Novel Antibiotic Combinations of Diverse Subclasses for Effective Suppression of Extensively Drug-Resistant Methicillin-Resistant Staphylococcus aureus (MRSA)

The emergence of multidrug-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), the chief etiological agent for a range of refractory infections, has rendered all β-lactams ineffective against it. The treatment process is further complicated with the development of resistance to glycopeptides, primary antibiotics for treatment of MRSA. Antibiotic combination therapy with existing antimicrobial agents may provide an immediate treatment option. Minimum inhibitory concentrations (MICs) of 18 different commercially available antibiotics were determined along with their 90 possible pairwise combinations and 64 triple combinations to filter out 5 best combinations. Time-Kill kinetics of these combinations were then analyzed to find collateral bactericidal combinations which were then tested on other randomly selected MRSA isolates. Among the top 5 combinations including levofloxacin-ceftazidime; amoxicillin/clavulanic acid-tobramycin; amoxicillin/clavulanic acid-cephradine; amoxicillin/clavulanic acid-ofloxacin; and piperacillin/tazobactam-tobramycin, three combinations were found to be collaterally effective. Levofloxacin-ceftazidime acted synergistically in 80% of the tested clinical MRSA isolates. First-line β-lactams of lower generations can be used effectively against MRSA infection when used in combination. Antibiotics other than glycopeptides may still work in combination.


Introduction
Hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) has been a predominant agent for skin and nosocomial infections for several years [1][2][3]. MRSA is responsible for about more than 20% of all bloodstream infections, and mortality rate is as high as 25-50% [4,5]. Methicillin resistance in Staphylococcus aureus is mediated by SCCmec gene, which encodes polypeptide penicillinbinding-protein 2a (PBP2a) [6,7] and also provides insertion sites for plasmids and transposons which assist in transmission of resistance to non-β-lactam antibiotics [8,9]. Emergence of β-lactamase-producing MRSA strains has rendered all β-lactams ineffective against its infections. Glycopeptides (mainly vancomycin) remain the major class of antibiotics for treatment of MRSA, but indiscriminate use of these antimicrobial agents has led to the emergence of vancomycin-resistant S. aureus (VRSA) [10]. Development of resistance and other complications such as infection recurrence, and treatment failure pose a serious hindrance in treatment of MRSA infections [4,11]. erefore, potential options for treatment of these refractory infections need to be explored. Since other therapeutic interventions such as phage therapies are still in development, antibiotic combination therapy with existing antimicrobial agents provides an immediate treatment option.
Usage of multidrug combinations has proven successful against infection caused by H. pylori, M. tuberculosis, A. baumannii, P. aeruginosa, and K. pneumonia [12][13][14][15][16]. Historically, vancomycin-aminoglycoside combination was successfully employed for endocarditis caused by MRSA, but due to increased possibility of renal impairment, this combination is not recommended anymore [17][18][19]. More recent studies, where vancomycin was used concurrently with beta-lactams improved in vitro results, were observed. In another study, antibiotics from different subclasses and generations of β-lactams (meropenem, piperacillin, and tazobactam) were successfully combined against MRSA infections [20].
In this study, we combined FDA approved antibiotics from different generations and classes to determine synergistic combinations which do not necessarily include vancomycin (or any other glycopeptide) against highly resistant MRSA. We anticipate that using antibiotic combinations of commercially available antibiotics from diverse subclasses has a potential for overcoming antibiotic-resistant infections and may serve as a powerful technique in reversing antibiotic resistance on top of producing bactericidal effects.

