Evaluation of the Synergistic Antibacterial Effects of Fosfomycin in Combination with Selected Antibiotics against Carbapenem–Resistant Acinetobacter baumannii

The spread of multi-drug resistant (MDR) pathogens and the lagging pace in the development of novel chemotherapeutic agents warrant the use of combination therapy as a reliable, cost-effective interim option. In this study, the synergistic effects of fosfomycin in combination with other antibiotics were assessed. Of the 193 isolates, 90.6% were non-susceptible to fosfomycin, with minimum inhibitory concentrations (MICs) of ≥128 µg/mL. Antibacterial evaluation of fosfomycin-resistant isolates indicated multi-drug resistance to various antibiotic classes. Combinations of fosfomycin with 12 commonly used antibiotics synergistically inhibited most fosfomycin-resistant isolates. The fractional inhibitory concentration index indicated that combining fosfomycin with either aminoglycosides, glycylcyclines, fluoroquinolones, or colistin resulted in 2- to 16-fold reduction in the MIC of fosfomycin. Time-kill kinetics further confirmed the synergistic bactericidal effects of fosfomycin in combination with either amikacin, gentamicin, tobramycin, minocycline, tigecycline, or colistin, with more than 99.9% reduction in bacterial cells. Fosfomycin-based combination therapy might serve as an alternative option for the treatment of MDR A. baumannii. Further steps including in vivo efficacy and toxicity in experimental models of infection are required prior to clinical applications.


Introduction
The emergence of multi-drug-resistant pathogens has limited treatment options with a consequent increase in mortality and extended hospital stays. With the rapid spread of drug resistance, especially among Gram-negative bacterial isolates, and the lag in the discovery of novel bioactive compounds, the fight against infectious diseases continues. The search for alternative chemotherapeutic agents effective for the management of multidrug resistant (MDR) pathogens including MDR Acinetobacter baumannii have become an urgent public health priority, warranting prospecting for novel active compounds [1]. Attempts at revitalizing old and already existing but abandoned agents through chemical modifications and combination therapies is indicated as an interim strategy for effective management of drug-resistant pathogens [2,3]. Antimicrobial combinations have been proposed to synergistically inactivate microbial cells through several mechanisms including enhanced bioavailability, inhibitor inhibition, sequential blockade, mutual stabilization, parallel pathway inhibition, and regulation modulation [4].
Acinetobacter baumannii, a Gram-negative bacterium of the family Moraxellaceae, is an opportunistic pathogen frequently associated with hospital-acquired infections and disease

Antibacterial Activities of Fosfomycin against A. baumannii Isolates
The broth microdilution results of fosfomycin activity on 191 A. baumannii isolates are presented in Figure 2 and Table S2. The MIC50 and MIC90 were recorded at 128 and 256 µ g/mL, respectively. Although fosfomycin is listed as a miscellaneous agent for the management of A. baumannii [6], there are currently no standard breakpoint figures for interpreting of antimicrobial activity of fosfomycin against A. baumannii. Several researchers have adopted CLSI breakpoint limits for Enterobacterales (susceptible, ≤64; intermediate, 128; resistant, ≥256) [26][27][28]. Based on the CLSI standards for Enterobacterales, the antibacterial effects of fosfomycin on 110 isolates were classified as intermediate, 63 isolates were resistant, while 18 isolates were susceptible. A similar high prevalence of fosfomycin resistance among A. baumannii isolates has been reported [26,[29][30][31].

Antibacterial Activities of Fosfomycin against A. baumannii Isolates
The broth microdilution results of fosfomycin activity on 191 A. baumannii isolates are presented in Figure 2 and Table S2. The MIC 50 and MIC 90 were recorded at 128 and 256 µg/mL, respectively. Although fosfomycin is listed as a miscellaneous agent for the management of A. baumannii [6], there are currently no standard breakpoint figures for interpreting of antimicrobial activity of fosfomycin against A. baumannii. Several researchers have adopted CLSI breakpoint limits for Enterobacterales (susceptible, ≤64; intermediate, 128; resistant, ≥256) [26][27][28]. Based on the CLSI standards for Enterobacterales, the antibacterial effects of fosfomycin on 110 isolates were classified as intermediate, 63 isolates were resistant, while 18 isolates were susceptible. A similar high prevalence of fosfomycin resistance among A. baumannii isolates has been reported [26,[29][30][31].

