Fosfomycin as Partner Drug for Systemic Infection Management. A Systematic Review of Its Synergistic Properties from In Vitro and In Vivo Studies

Fosfomycin is being increasingly prescribed for multidrug-resistant bacterial infections. In patients with systemic involvement, intravenous fosfomycin is usually administered as a partner drug, as part of an antibiotic regimen. Hence, the knowledge of fosfomycin pharmacodynamic interactions (synergistic, additive, indifferent and antagonistic effect) is fundamental for a proper clinical management of severe bacterial infections. We performed a systematic review to point out fosfomycin’s synergistic properties, when administered with other antibiotics, in order to help clinicians to maximize drug efficacy optimizing its use in clinical practice. Interactions were more frequently additive or indifferent (65.4%). Synergism accounted for 33.7% of total interactions, while antagonism occurred sporadically (0.9%). Clinically significant synergistic interactions were mostly distributed in combination with penicillins (51%), carbapenems (43%), chloramphenicol (39%) and cephalosporins (33%) in Enterobactaerales; with linezolid (74%), tetracyclines (72%) and daptomycin (56%) in Staphylococcus aureus; with chloramphenicol (53%), aminoglycosides (43%) and cephalosporins (36%) against Pseudomonas aeruginosa; with daptomycin (97%) in Enterococcus spp. and with sulbactam (75%) and penicillins (60%) and in Acinetobacter spp. fosfomycin-based antibiotic associations benefit from increase in the bactericidal effect and prevention of antimicrobial resistances. Taken together, the presence of synergistic interactions and the nearly total absence of antagonisms, make fosfomycin a good partner drug in clinical practice.


Introduction
Antimicrobial resistance (AMR) is a health issue of global concern, burdened with elevated costs and high morbidity and mortality rates. Limited therapeutic options and the increasing occurrence of resistance to last-resort antibiotics, i.e., colistin or carbapenems, make it necessary to reassess the role of "old" drugs while waiting for new antibiotics available on the market. Fosfomycin (FOS) is an inhibitor of the synthesis of the bacterial wall acting with a unique mechanism of action.
To carry out its action, FOS enters in the bacterial cell through the L-alpha-glycerophosphate and the hexose-6-phosphate transporter systems, interfering with the formation of the peptidoglycan precursor uridine diphosphate N-acetylmuramic acid (UDP-MurNAc) [1].
FOS, after being discovered in 1969 [2], has long been prescribed orally for low urinary tract infections (UTIs) and only recently has been repurposed, also intravenously and in combination, as a meropenem-and colistin-sparing agent to treat other infections (complicated UTIs, severe soft tissue infections, osteomyelitis, prostatitis, etc.) [1,[3][4][5]. The excellent distribution in body sites, the safety and tolerability profile, as well as its affordability, make FOS a therapeutic option worth considering to treat multidrug-resistant (MDR) bacterial infections [6,7].
FOS is generally prescribed in association with at least another active agent. The association benefits from increase in the bactericidal effect of FOS, prevention of AMR, limitation of side effects thanks to lower dosages. Examples of commonly used empirical combination regimens including FOS are: Carbapenems + FOS, colistin + FOS, ceftolozane/tazobactam + FOS and tigecycline (TIG) + FOS.
We performed a systematic literature review concerning in vitro and in vivo studies to evaluate the synergistic effect of FOS in combination with other antibiotics and offer an overall view with clinically practical tables divided by antibiotic class.

