In Vitro Synergistic Activity of Combinations of Tetrahydroisoquinolines and Treatment Antibiotics against Multidrug-Resistant Salmonella

The global burden of Salmonella infections remains high due to the emergence of multidrug resistance to all recommended treatment antibiotics. Tetrahydroisoquinolines (THIQs) have demonstrated promising activity against multidrug-resistant (MDR) Salmonella Typhi. Hence, their interaction with treatment antibiotics was investigated for possible synergy. Twenty combinations of five THIQs (1, 2, 3, 4, and 5) and four antibiotics were tested against each of 7 Salmonella isolates by the checkerboard method giving a total of 140 assays performed. Fractional inhibitory concentration indices (FICIs) were calculated, and isobolograms were plotted. In terms of FICI, synergism ranged from 0.078 to 0.5 and the highest magnitude (0.078) was recorded for chloramphenicol-THIQ 1 combination. In a total of 140 antibiotics-THIQs combination assays, 27 were synergistic (17%), 42 were additive (30%), 11 were antagonistic (7.8%), and 60 were indifferent (42%). The synergistic activity recorded for each antibiotic class in combination based on the total of 7 bacterial isolates tested ranged from 14.29% to 71.43%; the highest percentage was recorded for two combinations (chloramphenicol or sulphamethoxazole with THIQ 1). Ciprofloxacin-THIQ 1 combination showed additivity on all bacteria isolates tested (100%). Overall, THIQ 1 was the most synergistic and most additive in combination with three antibiotics (ampicillin, chloramphenicol, or sulphamethoxazole-trimethoprim). Some combinations of the THIQs and treatment antibiotics have shown high synergism which could potentially be efficacious against multidrug-resistant S. Typhi, hence this interaction should be further studied in vivo.


Introduction
Te morbidity and mortality of salmonellosis in humans remain high [1].It is caused by both typhoidal and nontyphoidal Salmonella serovars [2].Salmonella is spread mainly by the consumption of contaminated food or water, poultry (chicken), and animal products such as eggs.It is transmitted through the faecal-oral route.Te spread of Salmonella can be prevented through several strategies including vaccination and more efectively by improved water and food hygiene and proper cooking of food.Prevention of Salmonella infection from eggs and chickens to humans can be achieved by inclusion of antibiotics in animal feed and water and thorough washing of hands after handling and properly cooking these items before consumption [3].
Initially, treatment of Salmonella infections made use of penicillins, phenicols, cephalosporin, antifolates, and macrolides [4] and later following the emergence of resistance, fuoroquinolones and third generation cephalosporins were used as alternatives.However, high multidrug resistance to all the above mentioned treatment antibiotics has been reported worldwide, especially for S. Typhi [5].Mild Salmonella infections are treated by electrolyte replacement and rehydration but severe cases require antibiotics [3].Presently, the recommended treatment antibiotics are ciprofoxacin, ceftriaxone, and azithromycin, taking into consideration the local pattern of resistance [6].However, genetic resistance to these antibiotics has been reported with decreased susceptibility and treatment failure due to MDR strains [7].A recent study detected a high level of multidrug resistance to frst-line antibiotics encoded by resistance marker genes (tem, sul1, and dfrA1) and also found low to moderate resistance to fuoroquinolones alongside single and double point mutations in the quinolone resistance determining region (QRDR) in gyrA [8].Te decreasing efcacy to the antibiotics in current use justifes the urgent search for new safe efcacious antibacterials and/or alternative strategies of their use against MDR Salmonella infection.
Tere are several reports of synergism in combination studies of natural or synthetic compounds with treatment drugs against various diseases including bacterial infections [9] and also studies on treatment drugs only such as antibiotic combinations [10].So, combination therapy is presently one of many strategies used to counter antibiotic resistance.Several fxed dose combinations of antibiotics are in clinical use [11].Tese combinations ofer higher efcacy against multidrug-resistant bacterial strains due to their synergistic efects; they also have a broader spectrum of activity and reduce the risk of emergence of resistance during treatment [12].Findings from antibacterial combination studies to counter MDR strains have been promising.Higher efcacy has been recorded in such studies compared to monotherapy where emergence of resistance to a single drug is more likely to occur [13].Reports of combination of aminoglycosides with other antibiotics or plant-derived compounds have shown enhanced bactericidal activity against Salmonella enterica [14][15][16].Also, synergistic activity has been recorded for some of the frst-line anti-Salmonella antibiotics (ampicillin, chloramphenicol, and cefotaxime) in combination with an aminoglycoside (gentamycin) against MDR Staphylococcus aureus [17].
Tetrahydroisoquinolines (THIQs) are aromatic alkaloids which occur widely in nature.Teir semisynthetic and synthetic derivatives have a broad range of bioactivity [18].Te tetrahydroisoquinoline contains a nitrogen heterocycle found in many approved drugs in clinical use for several diseases.Medicinal chemistry exploration of this scafold has yielded analogues with antitubercular, antibacterial, antifungal, and antiviral action [19].In a recent study of seventeen THIQs, six demonstrated a moderate but structurerelated antibacterial activity against MDR S. Typhi strains comparable to some of the treatment antibiotics [20].Te fndings on synergistic interactions mentioned above [9,10] support the search for new efcacious antibacterial treatments against resistant strains using combination studies.Hence, this study investigated the interaction between these moderately active THIQs and some of the treatment antibiotics against MDR S. Typhi.