Antibacterial Effects of Fosfomycin-Resistant Isolates
The results of the antibacterial effects of fosfomycin against the five highly resistant isolates (MIC ≥ 512 µg/mL), evaluated by the agar dilution technique, are presented in

Antibacterial Effects of Fosfomycin-Resistant Isolates
The results of the antibacterial effects of fosfomycin against the five highly resistant isolates (MIC ≥ 512 µg/mL), evaluated by the agar dilution technique, are presented in Table 1. The results indicated agreement between the broth microdilution and agar dilution methods for all the isolates, and the standard strain ATCC 19606 was used (Table 1). Minimum inhibitory concentrations were obtained for all isolates except ST26. In addition, in the presence of the efflux pump inhibitor compound CCCP, a two-to four-fold reduction in the MIC of isolates was observed (Table 1). Table 1. Minimum inhibitory concentrations of fosfomycin against carbapenem-and fosfomycinresistant Acinetobacter baumannii isolates evaluated using the agar dilution method and broth microdilution with and without carbonyl cyanide 3-chlorophenyl hydrazine (CCCP).

Susceptibility Profile of Carbapenem-and Fosfomycin-Resistant A. baumannii Isolates
The antibiogram of the highly fosfomycin-resistant isolates were evaluated against 12 conventional antibiotics including carbapenems, aminoglycosides, glycylcyclines, fluoroquinolones, polymyxin (colistin), and the folate pathway antagonist (trimethoprim/ sulfamethoxazole) ( Table 2). The isolates showed multi-drug resistance to antibiotics across the various classes. All the isolates were highly resistant to carbapenems and trimethoprim/sulfamethoxazole but were susceptible to amikacin. The results suggested that antibiotics of the aminoglycoside class were more effective, with 60% susceptibility against the isolates. In addition, four of the isolates were resistant to colistin, a last-resort antimicrobial agent for the treatment of carbapenem-resistant A. baumannii, with MIC values ≥2 µg/mL. Antibiotics of the glycylcycline class also exhibited good activity against the resistant isolates. Three isolates and the standard strain ATCC 19606 were susceptible to minocycline, with MIC values ranging from 0.25 to 1 µg/mL, while four isolates were susceptible to tigecycline. Furthermore, antibiotics of the fluoroquinolone class showed limited activity against the isolates. All the isolates, including ATCC 19606, were resistant to ciprofloxacin, except isolate SK12 with an intermediate activity, whereas two isolates and the standard test strain were resistant to levofloxacin.

Ethidium Bromide Uptake
Furthermore, uptake of ethidium bromide, an indicator of AdeABC efflux inhibition, was investigated [32] (Figure 3). Binding of EtBr to double-stranded DNA resulted in a substantial increase in fluorescence for CCCP-treated cells compared with untreated cells. Isolates SK01, SK12, and ST26 showed a significant increase in EtBr uptake after de-energizing with CCCP (p < 0.05). The results suggested the likely presence of efflux pumps, as a possible mechanism mediating the resistance of the isolates to fosfomycin. susceptible to tigecycline. Furthermore, antibiotics of the fluoroquinolone class showed limited activity against the isolates. All the isolates, including ATCC 19606, were resistant to ciprofloxacin, except isolate SK12 with an intermediate activity, whereas two isolates and the standard test strain were resistant to levofloxacin.