Materials and Methods
This systematic review was carried out following the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA).
On 14 April 2020 we performed a MEDLINE/PubMed search using the search string "Fosfomycin" 1232 papers, from inception to 14 April 2020, were identified. Of these, 870 were excluded by title screening, 84 by abstract screening, 28 after full-text reading. Fifty-eight papers were excluded because written in a language different from English. 7 papers were excluded because full text was not available either online or in paper version. 185 papers were reviewed and discussed independently by seven authors (RMA, RP, AL, SDB, VV, LP, MF).
Common criteria for the evaluation of susceptibility and synergism were adopted by all authors. Susceptibility. Susceptibility to FOS for Enterobacterales and Staphylococcus spp. was determined, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, when the minimum inhibitory concentration (MIC) was ≤ 32 µg/mL. Enterococcus spp. were considered susceptible when exhibiting a MIC ≤ 64 µg/mL, according to the Clinical & Laboratory Standards Institute (CLSI) breakpoints. FOS breakpoints are not defined either by EUCAST or CLSI for Pseudomonas spp., Acinetobacter spp. and Streptococcus spp. Based on literature data, susceptibility was defined as a MIC ≤ 128 µg/mL for Pseudomonas spp. (ECOFF value), MIC ≤ 32 for Acinetobacter spp. and ≤64 µg/mL for Streptococcus spp. [8,9].
For all the antibiotics tested in combination, EUCAST breakpoints was considered at first and CLSI breakpoints were considered when EUCAST breakpoints were not available. Breakpoints adopted are specified in each paragraph.
For in vitro studies using a method different from checkerboard or time-kill assay, or in case data on effective concentrations were not available, synergism was evaluated according to the authors' judgment.
For studies performed in vivo, synergism was established with the same ratio of effective concentrations considered for checkerboard assays or with the same log kill considered for time-kill assays. When these data were not reported in the paper, synergism was evaluated according to the authors' judgment.

Results
For a better comprehension, a table with reviewed papers and a summary of most relevant results is proposed for each antibiotic class.

Penicillins
Twenty-eight papers evaluating FOS in combination with penicillins, penicillins + β-lactamase inhibitors, penicillinase-resistant penicillins were reviewed (Table 1). Breakpoints for penicillins were inferred from EUCAST breakpoints [10]. Penicillins are β-lactam antibiotics that acts through the inhibition of enzymes needed for peptidoglycans cross linking. Effect of FOS in combination with penicillins varied greatly according with the bacterial species considered. The highest rates of synergistic effect were observed against Enterobacterales and Acinetobacter spp. Despite this, Avery et al. [11] reported high rates of indifferent effect of FOS + piperacillin/tazobactam (PIP/TAZ) against PIP/TAZ-resistant Enterobacterales. Antagonistic effect was observed against one isolate of S. aureus with the combination FOS + methicillin [12] and against 6 biofilm-producer Enterococcus faecalis isolates with the combination FOS + ampicillin [13]. Four studies [14][15][16][17] performed in vivo experiments, with no substantial differences in results when compared with results obtained in vitro.
The combination of cephalosporins or cephalosporins + β-lactamase inhibitors + FOS appears to be clinically appealing especially against infections sustained by Enterobacterales and Pseudomonas spp.
Synergism rates were not unanimous on all studies, but antagonistic effect was observed only in 2 isolates of P. aeruginosa in the study by Pruekprasert et al. [22] and in 1 isolate of S. aureus in the study by Quentin et al. [35]. No evident differences in the synergistic effect was observed depending on the carbapenem tested. The association FOS + carbapenem often resulted, when reported, in FOSand/or carbapenem-susceptibility restoration. Three authors performed in vivo experiments using methicillin-resistant Staphylococcus aureus (MRSA) isolates: in two studies [28,36] the results in vivo were concordant with those found in vitro, while in the third study the combination in vivo resulted less effective [37].
From the clinical point of view the combination of carbapenems + FOS against Enterobacterales, P. aeruginosa end Acinetobacter spp. appears appealing.

Monobactams
Five papers evaluating FOS in combination with aztreonam (ATM) were reviewed (Table 4). ATM is a synthetic antibiotic whose susceptibility is often preserved also in those strains which are resistant to other β-lactam antibiotics. The mechanism of action is similar to penicillins. ATM breakpoints are ≤1 µg/mL for Enterobacterales and ≤0.001 µg/mL for Pseudomonas spp. [10].
The largest study evaluating FOS in combination with ATM on Enterobacterales isolates [33] reported an indifferent effect on most (64.6%) isolates. The combination was reported to have an additive effect on most isolates of P. aeruginosa [33,38], sometimes leading to ATM susceptibility restoration [33,39]. There were no in vivo studies evaluating this combination.