Materials and Methods
Isolates of S. aureus were acquired from major hospitals of Rawalpindi, Lahore, and Peshawar, and then screened for methicillin and vancomycin resistance. Further susceptibility testing by the Kirby disk diffusion method was performed, and by using clinical breakpoints from the Clinical and Laboratory Standards Institute (CLSI), an extensively drug-resistant (XDR) MRSA isolate (LR-2) was selected for antibiotic synergy testing [21]. A total of 18 different commercially available antibiotics (Table 1) were used for 90 possible pairwise combinations (Figure 1(a)) and 64 triple combinations (antibiotic from varying class/penicillin/inhibitor) (Figure 2(a)). Each experiment was conducted in duplicate, and mean values were calculated for each combination.
XDR MRSA isolate was cultured in cation-adjusted Muller-Hinton Broth (MHB) for 24 hours at 37°C. Minimum inhibitory concentration (MIC) was determined by the broth microdilution method as described by Wiegand et al. [22]. Stock solutions of antimicrobial agents were prepared by calculating the amount of antibiotic to be used: (amount of drug in each tablet (y))/10 mg � (amount of active agent in each tablet)/(amount of drug needed to produce 10 mg/ml solution (x)). e antibiotic-containing media were serially diluted in 96-well plate to create an array of 10-fold dilution across the row. Log-phase culture of MRSA was diluted in MHB to produce 1/50 dilution of bacterial suspension and inoculated in the 96-well plate. Column 11 and 12 were kept as negative and positive control, respectively. Plates were incubated for 24 hours at 37°C. Wells showing no visible turbidity after 24 h were subcultured on plate count agar for colony counting. Minimum inhibitory concentration (MIC) was considered as the lowest concentration inhibiting 99% of bacterial growth. Each experiment was performed in duplicate. Synergistic combinations were determined by the fractional inhibitory concentration index (FICI) method [23][24][25][26]. FICI calculation involves MIC of the antimicrobial agents in combination divided by the MIC of the agents alone to determine antibiotic interactions, as shown in the following formula: whereby FICI ≤0.5 was interpreted as "synergistic," FICI � 1-4.0 was considered "indifferent," and FICI >4.0 was inferred "antagonistic." For further analyses, five highly synergistic combinations against MRSA isolate (LR-2) were selected. e criteria for selection required antibiotics in combination to belong to lower generation with simple mechanism of action, and low FICI values. To further confirm the synergistic mechanism of action, Time-Kill assays were performed on selected combinations according to a formerly described protocol [27] ( Figure 3). Briefly, MHB was inoculated with test organism and incubated to midlog phase. Aliquots containing antibiotic combination (two or three drug combination) and test MRSA at density of 10 6 CFUs were incubated at 37°C. At times 0 h, 2 h, 4 h, 6 h, and 8 h, the inoculum was withdrawn, serially diluted from 10 3 to 10 7 , and 100 μl of each dilution was plated on a nutrient agar medium and incubated at optimal growth condition of S. aureus to determine colony counts [27]. Killing kinetics were interpreted as "synergistic" when ≥2-log10 reduction in growth was observed in the colony forming units (CFU) at 24 h with antibiotic combination compared to most active drug alone, and "indifferent" when <2-log10 decrease was observed.
Synergistic drug combinations were then tested on other randomly selected methicillin-resistant S. aureus isolates, and their MIC and FICI values were determined to validate drug combination synergy.
All assays were conducted in duplicate, and mean MIC values were calculated along with standard deviations. e FICI values were calculated from the mean MIC values obtained in individual and combination assays. e standard deviation is represented in graphs on the error bars.

Results
Results obtained from the Kirby-Bauer disk diffusion method on MRSA isolate (LR-2) showed complete resistance to penicillin, cephalosporin, fluoroquinolones, aminoglycosides, and glycopeptides and partial resistance to carbapenem, tetracycline, and certain miscellaneous agents. erefore, MRSA isolate (LR-2) was classified as XDR [28]. Individual MIC assays showed insensitivity to all antibiotics from Table 1 except for imipenem (MIC � 4 μg/ml). Double and triple antibiotic combinations from diverse subclasses were tested against MRSA LR-2 to identify the combinations that were synergistic when administered concomitantly. Figure 4 shows the interactions between antibiotics indicating synergistic or indifferent relationship.
Triple combination of amoxicillin/clavulanic acid with cephalosporin yielded a peculiar result when synergy was observed with cephradine (1 st generation cephalosporin) with an FICI value of 0.281 (Figure 2(b)). is finding was interesting because if proven successful in further experimentation, an ineffective lower generation antibiotic [30] can be brought in use again. Sensitivity was observed when amoxicillin/clavulanic acid was combined with fluoroquinolones (FICI 0.25) (Figure 2 e combinations selected for further testing included levofloxacin-ceftazidime; amoxicillin/clavulanic acidtobramycin; amoxicillin/clavulanic acid-cephradine; amoxicillin/clavulanic acid-ofloxacin; and piperacillin/tazobactam-tobramycin. Levofloxacin combination with cephalosporin was selected instead of moxifloxacin combination because it is a lower-generation gluoroquinolone. Amoxicillin/clavulanic acid and cephradine was selected because both antibiotics other than the inhibitors are of lower generation. Amoxicillin/clavulanic acid showed FICI values lower than 0.5 with fluoroquinolones and aminoglycosides, so one combination from each group was selected. Ofloxacin in combination to amoxicillin/clavulanic acid made this list because it is the lowest-generation aminoglycoside. However, tobramycin though is not a low generation aminoglycoside but showed good potential with other penicillin-β-lactam inhibitor combinations. Hence, tobramycin in amalgamation with amoxicillin/clavulanic acid and with piperacillin-tazobactam was selected. Time-Kill kinetics illustrate up to 8-fold reduction in growth compared to most active drug alone which means a synergistic relation exists between levofloxacin-ceftazidime; amoxicillin/clavulanic acid-tobramycin; and piperacillintazobactam-tobramycin (Figures 3(a)-3(e)). Bactericidal effect was observed within 8 hours of incubation in all three combinations. e combination showing >2-log10 reduction when tested on other randomly selected MRSA isolates,