Ethidium Bromide Uptake
Furthermore, uptake of ethidium bromide, an indicator of AdeABC efflux inhibition, was investigated [32] (Figure 3). Binding of EtBr to double-stranded DNA resulted in a substantial increase in fluorescence for CCCP-treated cells compared with untreated cells. Isolates SK01, SK12, and ST26 showed a significant increase in EtBr uptake after de-energizing with CCCP (p < 0.05). The results suggested the likely presence of efflux pumps, as a possible mechanism mediating the resistance of the isolates to fosfomycin. inimum inhibitory concentrations of fosfomycin-resistant A. baumannii isolates against conventional antibiotics.

Synergistic Effects of Fosfomycin-Antibiotics Combination
Several pathogenic bacteria have evolved mechanisms to neutralize or evade the effects of antibiotics. Presently, management of infections caused by A. baumannii relies on the use of last-resort antibiotics or administration of multiple antibiotic combinations. Thus, fosfomycin was combined in vitro with 12 conventional antibiotics and evaluated for synergistic effects. The results were interpreted based on the fractional inhibitory concentration index (Table 3).    The antibacterial activities of fosfomycin in combination with carbapenems (imipenem, doripenem, and meropenem) were mainly additive, except the doripenem combination, with FICI vales of 0.31 to 0.50. The fosfomycin and imipenem combination was not synergistic on any isolate, whereas fosfomycin with meropenem showed an FICI range of 0.38 to 0.50 for the isolate SK12. Combinations of fosfomycin with aminoglycosides (gentamicin, amikacin, and tobramycin) presented synergistic effects, with FICI values ranging from 0.25 to 0.5. The results showed that fosfomycin plus gentamicin was synergistic against four isolates, while the fosfomycin plus amikacin combination and the fosfomycin plus tobramycin combination showed synergistic antibacterial effects against two and three isolates, respectively. Similarly, fosfomycin plus glycylcycline combinations (tigecycline or minocycline) displayed synergistic effects against three isolates each, with FICI values ranging from 0.14 to 0.5 and 0.25 to 0.5 for the tigecycline and minocycline combination, respectively. Furthermore, the results revealed the synergistic effects of fosfomycin plus fluoroquinolone (ciprofloxacin or levofloxacin) combinations against two isolates each, and FICI values of 0.31 to 0.5 for the ciprofloxacin combination and 0.25 to 0.5 for the levofloxacin combination. Fosfomycin plus colistin in combination yielded synergistic effects against three isolates with FICI values of 0.25 to 0.5, whereas the combination of fosfomycin and trimethoprim/sulfamethoxazole yielded no synergism.

Time-Kill Kinetics of Combinations of Fosfomycin on A. baumannii
The time-dependent killing of fosfomycin in combination with amikacin, gentamicin, tobramycin, doripenem, colistin, levofloxacin, ciprofloxacin, tigecycline, and minocycline was monitored over an 18-h exposure (Figure 4). The results revealed the substantial synergistic effects of fosfomycin in combination with all three aminoglycosides (amikacin, gentamicin, and tobramycin) ( Figure 4A Figure 4C). At the concentrations used, the combinations of fosfomycin with either doripenem, levofloxacin, or ciprofloxacin were not synergistic against the A. baumannii isolate (TR 122). However, the combination with fosfomycin displayed an additive effect and enhanced the antibacterial activities of doripenem, levofloxacin, and ciprofloxacin with >3 log reduction in CFU/mL when compared with the single antibiotic MIC of doripenem or levofloxacin, and a 1-3 log reduction in CFU/mL for ciprofloxacin. Combinations of fosfomycin and glycylcycline antibiotics (tigecycline and minocycline) demonstrated synergistic bactericidal (>3 log reduction in CFU/mL) effects at 1