Quinolones
Twenty-nine papers evaluating FOS in combination with quinolones were reviewed (Table 5). Quinolones are bactericidal antibiotics that directly inhibit bacterial DNA synthesis. Breakpoints for quinolones were inferred from EUCAST breakpoints [10]. Synergism rates were not unanimous on all studies for isolates of P. aeruginosa. In 1 in vivo study synergism rate was 100% according to Mikuniya et al. [40]. Antagonism was observed in 1 in vivo [41] and 1 in vitro studies [39]. For E. coli isolates there was a weak synergism. In a recent in vitro study there was complete FOS and ciprofloxacin susceptibility restoration [42]. The combinations showed different synergistic rates for Staphylococcus spp. isolates with 100% synergistic rate in 1 in vitro study [43] and in 1 in vivo study [44]. No antagonism was observed for E. coli and Staphylococcus spp. isolates. There were some differences in the synergistic effect depending on the quinolone tested. The most frequent effect of FOS + ciprofloxacin was indifferent even though it showed in vitro 95% synergistic effect with S. aureus [45] The combination with levofloxacin showed mainly an additive effect in P. aeruginosa [38,39,46] and in Acinetobacter spp. [38] isolates.
In summary good additive/synergistic effect rates are reported when quinolones + FOS are used against S. aureus and P. aeruginosa isolates. cases additive effect or, less frequently, synergistic effect [14]. When this combination was tested against Streptococcus spp. synergistic effect was observed against 15% of isolates, while additive (27%) or indifferent (58%) was seen against the remaining [14]. Some studies evaluated FOS + AZT combination, reporting indifferent effect in 100% of cases, either when tested against N. gonorrhoeae (2 studies) [54,57] or against S. epidermidis (1 study) [58]. Finally, FOS + CLT and FOS + MDM combinations were evaluated against S. pseudointermedius and P. aeruginosa respectively; in both cases additive or synergistic effect was demonstrated in vitro or in vivo experiments [59,60]. No antagonistic effect was observed for any combination against any isolate.
From the clinical point of view the combination of macrolides + FOS appears the less appealing.
In summary the combination of VAN + FOS resulted in good synergistic effect rates against Enterococcus spp. isolates and seems to be the most clinically relevant combination.

Tetracyclines
Ten papers evaluating FOS in combination with tetracyclines, mostly with minocycline (MIN) and in few cases with doxycycline (DOX) or tetracycline (TEC), were reviewed (Table 9). Tetracyclines are a large class of antibiotics that acts binding the 30S ribosomal subunits, inhibiting bacterial proteins synthesis. They have broad-spectrum activity, being active against many Gram-positive bacteria, Gram-negative, and atypical bacteria [64]. Almost all studies evaluated in vitro FOS + MIN combination against different bacterial species. When evaluated against Enterobacterales (20 strains), FOS + MIN proved to have additive effect most of the time (65% of isolate), but only in few cases synergistic effect [38]. Similar results were observed when it was tested against multidrug-resistant P. aeruginosa [38] and A. baumannii isolates; furthermore, in the last case, complete restoration of susceptibility of MIN was reported [65]. Only one study evaluated FOS + TEC combination against Enterobacterales (100 isolates), observing indifference in almost 100% of cases [66]. 2 studies evaluated FOS + MIN combination against vancomycin-resistant E. faecium or E. faecalis (51 strains), reporting most often indifferent effect and some sporadic case of synergism [13,67]. Otherwise, FOS + DOX combination was tested once against 24 isolates of vancomycin-resistant E. faecium, demonstrating to have synergistic or additive effect in most of cases [68]. Finally, when FOS + MIN was tested against MRSA (152, strains, 3 studies) proved to have synergistic effect in numerous cases [18,69,70]. No study reported any case of antagonism.
The combination of minocycline + FOS against A. baumannii appears interesting.

