Combination therapy in severe Acinetobacter baumannii infections: an update on the evidence to date.

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Combination therapy in severe Acinetobacter baumannii infections: an update on the evidence to date

Emanuele Durante-Mangoni1, Riccardo Utili*,1 & Raffaele Zarrilli2

Abstract: Acinetobacter baumannii is a drug-resistant Gram-negative pathogen increasingly causing hospital-acquired infections in critically ill patients. In this review, we summarize the current mechanisms of antimicrobial resistance in A. baumannii and describe in detail recent in vitro and in vivo experimental data on the activity of antimicrobial combinations against this microorganism. We then introduce the rationale for the use of combination antibiotic therapy in resistant A. baumannii infections. Finally, we present and critically discuss both uncontrolled clinical studies and the few randomized clinical trials of combination antimicrobial therapy for these infections, with a special focus on ongoing multinational trials and optimal approach to future research in this field. Acinetobacter baumannii is a glucose nonfermentative Gram-negative coccobacillus that has increasingly been responsible for healthcare-associated infection with elevated morbidity and mortality [1,2] . These infections are difficult to treat as the causative strains often show broad antimicrobial resistance. According to the actual antimicrobial susceptibility profile, A. baumannii strains can be classified as multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) if they are resistant to three or more, all but one or two, and all classes of potentially effective antimicrobial agents, respectively [3] . Antimicrobial agents and categories used to define MDR, XDR and PDR strains according to Margiorakos et al. [3] are shown in Table 1.

Keywords

• Acinetobacter baumannii • antimicrobial resistance • clinical trials • combination

therapy

Antimicrobial resistance in A. baumannii MDR and XDR in A. baumannii is now an emerging issue worldwide. Strains responsible for epidemics are resistant to carbapenems and show intermediate resistance to tigecycline, but usually retain susceptibility to colistin [1–3,35–39] . Although the mechanisms of resistance to fosfomycin in A. baumannii strains have not been elucidated yet, approximately 48% of A. baumannii strains isolated from community and hospital-acquired urinary tract infections in Turkey were resistant to fosfomycin as assessed by disk diffusion (Box 1) [40] . Indeed, genomic and genetic studies show that A. baumannii possesses multiple mechanisms of resistance to all existing antibiotic classes as well as a striking capacity to acquire new determinants of resistance [23,36,41] . A. baumannii strains are able to hydrolyze penicillins (benzylpenicillin, ampicillin, ticarcillin and piperacillin) because they possess an intrinsic class D oxacillinase belonging to the OXA-51like group of enzymes [7] . These microrganism may also break down antipseudomonal penicillins + β-lactamase inhibitors and cephalosporins through an AmpC cephalosporinase [5,6] , as well as third-generation cephalosporins through the expression of TEM, SHV, CTX-M, GES, SCO, PER and VEB families of class A extended-spectrum β-lactamases (Table 1) [8–12] . The resistance Internal Medicine, University of Naples S.U.N. & AORN dei Colli, Monaldi Hospital, Via L. Bianchi, Naples, Italy Department of Public Health, University of Naples Federico II, Naples, Italy *Author for correspondence: Tel.: +1 39 081 7062475; [email protected]

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10.2217/FMB.14.34 © 2014 Future Medicine Ltd

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Review Durante-Mangoni, Utili & Zarrilli Table 1. Mechanisms of antimicrobial resistance in multidrug-resistant, extensively drug-resistant and pandrug-resistant Acinetobacter baumannii. Antimicrobial category

Antimicrobial agent

Major resistance mechanism

Penicillins + β-lactamase inhibitors Antipseudomonal penicillins + β-lactamase inhibitors Extended-spectrum cephalosporins

Ampicillin–sulbactam

blaTEM-1 overexpression

Piperacillin–tazobactam Ticarcillin–clavulanic acid Cefotaxime Ceftriaxone Ceftazidime Cefepime Imipenem Meropenem Doripenem