Discussion
Antimicrobial drug resistance is ranked as one of the top 10 global health threats facing humanity [33]. Clinical management of hospital-related infections caused by pathogenic Gram-negative Enterobacterales and A. baumannii uses administration of carbapenems as a last-resort remedy. However, the emergence of carbapenem resistance due to the production of carbapenamase enzymes limits the continued usage of carbapenem antibiotics. Antibacterial testing against carbapenems revealed greater than 90% resistance. Similar results were reported for A. baumannii isolates obtained from tertiary hospitals within Thailand [34]. Considering the severity of carbapenem resistance and the dangers it poses, carbapenem-resistant Enterobacterales and A. baumannii are classified in the critical tier of antimicrobial-resistant pathogens [5].
As treatment options continue to dwindle, the use of combination therapies has become a reliable strategy for the management of drug-resistant pathogens. In addition, antimicrobial combinations subdue the development of resistance by limiting the mutant selection window of single agents [35]. Revitalization of old, abandoned, and somewhat ineffective antibiotics, through arrays of chemical modification, antibiotic hybridizing, or as adjunctive therapies, promises reliable outcomes [36,37]. Although developed nations are restricted by legislations regarding the choice and usage of antibiotics, clinical practitioners in most developing nations are at liberty to administer combinations of antibiotic medications based on experience of previous efficacy.
Fosfomycin, an old phosphonic acid derivative, exhibits enhanced tissue penetration due to its low molecular weight (138 Da) [38] and relatively mild side effects. The in vitro and in vivo antimicrobial effects of fosfomycin monotherapy and in combination have been demonstrated for Gram-positive MDR bacterial isolates including methicillin-resistant Staphylococcus aureus [39,40] and Gram-negative Enterobacterales [41,42]. However, A. baumannii is reported to be intrinsically resistant to fosfomycin monotherapy [43]. The present study investigated the antibacterial effects of fosfomycin alone and the combinatory effects of fosfomycin with regularly used antibiotics as a last-line option for the treatment of infections caused by carbapenem-resistant A. baumannii. Fosfomycin alone showed MIC values ranging from 32 to >2048 µg/mL against the 191 isolates. Currently, fosfomycin is listed as a miscellaneous agent for the manage-ment of A. baumannii; however, breakpoints have not yet been established. Based on fosfomycin breakpoint values for Enterobacterales, the isolates showed a high rate of resistance to fosfomycin, with 57.59% classified as intermediate and 32.98% as resistant. Similar antibacterial effects of fosfomycin against A. baumannii have been previously reported [26,31,43]. Several factors, including the presence of efflux pumps [31], resistance-encoded plasmids [44], and mutations in the ampD and anmK genes, encoding enzymes of the peptidoglycan recycling pathway [43], have been reported to mediate A. baumannii resistance to fosfomycin. However, the mechanism of re-sistance is still poorly understood. The MIC values of fosfomycin decreased two-to fourfold, while uptake of ethidium bromide increased in the presence of CCCP. Previous studies have reported reductions in MIC values in the presence of CCCP as a positive indicator of efflux pump-mediated resistance [20,45,46]. Carbonyl cyanide 3-chlorophenyl hydrazone is a known proton motive force and resistance-nodulation-division efflux pump inhibitor that promotes the transport of molecules across the bacteria membrane.
Combinations of fosfomycin with other antibiotics yielded synergistic antibacterial effects against the tested isolates. In particular, combinations of fosfomycin with aminoglycosides demonstrated enhanced bactericidal effects when compared with antibiotics of other classes. This demonstrates the role of a multi-target mechanism in the management of antibiotic-resistant pathogens. Similar findings were reported for combination therapies of fosfomycin and aminoglycosides (amikacin or gentamicin) against MDR bacterial isolates including Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli [20,47,48]. Aminoglycosides generally inhibit protein synthesis by binding to the A-site on the 16S ribosomal RNA of the 30S ribosome [49], whereas fosfomycin inhibits the MurA enzyme and UDP-N-acetylglucosamine-enolpyruvyltransferase, involved in peptidoglycan synthe-sis [50]. In addition, antibiotics of the glycylcycline group (minocycline and tigecycline) and colistin in combination with fosfomycin exhibited synergy against the test isolate. Both the checkerboard techniques and the time-kill assays used to evaluate the in vitro efficacies of the antibiotic combinations showed that fosfomycin with either of amikacin, gentamicin, tobramycin tigecycline, minocycline, or colistin could be used for the management of the carbapenem-resistant A. baumannii. The time-kill kinetics further showed additive effects for combinations of fosfomycin and fluoroquinolones (ciprofloxacin or levofloxacin), with less reduction of bacterial count (CFU/mL) compared with fosfomycin monotherapy at MIC. In addition, combinations of fosfomycin with carbapenem (imipenem, meropenem or doripenem) displayed synergistic effects, with two-to fourfold reductions in fosfomycin MICs, as demonstrated by the checkerboard technique. Contrarily, when the doripenem plus fosfomycin combination was assayed with time-kill kinetics, the combination failed to meet the criteria for synergy. Similar inconsistencies between the FICI and time-kill techniques have been reported by previous researchers [51,52]. This observation might be due to the presence of persistent cells that remain viable when the level of antibiotics drops [53]. The addition of carbapenem did not yield effective experimental outcomes, thus co-administration or adjunctive therapy of fosfomycin in A. baumannii-infected patients receiving carbapenems might not potentiate favorable clinical outcomes. However, combination therapy might completely kill A. baumannii if a longer treatment time is used [53].