Polymyxins
Thirty-two papers evaluating FOS in combination with polymyxins were reviewed (Table 10). Polymyxins are bactericidal drugs that bind to lipopolysaccharide (LPS) and phospholipids in the outer cell membrane of Gram-negative bacteria and leads to disruption of this. Twenty-eight papers evaluated colistin. Colistin breakpoints are ≤ 2 µg/mL for Enterobacterales, Acinetobacter spp. and Pseudomonas spp. according to the EUCAST [10]. Synergism rates were not unanimous on all studies but was reported in 23/29 papers. Synergisms rate were 100% in 2 in vitro studies against K. pneumoniae [50,71] and 2 in vivo studies respectively against A. baumannii and E.coli [72,73]. The overall effect was indifferent on most isolates of P. aeruginosa and Enterobacterales. Antagonism was reported in vitro against K. pneumoniae and A. baumannii. In particular the combination was antagonist in 100% of all K. pneumoniae OXA-48 isolates according to Evren et al. [74].
Four papers evaluated polymyxin B. Polymyxin B breakpoints for Enterobacterales, Acinetobacter spp. and Pseudomonas spp. are ≤ 2 µg/mL according to CLSI. Synergism was observed in 100% of in vitro isolates of CP K. pneumoniae according to Bulman et al. [75]. FOS + polymyxin had a prevalent addictive effect in vitro against Pseudomonas spp. [76] and A. baumannii [65]. In a study there was a complete polymyxin B susceptibility restoration [65]. No antagonistic effect was observed either in in vitro or in vivo studies.
The combination of polymyxins and FOS appears a good option against Enterobacterales and P. aeruginosa strains.
When evaluated against S. aureus isolates, the combination FOS + DAP had a synergistic effect in vitro against 37-100% of isolates (synergistic effect of the combination against 100% of the tested isolates was reported in 4 in vitro studies [63,[79][80][81] and 2 in vivo studies [37,79]). DAP showed excellent synergistic activity in association with FOS against Enterococcus spp., resulting in synergistic effect in all 34 tested isolates (4 studies). FOS + DAP also exhibited a greater efficacy against E. faecalis biofilm formation than FOS or DAP alone. Efficacy in vivo sometimes differed from the results obtained in vitro, resulting in greater [37] or less [82] efficacy. No antagonistic effect was observed either in in vitro or in vivo studies.
The combination of daptomycin + FOS has good synergistic effect rates against S. aureus and Enterococcus spp. and deserves clinical interest.

Tigecycline
Fourteen papers evaluating FOS in combination with TIG were reviewed (Table 12). TIG is the first glycylcycline antibiotic, a broad-spectrum class of bacteriostatic derivate from tetracyclines, that acts binding the 30S ribosomal subunits, inhibiting bacterial proteins synthesis. It is only available for intravenous administration and shows activity against either Gram-positive or Gram-negative or atypical bacteria [64]. Its breakpoint are ≤0.5 mg/L both for S. aureus and Enterobacterales and ≤0.25 mg/L for Enterococcus spp. [10].
According to the literature the combination of TIG + FOS appears to be particularly interesting (good synergistic effect rates) against Enterobacterales and Enterococcus spp.

Linezolid
Thirteen papers evaluating FOS in combination with linezolid (LZD) were reviewed (Table 13). LZD is a synthetic antibiotic which binds rRNA on both 30S and 50S ribosomal subunits, inhibiting bacterial proteins synthesis [92]. It is used for Gram-positive infections treatment, including MRSA and E. faecium vancomycin-resistant (VREF) infections [93]. Its breakpoint is ≤4 µg/mL both for S. aureus and E. faecium.
When evaluated against S. aureus isolates (9 studies), combination FOS + LZD had a synergistic effect in vitro approximately in 95% of cases (synergistic effect of the combination against 100% of the tested isolates was reported in 6 in vitro studies [36,43,63,94,95]) and even against staphylococcal biofilm cultures [69]; furthermore, the only 2 in vivo studies performed proved FOS + LZD combination to have higher efficacy than FOS or LZD alone [36,95]. One study evaluated the combination on 2 strains of S. epidermidis proving synergism on both [43]. Otherwise, in the 4 studies in which it was tested against E. faecium, this combination showed in most cases additive effect and only few cases of synergism. In no case was reported synergistic effect against E. faecalis (2 studies). No antagonistic effect was observed either in in vitro or in vivo studies.
The good synergistic effects reported make LZD + FOS a promising combination against staphylococci.