Test Microorganisms. Six MDR clinical isolates of S.
Typhi (BU02, BU05, BU07, BU09, BU11, and BU70) were isolated from patient specimens obtained from health facilities in the South West Region, Cameroon.Tey had been characterized in an earlier study using cultural, biochemical, and molecular techniques, and the data on their susceptibility were published [8].One control strain (S.Typhimurium ATCC 14028) obtained from the American Type Culture Collection, Manasses, USA, was included making a total of seven strains used in this study.Stocks of isolates were stored in 50% glycerol in Muller-Hinton broth (MHB) at −20 °C.

Chemicals and Reagents.
Te materials, chemicals, and reagents used were Salmonella-Shigella agar, Mueller-Hinton agar, and broth from Lioflchem (Italy).Commercial antibiotics discs (ampicillin, chloramphenicol, sulphamethoxazole-trimethoprim, and ciprofoxacin) were purchased from Abtek Biologicals Ltd., UK.Dimethylsulfoxide (DMSO) was purchased from Sigma-Aldrich (USA).Te tetrahydroisoquinolines were synthesized and obtained as solids in the Department of Chemistry, University of Buea, characterized as previously reported and stored at room temperature [20].

Tetrahydroisoquinolines.
Five tetrahydroisoquinolines (THIQs 1, 2, 3, 4, and ) used in this study were synthesized using the Pictet-Spengler reaction as described in detail [20].In brief, a mixture of 3,4-dihydroxyphenethylamine hydrochloride (1 equiv), substituted benzaldehydes (1 equiv), and triethylamine (1 mL) in ethanol (10 mL) was stirred, heated under refux (6-10 hours), and concentrated to remove the solvent.Te residue was diluted with methylene chloride (100 mL) and distilled water (100 mL), giving a solid precipitate as the fnal product.Te solid was collected by fltration, washed with acetone, and air-dried.Te synthesis and structural characterization including all spectroscopic data of these fve THIQs have been published in an earlier study of their antibacterial activity [20].Teir chemical structures are shown in Figure 1.Stock solutions of each compound were prepared by dissolving in dimethylsulfoxide (DMSO) as described in [20] and were further diluted in MHB before use in the experiments.

Determination of the Antibacterial Activity of
Compounds.Te diameters of zones of inhibition and the minimum inhibitory concentrations (MICs) of the antibiotics and selected THIQs had earlier been determined using 2 Advances in Pharmacological and Pharmaceutical Sciences disc difusion and microdilution methods in a separate study, and the data were published in detail [20].In brief, stock solutions (1.024 mg/mL) were prepared as described in the cited work and incubated at fnal concentrations from 1 to 512 μg/mL with Salmonella bacterial cells (5 × 10 5 CFUs/ mL fnal density) in a 96-well microtitre plate in duplicates.
Positive and negative controls were included and the optical densities (ODs) were read at 595 nm using a microplate reader (Emax microplate reader, Molecular Devices, USA).Te plate was incubated at 37 °C (DHP-9052, England) for 24 h, read visually for inhibition, and the OD read again at 595 nm.MIC was taken as the lowest concentration well with more than 50% inhibition of bacterial growth.MICs of selected antibiotics (ampicillin, chloramphenicol, sulphamethoxazole-trimethoprim, and ciprofoxacin) were determined as described above.