Bacterial Strains
The study included 193 A. baumannii isolates collected from patients admitted to hospitals within Southern Thailand. All isolates were presumptively identified as Acinetobacter species using standard biochemical tests as Gram-negative, oxidase-negative, nonmotile, non-fermenting coccobacilli [54], and further identified as A. baumannii by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Acinetobacter baumannii ATCC 19606 was used as a quality control. All the bacterial cultures were stored in tryptic soy broth (TSB) supplemented with 40% glycerol and kept at −80 • C.

Resistance to Carbapenems
The resistance of the 193 A. baumannii isolates to carbapenem was assessed by disc diffusion assay as recommended [55], using a 10-µg disc of imipenem and meropenem. The isolates were cultured to log phase and the cultures were adjusted to 10 6 CFU/mL in phosphate buffer solution. An aliquot (100 µL) of adjusted culture was evenly spread on Mueller-Hinton agar, and the disc was properly positioned. Plates were incubated at 35 • C for 16 to 18 h, and the zone of inhibition was measured and interpreted.

Screening for Fosfomycin Resistance
The minimum inhibitory concentrations (MICs) of fosfomycin on the 193 isolates was determined by the broth microdilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [55]. Briefly, serial two-fold dilutions of antibiotics were prepared in cation-adjusted Mueller-Hinton II broth. Aliquots (100 µL) of the diluted bacterial suspension (1 × 10 6 CFU/mL) were exposed to 100 µL of varying antibiotic concentrations and incubated at 37 • C for 18 h. MIC was expressed as the lowest concentration of the antibiotic without microbial growth as indicated by the resazurin test. MIC 50 was defined as the lowest concentration of fosfomycin that inhibited 50% of the isolates, and MIC 90 as the lowest concentration that inhibited 90% of the isolates.
Additionally, in line with the recommendations for antimicrobial testing of fosfomycin [55], the antibacterial activities of fosfomycin were evaluated on highly resistant isolates with MIC ≥ 512 µg/mL. Mueller-Hinton agar was supplemented with 25 µg/mL of glucose-6-phosphate and varying concentrations of fosfomycin (128-2048 µg/mL). Plates were dried overnight and inoculated with 10 4 CFU of each isolate. MIC was recorded as the lowest fosfomycin concentration that completely inhibited growth, disregarding a single colony or a faint haze caused by the inoculum. The MICs of fosfomycin in the presence of 12.5 and 25 µg/mL carbonyl cyanide 3-chlorophenyl hydrazone (CCCP) (Sigma-Aldrich, USA) was evaluated.