ADC class C β-lactamase [5,6] overexpression ADC class C β-lactamase [5–12] VEB-1, VEB-2, PER-1, PER-2, TEM-92, TEM-116, SHV-12, CTX-M-2, CTX-M-3 OXA-51-like class D β-lactamase Class B carbapenemase (IMP, VIM, [7,13–19] SIM, NDM) Class A carbapenemase (KPC, GES) Class D carbapenemase (OXA-23, OXA-24/40, OXA-58, OXA-143 clusters) Changes in outer membrane [20–22] proteins (OprD-like OMPs and CarO) Mutations in gyrase subunit [23–25] topoisomerase IV subunit Aminoglycoside-modifying [26,27] enzymes 16S rRNA methyltransferase

Antipseudomonal carbapenems

Antipseudomonal fluoroquinolones Aminoglycosides

Folate pathway inhibitors Tetracyclines

Polymyxins

Rifampicin

Ciprofloxacin Levofloxacin Gentamicin Tobramycin Amikacin Netilmicin Trimethoprim– sulfamethoxazole Tetracycline Doxycycline Minocycline Tigecycline Colistin Polymyxin B

Dihydropteroate synthase Dihydrofolate reductase Efflux pumps overexpression

Mutations in pmrAB two component regulator and in genes of lipid A biosynthesis Mutations in rpoB Rifampicin ADP-ribosylating transferase Arr-2 Efflux pumps overexpression

Ref. [4]

[2,23] [2,28]

[29–33]

[34,35]

ADC: Acinetobacter-derived cephalosporinase; OMP: Outer membrane protein.

to extended-spectrum cephalosporins except cefepime is also contributed to by class C β-lactamase [5] . The resistance to sulbactam, a β-lactam showing intrinsic and clinically relevant antimicrobial activity against Acinetobacter spp., has been positively correlated with the presence and level of expression of blaTEM-1 [4] . The resistance to carbapenems in A. baumannii is more frequently caused by the expression of class B metallo β-lactamases (VIM-, IMP-, SIM- and NDM-types), class A carbapenemases (KPC, GES) and class D β-lactamases [7,13–19,36] . Carbapenem-hydrolyzing class D β-lactamases (CHDLs) major gene clusters identified in A. baumannii include the blaOXA-23-like, blaOXA24/40-like, blaOXA-58-like and blaOXA-143

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genes [7,36] . The chromosomal blaOXA-51-like gene may confer carbapenem resistance when overexpressed [7] . Intermediate susceptibility or resistance to carbapenems in A. baumannii is also contributed by the modification of penicillin-binding proteins and porins or upregulation of the active drug efflux ATP binding cassette (AdeABC) system (Table 1) [20–22] . Mutations in DNA topoisomerase II and/or DNA topoisomerase IV genes are responsible for resistance to fluoroquinolones observed in A. baumannii epidemic strains [23–25] . The resistance to aminoglycosides is due to the presence of several aminoglycosidemodifying enzymes, which inactivate different classes of aminoglycosides and are frequently found in combination in the same strain [26] ,

future science group

Combination therapy in severe A. baumannii infections: an update on the evidence to date or to the 16S rRNA methyltransferase ArmA enzyme, which causes resistance to different aminoglycosides simultaneously [27] . Mutations in dihydropteroate synthase and dihydrofolate reductase genes confer resistance to trimethoprim-sulfomethoxazole [2,41] . Drug removal by active efflux is the mechanism responsible for resistance to tetracyclines in A. baumannii. In particular, narrow-spectrum pumps of the major facilitator superfamily confer resistance to tetracycline (TetA, TetB) and minocycline (TetB) [2] . Also, AdeABC, AdeFGH and AdeIJK pumps of the resistance-nodulation cell division (RND) type confer resistance to tigecycline when overexpressed. RND-type efflux pumps may also contribute to resistance to aminoglycosides, β-lactams, chloramphenicol, erythromycin, trimethoprim, tetracycline and fluoroquinolones [28] . Resistance to polymyxin antibiotics can be caused by mutations in the PmrA/B two-component regulator involved in phosphorylation that results in the addition of phosphoethanolamine to hepta-acylated lipid A, leading to the LPS modifications that directly confer the colistin resistance. [29,30] . Point mutations in the pmrA1 and/or pmrB genes were identified in several colistin-resistant XDR A. baumannii strains isolated in patients treated with colistin [31,32] . Also, resistance to polymixins in A. baumannii has been associated with mutations within either lpxA, lpxC or lpxD lipid A biosynthesis