Rifampin
Fourteen papers evaluating FOS in combinations with rifampin were reviewed (Table 14). Rifampin breakpoints are ≤0.06 µg/mL for Staphylococcus spp., Streptococcus spp. and ≤0.125 µg/mL for S. pneumoniae. Rifampin inhibits bacterial DNA-dependent RNA polymerase with a concentration related effect. It is used for the treatment of intracellular pathogens and it has a broad-spectrum antibacterial activity. Rifampin breakpoints are not defined either by EUCAST or by CLSI for Acinetobacter spp., Enterobacterales and Enterococcus spp. Based on literature data, susceptibility was defined as a MIC ≤ 1 µg/mL for Enterococcus spp. [71]. Rifampin showed synergistic activity in association with FOS against Enterococcus spp., resulting in synergistic effect in 20−100% of cases. High activity was reported in vitro and in vivo in a recent paper where FOS + RIFA also exhibited a greater efficacy against E. faecalis biofilm formation [90]. When evaluated against S. aureus isolates, the combination FOS + rifampin had a synergistic effect in vitro against 34−100% of isolates. Synergistic effect of the combination against 100% of the tested isolates was reported in 3 in vitro studies [43,90,96] and 2 in vivo studies [37,96]. Antagonistic effect was observed only in 33% of isolates in the study by Quentin et al. [35] where the antibiotic combination was antagonist for the isolates susceptible and intermediate to rifampin and indifferent for those resistant. No antagonistic effect was observed in other studies.
In clinics RIF + FOS should be considered (usually with a third agent) against S. aureus sustained infections, especially when biofilm production is likely.

Miscellanea
Two papers evaluating FOS in combination with metronidazole (MTZ) were reviewed (Table S1). MTZ is a bacteriostatic antimicrobial, active on bacteria (mainly anaerobic) and parasites. When evaluated in vitro against Helicobacter pylori, combination FOS + MTZ had a prevalent indifferent effect, an additive effect in only 21% of cases and an antagonist effect in 4% [97]. In vivo study showed a significantly decrease mortality and increase cure rates if the animal treated with MTZ + FOS [98].
One paper evaluating FOS in combination with spectinomycin (SCM) was reviewed (Table S1). SCM is an aminocyclitol aminoglycoside antibiotic with bacteriostatic activity, used to treat gonorrhea. In vitro study reported that antimicrobial combinations of SMC + FOS no synergistic effect was found [54].
One paper evaluating FOS in combination with sulbactam (SLB) was reviewed (Table S1). SLB is an irreversible β-lactamase inhibitor capable to binding to penicillin-binding proteins and with weak antimicrobial activity. When evaluated in vitro against A. baumannii OXA-23, combination FOS + SLB had a synergistic effect in 75% of case, and an indifferent effect in 25% of cases [99].
One paper evaluating FOS in combination with lincomycin (LNM) was reviewed (Table S1). LMN is a protein synthesis inhibitor with activity against gram positive and anaerobic bacteria. When evaluated in vitro against S. aureus, combination FOS + LNM had a synergistic effect in 81% of case and an additive effect in 25% of cases [14].
One paper evaluating FOS in combination with nitroxoline (NTX) was reviewed (Table S1). NTX is a urinary antibacterial agent active against susceptible Gram-positive and Gram-negative organisms. In vitro study, NTX was synergistic with FOS in only 12% of cases and in other cases shoed an indifferent effect (88%) [66].
Two papers evaluating FOS in combination with quinupristin/dalfopristin (Synercid) were reviewed (Table S1). Synercid is a protein synthesis inhibitor used to treat infections by staphylococci and by vancomycin-resistant strain. When evaluated in vitro against methicillin resistant or susceptible Staphyloccoccus spp., combination FOS + Synercid had a synergistic effect in 100% of case [43,100].
Three papers evaluating FOS in combination with fusidic acid (FSA) were reviewed (Table S1). FSA is a bacteriostatic antibiotic with acts as a bacterial protein synthesis inhibitor. When evaluated in vitro against MRSA, combination FOS + FSA had a various behavior, showing a synergistic effect in 88-100% of case or an indifferent effect in 100% of cases. No antagonism was found [69,101,102].
Four papers evaluating FOS in combination with chloramphenicol (CHL) were reviewed (Table S1). CHL is a synthetic broad-spectrum antimicrobial, mainly bacteriostatic, active on numerous Gram-positive and Gram-negative, aerobic and anaerobic bacteria; it acts binding 50S ribosomal subunit, inhibiting bacterial protein synthesis [103]. Its breakpoint is ≤ 8 mg/L both for S. aureus and Enterobacterales [10]. When evaluated in vitro against either Enterobacterales (468 isolates, 4 studies), combination FOS + CHL had synergistic effect approximately in 40% of cases, while additive effect in 35% and indifferent effect in the remaining cases [14,66,104,105]. Furthermore, one study tested this combination against S. aureus, with similar results (synergistic effect against 44% of isolates) [14]. No antagonistic effect was observed.
Three papers evaluating FOS in combination with trimethoprim-sulfamethoxazole (TMP-SMX) were reviewed (Table S1). TMP-SMX is a fixed combination of 2 antimicrobials that inhibits bacterial synthesis of tetrahydrofolate, a necessary cofactor for bacterial DNA synthesis. It is available in oral or intravenous preparation and it is mainly used for treatment of urinary and respiratory infections [106]. Its breakpoint is ≤ 2 µg/mL both S. aureus and Enterobacterales [10]. When evaluated in vitro against either S. aureus (148 isolates) or Enterobacterales (120 isolates), combination FOS + TMP-SMX had indifferent effect approximately against 92% of isolates [12,38,66]. Only in few cases, against Enterobacterales, was reported synergistic or additive effect (1 study) [38] and even antagonistic effect was reported in 4 cases when tested against S. aureus [12].
Two papers evaluating FOS in combination with nitrofurantoin (NTF) were reviewed (Table S1). NTF is a synthetic antibiotic administered orally mainly for treatment of lower urinary tract infections.