Discussion
Rapid emergence of extensive drug resistance in Grampositive and Gram-negative bacteria necessitates identification of novel therapeutic approaches. Reliance on monoantibiotic therapeutic approach provided selection pressure for development of resistance. With the depleting arsenal of antibiotics and a gap in the successful development of alternative therapeutics, determining synergistic combination of currently available antibiotics could provide an effective alternative to curtailing the alarming increase in antibiotic resistance [31]. e purpose of this study was to find combinations from diverse subclasses of antibiotics that can produce synergistic effects in combinations against extremely drug-resistant MRSA isolates. We report that a novel combination of levofloxacin-ceftazidime (LVX/CAZ) acts synergistically to produce bactericidal effect on XDR MRSA isolate LR-2.

International Journal of Microbiology
Previously, fluoroquinolone-cephalosporin combination was shown to have synergistic effect against P. aeruginosa [32]. Our findings on pairwise combinations suggest that fluoroquinolones (especially levofloxacin and moxifloxacin) are very effective when combined with cephalosporin and carbapenem antibiotics.
ough monotherapy with fluoroquinolones is recommended to be minimized for treatment of S. aureus infections because of multidrug resistance [33], this finding provides a solution to overcome fluoroquinolone resistance in MRSA. e overall higher sensitivity ratios to amoxicillin/ clavulanic acid in combination with other antibiotics is not surprising since existing evidence on antimicrobial combinations suggests that despite being the first-line antibiotic, beta-lactam antibiotics in combination therapy are crucial for the treatment of obstinate S. aureus bacteremia [34]. Besides, amoxicillin/clavulanic acid is recommended in amalgamations to overcome vancomycin or daptomycin resistance in MRSA [35][36][37]. Amoxicillin/clavulanic acid in conjunction with cephradine, an ineffective lower-generation antibiotic against MRSA [30], was an interesting combination. is lower-generation antibiotic proved successful in Time-Kill studies and shows the potential to be used again as a therapeutic agent in combination. Although, amoxicillin/clavulanic acid-tobramycin trio showed >2-log10 decrease in XDR MRSA isolate (LR-2), it did not show synergistic interactions in other isolates. Amoxicillin/clavulanic acid/fluoroquinolone (ciprofloxacin) combination has also been proven successful before for treatment of Gram-negative infections of prosthetic joints [38]. However, it must be noted that most of these isolates showed low individual MIC values for tobramycin and amoxicillin/ clavulanic acid, and so the FICI values were mostly "indifferent." Synergy between piperacillin-tazobactam-meropenem combination is consistent with the findings of PR Gonzales and his colleagues [20]. e inability to confirm all synergistic combinations by Time-Kill assay was a limitation of our study. Due to this, we might have overlooked certain combinations that might     International Journal of Microbiology produce better results than the ones we screened. However, the main objective of our study to test and identify combinations from diverse subclasses that interacted synergistically, belonged to lower generations, produced bactericidal effects, and overcome the resistance for first-line antibiotics that could not be used anymore was achieved. Although coadministration of drugs is a promising approach to mitigate rapid evolution of antibiotic resistance, it may also lead to production of unexpected and unwanted outcomes as reported previously [39]. Although the FIC index was determined to ensure that a synergistic relationship exists between selected antibiotics when used in combination, further investigations may help optimize the management of antibiotic combinations.

Conclusion
We report that pairwise LVX/CAZ combination allows us to use first-line antibiotics that not only produce bactericidal effects against XDR MRSA but also address need of time by extending the lifespan of existing antibiotics. is combination will be immediately available for use since these drugs have already been approved by the FDA. Resistance to antibiotic combination though inevitable, evidence-based synergistic studies might diminish the emergence of resistance. For future prospects, we propose that these combinations should be further tested in vivo to determine if these amalgams are as effective in vivo as they were in vitro. We also suggest that investigations should be carried out on how these combinations, where target proteins of the antibiotics involved in amalgam, work synergistically at molecular and systemic level to give a better insight. is will help predict better and robust combinations in future.

Data Availability
Data produced and analyzed during this study are included in this article.