Review

pathway genes resulting in the complete loss of the lipid A component of lipopolysaccharide [33] . The phenomenon of heteroresistance to colistin in clinical isolates of A. baumannii that were apparently susceptible to colistin on the basis of MICs was also reported [42] . Decreased susceptibility to rifampicin, a drug often used in antimicrobial combination-based strategies against A. baumannii, has also been observed among A. baumannii strains [34,35] . Elevated rifampicin MICs are mostly caused by mutations in the RNA polymerase (RNAP) β-subunit rpoB target gene; a low to intermediate rifampicin MIC is dependent on altered permeability of A. baumannii strains to the drug [35] . Enzymatic modification by rifampin ADP-ribosyltransferase Arr-2 has been identified as additional mechanism of rifampicin resistance in Acinetobacter strains (Table 1) [34] . Antimicrobial combinations against A. baumannii: in vitro & in vivo experimental evidence The increase in antimicrobial resistance in A. baumannii strains, along with the paucity of novel antimicrobials against MDR/XDR Gramnegative pathogens, has led to the establishment of antimicrobial combinations using two or more antibiotics, even when MDR and XDR A. baumannii strains are not susceptible to the single antibiotics. Combination of antimicrobial

Box 1. Endemic extensively drug-resistant Acinetobacter baumannii strains in southern Europe. ●●

Within major hospitals of the city of Naples (Italy), and generally in the Mediterranean basin, major circulating strains of Acinetobacter baumannii show an XDR phenotype. They belong to a limited number of STs, including ST1, ST2, ST25 and ST78 [36]

●●

Carbapenem resistance occurs in over 95% of strains, with a MIC90 ≥32 mg/l, and is mostly due to expression of blaOXA-23 and blaOXA-58 genes [35,37–39]

●●

Most isolates show either susceptibility or low-level resistance to tigecycline (BSAC break point for susceptibility ≤1 mg/l), with a MIC90 of 2–4 mg/l [35,39]

●●

Nearly 100% of strains retain susceptibility to colistin (MIC ≤2 mg/l), with a

consistently low MIC90 (≤0.5 mg/l) either upon diagnosis and during colistin therapy. On-treatment emergence of colistin resistance or colistin MIC creep are rare/exceptional, as well as heteroresistance ●●

Nearly 15% of strains show high-level resistance to rifampicin (MIC ≥32 mg/l) as a

result of a chromosomal mutation within the rpoB gene. Lower-level resistance (MIC 6–24 mg/l) is found in 54% of strains, and is associated with efflux pump production. Approximately 30% of strains are fully susceptible (FSM break point for susceptibility ≤4 mg/l) [35] ●●

Nearly 48% of strains show resistance to fosfomycin [40]

BSAC: British Society for Antimicrobial Chemotherapy; FSM: French Society for Microbiology; MIC90: Minimum Inhibitory Concentration for 90% of strains; ST: Sequence type; XDR: Extensively drug resistant.