Non-Antibiotic Molecules
One paper evaluating FOS in combination with auranofin (AF) was reviewed (Table S2). AF is an orally active gold compound for the treatment of rheumatoid arthritis. When evaluated in vitro against Staphyloccoccus spp., combination FOS + AF had showed a reduction of bacterial load for both MSSA and MRSA strains. In vivo, this combination had showed a synergistically inhibition of abscess and inflammation formation. No interactions were showed against S. epidermidis MS [107]. Three paper evaluating FOS in combination with dilipid ultrashort cationic lipopeptides, tobramycin-efflux pump inhibitor (TOB-EPI) conjugates or amphiphilic lysine-tobramycin conjugates (ALT) against P. aeruginosa, were reviewed (Table S2). For all combinations, in vitro studies had showed a synergistic effect (100%). Furthermore, in presence of TOB-EPI or ALT conjugates MICs of FOS were dramatically reduced [108][109][110]. One paper evaluating FOS in combination with β-chloro-L-alanine (β-CLA) was reviewed (Table S2). β-CLA is an amino acid analog of FOS. When evaluated in vitro against MRSA, combination FOS + β-CLA had showed a synergistic effect on biofilm production [111]. One paper evaluating FOS in combination with plectasin NZ2114, compound capable to inhibits a cell wall biosynthesis, was reviewed (Table S2). When plectasin NZ2114 evaluated in vitro against E. faecalis, in combination with FOS it no show a synergistic effect [112]. One paper evaluating FOS in combination with 2 quinolone derivatives (A and B) was reviewed (Table S2). When evaluated in vitro against E. faecalis VRE and MRSA, combination FOS + A had always showed a synergistic effect, while FOS + B had showed a synergistic effect in 64% of cases and in other cases shoed an additive effect (36%) [113]. One paper evaluating FOS in combination with N-acetylcysteine (NAC), a mucolytic agent, was reviewed (Table S2). The in vitro analysis against E. coli, had showed a capable of NAC to reduce biofilm if used in combination with FOS. The most effective combination was that obtained using FOS at 2000 mg/L and NAC at 2 mg/mL [114]. One paper evaluating FOS in combination with sophoraflavanone G (SFG), a phytoalexins, was reviewed (Table S2). When evaluated in vitro against MRSA, combination FOS + SFG had showed a synergistic effect (100%) [115]. One paper evaluating FOS in combination with arenaemycin (ARM), also called pentalenolactones, was reviewed (Table S2). When evaluated in vitro against P. vulgaris and S. gallinarum, combination FOS + ARM had showed a synergistic effect (100%) [116]. One paper evaluating FOS in combination with chlorogenic acid (CHA) and caffeic acid (CFA) was reviewed (Table S2). When evaluated in vitro against Resistant Listeria monocytogenes, combination FOS + CHA had showed a reduction in the cell growth equal to 98% and FOS + CFA as to 85,2%. Moreover, CHA restored a FOS susceptibility in 100%, if 3 mg/L [117]. One paper evaluating FOS in combination with silver (AgNPs) and zinc oxide (ZnONPs) nanoparticles, are molecules known to affect bacterial membranes, was reviewed (Table S2). When evaluated in vitro against S. aureus, S. enterica, and E. coli, combination FOS + AgNPs or ZnONPs had showed a synergistic effect (100%) [118].