Determination of MIC and Interactions of THIQ-Antibiotic Combinations.
Te interactions of 5 THIQs with 4 antibiotics, i.e., 20 combinations each against 7 MDR Salmonella isolates giving a total of 140 assays, were assessed by the checkerboard method in 96-well microtitre plates as previously described [21].Stock solutions, 8 times the MIC (8MIC) of antibiotics and THIQs, were prepared by dissolving in dimethylsulfoxide (DMSO) as described [20] and further diluted in MHB.Working solutions of each test substance were prepared by dilution of the stocks in MHB in separate microtitre plates.Te antibiotic was serially diluted in 8 columns (2-9) (8MIC-MIC/16, 100 μL per well, respectively), with concentration decreasing from wells A to H of each column.Te THIQ was also diluted the same as the antibiotic in 8 rows (A-H) with concentration decreasing from wells 9 to 2 of each row.Ten, the checkerboard assay was set up in a fresh plate by adding 50 μL of diluted antibiotic to the corresponding well in a fresh plate, followed by 50 μL of THIQ and 100 μL of bacterial suspension giving fnal concentrations of 2MIC-MIC/64 and 5 × 10 5 CFUs/mL for test substance and bacteria cells, respectively, in the 8 × 8 matrix of diferent concentrations.Antibiotic and THIQ were each included alone in columns 1 and 10, respectively, to determine their MICs.
Te plates were incubated at 37 °C (DHP-9052, England) for 24 h, and the optical density (OD) was measured at 595 nm (Emax microplate reader).Te fractional inhibitory concentration (FIC) and FIC index (FICI) of each THIQantibiotic combination were calculated from the MICs of the THIQ or antibiotic alone and in combination according to the following equations: (1) In vitro interactions were determined algebraically using the formula above and the nature of the THIQ-antibiotic interaction based on the combined and individual antimicrobial activities were interpreted based on the following cutofs [22]: synergy: FICI ≤ 0.5, additive: 0.5 < FICI ≤ 1, indiference or no interaction: FICI: 1-4, and antagonism: FICI > 4.

Statistical Analysis.
Five THIQs were combined with four antibiotics giving a total of twenty combination pairs.For each combination, the proportion of the total of 7 MDR Salmonella strains which showed a given type of interaction was calculated using the following formula: where "x" is the number of each type of interaction against MDR Salmonella strains and "n" is the total number of MDR Salmonella strains tested (n = 7).Te overall percentage drug interaction type for all the 140 combination assays performed was calculated using the following formula: where x � number of drug interaction type and y � total number of drug combinations assays (140).
To determine the nature of their interaction, isobolograms were plotted using FICs of wells along the diagonal of the plate in which the concentrations of the two test compounds were decreasing and increasing, respectively, along the diagonal.FICs of antibiotic were plotted against the FICs of the THIQ and isobolograms which showed a concave curve indicate synergism; a convex curve indicates antagonism and linear curve shows additivity [23].All statistical analyses were performed using GraphPad Prism version 8.40 (GraphPad Software, USA).

Types and Efect of Interaction on the Minimum
Inhibitory Concentration.Te MICs of the antibiotics alone recorded in this study (16-256 μg/mL) confrm that the Salmonella isolates were multidrug resistant.Te antibacterial interactions were determined following the values of the FICI mentioned above [22].Of an overall total of 140 combination assays, 27 were synergistic (17%) based on FICI ranging from 0.078 to 0.5, with the lowest and most synergistic being chloramphenicol with THIQ 1 (0.078) (Table 1).
Te MICs of antibiotics in the combinations were considerably reduced compared to the MICs of the antibiotics alone.Te reductions were much greater in the synergistic than the additive interactions.Te highest reduction was observed for chloramphenicol with 1 and sulphamethoxazole-trimethoprim with 1, which both caused a 64-fold reduction in the MIC of the antibiotics to 2 and 4 μg/mL, respectively.Sulphamethoxazole-trimethoprim with 3, ampicillin with 1, and ciprofoxacin with 2 caused a 32-, 16-, and 16-fold decrease in the antibiotic MICs, respectively.Te lowest reduction was ciprofoxacin with 4. Te reductions in the MICs for the additive and indiferent combinations ranged from 1-to 2-fold (Table 1).