Ethidium Bromide Uptake Assay
Uptake of ethidium bromide (EtBr) in the presence and absence of CCCP was further investigated as described [31]. In brief, cells were grown to log phase, harvested, and washed thrice with a phosphate buffer solution. Cells were resuspended into PBS and adjusted to 0.3 OD at 600 nm. Afterwards, 1 mL of bacterial suspension was treated with EtBr at 2 µg/mL and incubated for 20 min; 0.4% (w/v) of glucose and 25 µg/mL CCCP were added and incubated for 30 min. The cell suspension was then aliquoted into 96-well plates and the fluorescence intensity was measured at excitation and emission of 513 and 600 nm. Cells treated with 0.1% dimethyl sulfoxide without CCCP treatment were used as controls for ethidium accumulation.

Checkerboard Technique
The effects of fosfomycin in combination with 12 other antibiotics (imipenem, meropenem, doripenem, amikacin, gentamycin, tobramycin, minocycline, tigecycline, ciprofloxacin, levofloxacin, colistin, and trimethoprim/sulfamethoxazole) on carbapenem-and fosfomycinresistant isolates of A. baumannii were evaluated using the checkerboard technique. Briefly, 100 µL of diluted bacterial suspension (1 × 10 6 CFU/mL) was added to wells containing 50 µL of subinhibitory concentrations of fosfomycin and 50 µL of subinhibitory concentrations of one of the 12 other antibiotics. The plates were incubated for 18 h at 37 • C. Inhibitory concentrations were determined as concentrations without growth as indicated by the resazurin test. The antibacterial effects of single antibiotics were tested as a control. The experiment was performed for three independent repeats. The effects of the antimicrobial combination were defined according to the fractional inhibitory concentration index (FICI) as shown in the following equation: The FICI results for each combination were interpreted as follows: FICI ≤ 0.5, synergism; 0.5 < FICI < 1, additive; 1 ≤ FICI < 2, indifference; FICI ≥ 2, antagonism. Escherichia coli ATCC 25922 was used as a standard control strain for the assays [56].

Time-Kill Assay
Time-kill assays was performed in cation-adjusted Mueller-Hinton broth using the checkerboard assay. Overnight culture of the isolate with the highest MIC of fosfomycin (TR122) was adjusted to~10 6 CFU/mL and treated with single antibiotics at MIC and a combination of fosfomycin and other antibiotics at 1 /2 MIC and 1 /4 MIC. Changes in bacterial population were monitored by plate count at 2, 4, 8, 12, and 18 h, and reported as log reductions in CFU/mL. Untreated cultures were included as a control, and the experiment was performed in triplicate. Antibiotic combination synergism was defined as a 2-log reduction in CFU/mL when compared with the most active single antibiotic, whereas bactericidal activity was defined as a ≥3 log reduction in CFU/mL when compared with the number of viable cells at time zero (0 h) [29].

Ethical Statement
This study was approved by the Institutional Review Board of the Faculty of Medicine, Prince of Songkla University (EC:54-080-14-1-2). The study was conducted at Songklanagarind Hospital, which is an 800-bed university hospital and referral medical center located in southern Thailand. The researchers were granted permission to extract the data from the database with a waiver of consent because of the observational nature of the study. All data were fully anonymized before the researcher accessed and analyzed them. The medical records of adult patients (age ≥ 18 years) seeking medical treatment between February and July 2019 and diagnosed with A. baumannii bacteremia were collected between February and December 2019 and used in the study.

Conclusions
Fosfomycin-based combination therapy might serve as an option for the treatment of MDR A. baumannii. Further steps including in vivo efficacy and toxicity in experimental models of infection are required prior to clinical applications.
Author Contributions: Experimental design, experimentation, manuscript writing, and data analysis, manuscript editing and revision: O.F.N.; experimentation, and data analysis: P.T.; supervision, editing, and contributing research materials: S.P.V.; providing funding, editing, and supervision: S.C. All authors have read and agreed to the published version of the manuscript.

Informed Consent Statement:
The researchers were granted permission to extract the data from the database with a waiver of consent because of the observational nature of the study. All data were fully anonymized before the researcher accessed and analyzed them. Data Availability Statement: Data is contained in the article.