Discussion
FOS is an inhibitor of bacterial wall synthesis with a unique mechanism of action. Its use in clinic is increasing as is often active against MDR bacteria. Intravenous FOS is often administered in combination with other antibiotics therefore the knowledge of pharmacodynamic interactions is of fundamental importance. In this review, we have investigated the role of FOS as partner drug, by analyzing literature studies in which it has been used in vitro and in vivo in combination with other antibiotics and evaluating the antimicrobial activity of combinations against the most common bacterial pathogens. From this huge data collection, no clinically significant antagonistic effect came out between FOS and any most common used antibiotics for the treatment of nosocomial infections.
Notably, 31.2% of synergistic interactions occurred in Enterobacterales (FOS in combination with 3 different antibiotics), followed by 31% occurred in S. aureus (FOS in combination with 4 different antibiotics) and 7.6% occurred Enterococcus spp. (FOS in combination with 5 different antibiotics).
When FOS is combined with molecules other than antibiotics, chlorogenic acid and caffeic acid appeared to be good partner drugs against L. monocytogenes.
Our tables (including the summarizing Table 15) could act as a useful consultation tool for clinicians using FOS both as empirical or targeted antibiotic regimen.

Conclusions
In conclusion, taken together, these data, the pharmacological characteristics (i.e., excellent distribution in body sites, the safety and tolerability profile) and the encouraging positive clinical outcome of treated patients highlight the role of FOS as partner drug (mostly intravenously) for the treatment of infections caused by common (including MDR) pathogens. In particular, the presence of synergistic interactions and the almost total absence of antagonisms, make FOS a good partner drug in clinical practice. Moreover, improving FOS-based combinations could act as a meropenem-and colistin-sparing agent, mostly contributing to prevent AMR, especially related to last resource antibiotics.

2001, Austria
Grif                                      Transmission Electron Microscopy, demonstrated more morphological alterations when using FOS + LZD, then using FOS or LZD alone. In vivo experiment showed higher survival rates of larvae when using FOS + LZD then LZD alone, but similar rates using FOS alone. [201] 33 20 One study [11] reported high rates of indifferent effect of FOS + 4 different cephalosporins against cephalosporin-R isolates.  Indifferent effect when tetracycline was tested, but one study showed additive or synergistic effect when using minocycline + FOS combination [38]. Indifferent effect when minocycline was tested, but one study showed additive or synergistic effect when using doxicycline + FOS combination [68].  Supplementary Materials: The following are available online at http://www.mdpi.com/2079-6382/9/8/500/s1, Table S1: Studies on combination between fosfomycin and different antibiotics. CB: checkerboard assay; TK: time-kill assay; ET: E-test, Table S2: Studies on combination between fosfomycin and molecules other than antibiotics. CB: checkerboard assay; TK: time-kill assay. Funding: This research received no external funding.