Interactions of THIQs Based on the Class of Antibiotics.
Te interactions were assessed using fractional inhibitory concentration indices (FICIs), isobolograms, and the proportion of each interaction against the MDR S. Typhi isolate.Te combination of the fve THIQs with the four antibiotics (20 combinations) resulted in at least one synergistic antibacterial efect per antibiotic class against a MDR S. Typhi clinical isolate.
Among all combinations tested, the overall percentage synergistic efects based on the total number of 7 MDR S. Typhi isolates ranged from 14.29 to 71.43%; additive efect ranged from 14.29 to 100%, antagonistic efect ranged from 71.43 to 85.71%, and no interaction efects ranged from 14.29 to 100% (Table 2).
Synergistic interactions were recorded for all four antibiotic classes.Te highest level of synergistic interactions in terms of proportion of MDR bacterial isolates on which it was exerted is 71.4%, which was recorded for two combinations, i.e., chloramphenicol or sulphamethoxazole with compound 1.Tis was followed by a moderate synergism for ampicillin with 1, sulphamethoxazole-trimethoprim with , and ciprofoxacin with 2 or 4, which all recorded 57.1% (Table 2).
Additive interactions were recorded for all four antibiotic classes.Te highest proportion of additive interactions recorded against all 7 isolates was 100% for ciprofoxacin with 1, followed by chloramphenicol with compound 4 (71.4%).Moderate synergism was observed for ampicillin with 1, chloramphenicol, or sulphamethoxazoletrimethoprim with , which all recorded 42.8%.Also, all the THIQs showed additivity with sulphamethoxazoletrimethoprim.
Antagonism was recorded in only one chemical class and for only two combinations (ciprofoxacin with 3 or ).Indiference (no interaction) was recorded in all antibiotic classes.Tese interactions are further illustrated in the isobolograms in Figure 2 for the most synergistic, additive, indiferent, and antagonistic THIQ-antibiotic combination against MDR S. Typhi.
Overall, in terms of the total of 20 THIQ-antibiotic combinations, seven (35%) and fourteen (70%) showed synergism and additivity, respectively (Table 2), with compound 1 being the most synergistic and additive in combination with three antibiotics (ampicillin, chloramphenicol, and sulphamethoxazole-trimethoprim).

