced the MIC of gentamicin toward bacteria ( Justino de Araújo et al., 2020 ). Considering that saponins can alter the local chemical environment of the cell membrane and may alter how bacteria absorb or act on antibiotics, the combination of glycyrrhizic acid and gentamicin was found to have therapeutic potential against local bacterial infections caused by vancomycin-resistant enterococci ( Schmidt et al., 2016 ). The Chinese herbal compound, plumbagin, stimulates the uptake of gentamicin by carbapenem-resistant klebsiella pneumonia (CRKp) by enhancing the efflux of the TCA cycle and the proton-motive force to achieve synergistic antibacterial activity. It can be combined with aminoglycoside antibiotics to enhance its efficacy and reduce its dosage ( Chen et al., 2020 ). Croton ceanothifolius essential oil (CcEO) can enhance the antibacterial activity of gentamicin and norfloxacin against E. coli , S. aureus , and P. aeruginosa . The presence of the two antibiotics acting within the bacterial cell indicated that the CcEO promotes the penetration of antibiotics into the bacterial cytoplasm and thus exerts a synergistic effect. By measuring the MIC of CcEO against multidrug resistant bacteria, it was found that the MIC was greater than 1,024 μg/ml in all strains, indicating that CcEO has no antibacterial activity. CcEO was analyzed by gas chromatography-mass spectrometry (GC/MS), and a total of 25 chemical components were identified. The main ingredients include bicyclogermacrene (26.3%), germanene D (14.7%), and E-caryophyllene (11.7%). The authors hypothesized that although CcEO cannot exert a direct antibacterial effect, it can synergistically enhance the effect of antibiotics on membrane permeability ( Araújo et al., 2020 ). Kaempferol 7-O-β-D-(6″-O -cumaroyl)-glucopyranoside flavonoids were isolated from croton leaves and studied for their antibacterial and synergistic antibacterial effects. It was found that although it did not have direct antibacterial activity against S. aureus , E. coli , and P. aeruginosa , when kaempferol 7-O-β-D-(6″-O-cumaroyl)-glucopyranoside at 128 μg/ml was combined with gentamicin, it showed a synergistic antibacterial effect on both S. aureus and E. coli ( Cruz et al., 2020 ). The hydroxyphenyl groups in flavonoids have affinity for proteins; therefore, these compounds act as inhibitors of bacterial enzymes and interfere with their synthesis pathways ( Alcaráz et al., 2000 ). The Hexanic Zea Mays L. Silk Extract (Poaceae or HEZN) is used widely in Brazilian folk medicine to treat genitourinary tract diseases. A study used standard E. coli ATCC 25922, S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 strains, as well as 27 multi-resistant strains of E. coli , 35 strains of S. aureus, and 31 strains of P. aeruginosa . The authors found that HEZM and its metabolites had an MIC ≥1,024 μg/ml for all tested strains, indicating that at least for these strains, HEZM and its metabolites have no obvious antibacterial activity. In order to study the regulatory effect of HEZM on antibiotics, a subinhibitory concentration (MIC/8) combined with amikacin and gentamicin was selected to test the resistance of multiple drug-resistant strains. In S. aureus and P. aeruginosa , HEZM combined with amikacin and gentamicin reduced the MICs of the antibiotics significantly. However, the binding of the extract with amikacin against E. coli significantly increased the MIC of this antibiotic, indicating that HEZM antagonized the effect of the antibiotic (A. B. L. Carvalho et al., 2019 ). Phytochemical analysis of HEZM revealed that it contained four types of important secondary metabolites, including: tannins, flavonoids, flavonols, and xanthones (A. B. L. Carvalho et al., 2019 ). Flavonoids, as common extracts in natural plants, are most frequently related to the synergistic effects of natural products. One of the classes of metabolites in the extract is xanthones, and a subgroup of xanthones, called oxygenated xanthones, can increase the solubility of cell membranes to exert antibacterial activity in norfloxacin-resistant S. aureus ( Xiao et al., 2008 ) . Studies have found that the effects of HEZM on AGAs are different in different gram-negative and gram-positive bacteria. The structural differences between gram-negative and gram-positive bacteria might also determine the effect of natural extracts on antibiotics (A. B. L. Carvalho et al., 2019 ).

Some antibiotics combined with photosensitizer-mediated photodynamic antimicrobial chemotherapy (PACT) have been found to be with better antibacterial effects against bacteria ( Branco et al., 2018 ; Pérez-Laguna et al., 2018 ; Tavares et al., 2018 ). PACT mediated by photosensitizers will produce ROS under specific wavelength light stimulation, which induces phototoxic damage to bacteria ( Pérez-Laguna et al., 2019 ). PACT is not limited to its antibiotic resistance pattern to eliminate microorganisms and is also effective against microorganisms in a biofilm state ( Giuliani, 2014 ; García et al., 2015 ). Rose bengal (RB) is a xanthine dye used as a photosensitizer for PACT ( Pérez-Laguna et al., 2017 ). The study found that RB-PACT combined with gentamicin has a synergistic antibacterial effect on planktonic S. aureus , while a synergistic effect is observed only when the maximum concentration tested of RB-PACT and gentamicin (64 μg/ml and 40 μg/ml, respectively) was used in biofilm ( Pérez-Laguna et al., 2018 ). Its mechanism of synergistic antibacterial effect is considered to be that after PACT damages cells, it promotes the entry of gentamicin into bacteria, and gentamicin binds to the 30S subunit of the bacterial ribosome to impair protein synthesis, resulting in bacterial death ( Barra et al., 2015 ; Pérez-Laguna et al., 2018 ). The photobacterial agent toluidine blue (TB) is a hydrophilic cationic alkaline photosensitizer (PS). TB has high affinity for bacterial membranes and is believed to cause membrane damage ( Wainwright et al., 2016 ). Further studies found that TB-PACT combined with gentamicin had a more significant synergistic antibacterial effect against S. aureus and MRSA than either treatment alone ( Liu et al., 2020 ). Antimicrobial photodynamic therapy (aPDT) is considered to promote the passage of antibacterial drugs by destroying the outer membrane of bacterial cells using light irradiation. Blue light-emitting diode (LED) light irradiation affects the growth of both gram-negative and gram-positive bacteria. When blue LED light was used in combination with the AGAs such as amikacin and gentamicin, it showed a synergistic antibacterial effect on E. coli and S. aureus , and promoted the inactivation of the bacteria, which might be attributed to the antibacterial effect induced by oxidative stress ( Silva et al., 2019 ).

HZ conceived and designed the conception of review article, and made the amendments of the manuscript. NW reviewed the relevant literature and wrote the manuscript. JL, FD, and YH supplemented the content of the manuscript. All authors read, revised and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.