Discussion
Various approaches are presently being used to counter increasing resistance in Salmonella including combination 4 Advances in Pharmacological and Pharmaceutical Sciences n � number of MDR Salmonella isolates which showed a given interaction; * Distribution of interaction type based on the total number of THIQ-antibiotic combination assays against 7 MDR Salmonella isolates.studies of active molecules.Tis study investigated the activity of combination treatment antibiotics with fve tetrahydroisoquinolines (THIQs), which have shown moderate antibacterial activity against MDR S. Typhi strains.All fve THIQs showed synergistic interaction with at least one class of treatment antibiotic.Overall, compound 1 was the most synergistic; it recorded synergistic activity with three antibiotic classes and the highest level of synergistic interactions (71.4%) against the total number of MDR S. Typhi isolates targeted.Tis is the frst report of the synergistic activity of THIQs in combination with Salmonella treatment antibiotics.
Te fndings from this study confrm the multidrugresistant nature of the Salmonella isolates as both THIQs and antibiotics recorded MICs in the same ranges as previously reported [8,20] (Table 1).All THIQ-antibiotic combinations tested showed synergistic interactions against at least one isolate as seen in Table 2. THIQ 1 was the most synergistic in combination with chloramphenicol (Figure 2(a)) or sulphamethoxazole-trimethoprim and was not synergistic with ciprofoxacin.Furthermore, there was a massive reduction in the MIC of antibiotics, chloramphenicol, or sulphamethoxazole-trimethoprim, in combination with THIQ 1, to the level recorded against sensitive Salmonella strains.Tis high reduction (64 fold) of the MIC of the treatment antibiotic further demonstrates the strong synergism in the combinations and indicates that THIQ 1 is a potential partner antibacterial which could be used in combination with some treatment antibiotics in the management of MDR Salmonella infections with resultant reduction in morbidity and mortality.However, further studies are required to establish this.Te other four THIQs (2, 3, 4, and ) showed lower levels of synergism against the MDR S. Typhi strains in various combinations with ciprofoxacin and sulphamethoxazole-trimethoprim as shown in Table 2. Te observed synergistic antibacterial activity could be due the action of the antibiotic and the THIQ at diferent targets in the bacterial cell; the diferences in the level of synergism of THIQ 1 and the others could be attributed to their structures.As seen in Figure 1, when THIQ 1 with para-chloro substitution was combined with tested antibiotics, a synergistic efect with ampicillin, chloramphenicol, or sulphamethoxazole-trimethoprim was observed.Te likely mechanism of action for the THIQ 1chloramphenicol combination could involve the inhibition of cell wall synthesis or some other essential process in the bacteria while chloramphenicol acts by its known mechanism of inhibition of protein synthesis [24].In a previous work of seventeen THIQs [20] tested against MDR Salmonella strains including those used in this study, THIQ 1 showed the highest activity against MDR Salmonella strains; this activity was attributed to the electron-withdrawing property of the chloro substituent at the paraposition of the pendant phenyl [20].Tis property may also account for the high synergism observed for this compound.
Additivity was also recorded in several combinations with four classes of antibiotics.High levels (70-100%) of additive interactions were recorded for THIQ 1 and 4 with three classes of antibiotics (Table 2, Figure 2(b)).Additive interactions suggest that the molecules in the combination may be sharing the same target sites, hence acting by the same mechanism in the bacterium, in which case both THIQ 1 and ciprofoxacin may be acting by inhibiting the bacterial DNA gyrase synthesis [24].THIQ 1 also showed relatively lower additive interactions for all three antibiotic classes with which it was synergistic, further indicating that THIQ 1 may be acting at two diferent targets.
Antagonism was very low and was observed only for combinations of THIQs 3 (Figure 2(d)) and with ciprofoxacin.THIQ 3 showed the highest indiference of 100% in combination with ampicillin or chloramphenicol.Te likely mechanism of action here could be blocking of the site of action of the antibiotics by the THIQ in the cell wall for ampicillin and protein synthesis for chloramphenicol [24].Antagonism suggests the THIQ prevents binding of antibiotic to its target while noninteraction suggests nonbinding of THIQ to the antibiotic target.Te indiference and antagonism observed in THIQ 3 could be due to the presence of the di substitution (3,4-dichloro-) and THIQ could be due to the presence of the methoxy (-OCH 3 ) substituent on the pendant phenyl with lower electron-withdrawing at the paraposition resulting in decreased activity against MDR S. Typhi.
Overall, when all the interactions are considered, THIQ 1 was most synergistic and also showed relatively lower additive interaction, no antagonism, and no indiference; it showed the highest synergism with phenicols and antifolates.THIQ 4 was the most additive but showed indiference to a relatively lesser extent.However, 4 showed 100% additivity with ciprofoxacin; hence, it did not show synergism, antagonism, or indiference in this combination.Te additivity observed in THIQ 4 combination with antibiotic may be due to the moderate electron withdrawing by the trifuoromethyl substituent at the paraposition as explained in [20].
Several studies of antibacterial compounds in combination with standard antibiotics have demonstrated improved activity against resistant strains of S. Typhi.However, most of these studies did not use the frst-line treatment antibiotics.Miladi et al. [25] reported synergistic efect between thymol and nalidixic acid against nalidixic-resistant S. Typhimurium strains with the lowest MIC values ranging from 32 to 128 μg/mL.
A study reported that the combination of erythromycin and epicatechin gallate against bioflm-forming MDR S. Typhimurium showed synergistic antibacterial efects with FIC indices of 0.5 [26].Hence, it can be a potential treatment for S.Typhimurium-associated diarrhoea and its transmission from animals to humans.

6
Advances in Pharmacological and Pharmaceutical Sciences Tetrandrine at subinhibitory concentrations in combination with colistin showed increased activity against most of the MCR-mediated colistin-resistant Salmonella typhimurium, with a FIC index (FICI) of 0.375-0.625 and the colistin MIC fold change ranged from 1 to ≥1024.Tey equally reported that the combination of tetrandrine and colistin demonstrated synergistic interactions in 81.8% (9/ 11) of the resistant S. Typhimurium isolates tested [27].Hence, tetrandrine can serve as a potential colistin adjuvant against MCR-positive Salmonella.
In terms of strength, this study is the frst to report synergistic activity of THIQs in combination with frst-line antibiotics against MDR Salmonella strains.Te practical value of this fnding is that the highly synergistic combinations can potentially be used to treat MDR Salmonella infections.As limitation, the study design was based on previously published data and needed to assess reproducibility and validation of results.However, this limitation will be addressed in further exploitation of the fndings of this work.

Conclusion
Tis study has revealed high synergistic and additive activities in combinations of tetrahydroisoquinoline with specifc classes of treatment antibiotics against multidrug-resistant Salmonella.Tese active anti-Salmonella combinations are a potential alternative treatment for MDR Salmonella infections.Tese active combinations should be further tested in vivo to assess their efcacy and THIQs should also be studied in combination with other antibiotics classes.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.Advances in Pharmacological and Pharmaceutical Sciences

Table 1 :
Minimum inhibitory concentrations (MICs) and fractional inhibitory concentration indices (FICIs) showing efects of antibiotics in combination with THIQs.