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Review Durante-Mangoni, Utili & Zarrilli agents with different mechanisms of action may exercise a greater pharmacodynamic (PD) effect or synergism; however, occasionally a reduced overall effect may be observed, namely antagonism. Results of synergy testing may be significantly affected by the actual method used and the experimental conditions applied. In this section, we will discuss the in vitro and in vivo experimental evidences of antimicrobial combinations against A. baumannii that have served as the basis for combination therapies in XDR A. baumannii infections (Table 2) . A number of in vitro and animal studies have evaluated the activity of carbapenems that are combined with other antimicrobials. In vitro synergy against MDR and carbapenem-resistant A. baumannii has been demonstrated when imipenem is combined with rifampin [45–47] . Also, imipenem combined with rifampin or sulbactam has been evaluated in two different in vivo models of infection: the mouse pneumonia model [43–45] and the rabbit meningitis model [43,44] . The combination of imipenem and rifampin prevented the occurrence of rifampin-resistant A. baumannii strains [43,45] and reduced bacterial cell counts [44,45] in the mouse pneumonia model, but did not increase survival of the animals compared with monotherapies in the mouse pneumonia or rabbit meningitis models of infection [43–45] . Concordantly, no differences were found in survival of animals between combination of imipenem and sulbactam compared with single-agent therapies in the mouse pneumonia and rabbit meningitis models of infection [43,44] . The combination of a carbapenem such as imipenem, meropenem or doripenem and colistin resulted in high synergy rates in vitro [46–49] . Also, the combination of doripenem and colistin was more active than either drug alone against XDR colistin-resistant A. baumannii strains in the Galleria mellonella moth model of infection [49] . Moreover, the combination of sulbactam with colistin and doripenem achieved higher bactericidal activity than colistin–doripenem alone against colistin-resistant A. baumannii isolates collected from patients after colistin–doripenem treatment (Table 2) [50] . Several studies have also assessed the activity of rifampin combined with other antimicrobials. The combination of rifampin with sulbactam resulted in in vitro synergy against MDR A. baumannii [47] and prevented the occurrence of rifampin-resistant A. baumannii strains but did not increase animal survival in the mouse

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pneumonia model [43,44] . Also, rifampicin has shown additive bactericidal activity and synergy when combined with colistin in time-kill studies [46,47,51] and in an in vitro pharmacokinetic (PK)/PD model using a three-combination regimen achieving clinically relevant concentrations of 0.5, 2 or 5 mg/l colistin and 5 mg/l rifampin every 24 h [52] . However, the effectiveness of rifampin and colistin combination in vitro is dependent on the level of rifampin MIC of the isolates, an additive effect and/or synergism being observed in strains with lowintermediate rifampicin MIC but not in strains with elevated rifampin MIC (>256 mg/l) and mutations in the RNAP β-subunit rpoB target gene [35] . Also, combined therapy of rifampin and colistin reduced bacterial cell counts in the mouse pneumonia model [44,45] , but did not increased survival of the animals compared with monotherapies in the mouse pneumonia, rabbit meningitis or rat thigh models of infection (Table 2) [43–45,53] . Mounting evidence demonstrated the activity of glycopeptide-containing regimens against MDR and XDR A. baumannii strains. The increased susceptibility of A. baumannii strains to glycopeptides in the presence of colistin is believed to be mediated via a permeabilizing effect of colistin on the A. baumannii outer membrane, simplifying the entry of glycopeptide molecules, which are usually excluded owing to their size. The combination of either vancomycin or teicoplanin with colistin resulted in in vitro synergy against XDR A. baumannii and inhibited regrowth observed with colistin alone [54,55] . The above antimicrobial combinations were more active than either drug alone in the Galleria mellonella moth model of infection [49,54] . In particular, the combination of vancomycin with colistin was more active than either drug alone against colistin-resistant A. baumannii strains [49] . The activity of the combination of vancomycin and doripenem against colistin-resistant A. baumannii strains was also demonstrated both in vitro and in vivo [49] . The in vitro synergism of the combination of the novel lipoglycopeptide telavancin with colistin against a susceptible A. baumannii strain [56] and the activity of the combination against an MDR carbapenem-resistant A. baumannii strain in the Galleria mellonella moth model of infection was also demonstrated [57] . Also, the combination of the cyclic lipopeptide daptomycin with colistin resulted in in vitro synergy against MDR


Combination therapy in severe A. baumannii infections: an update on the evidence to date

Review

Table 2. Antimicrobial combinations against Acinetobacter baumannii: in vitro and in vivo experimental evidences. Combination

Study method

Results/comments

Imipenem and sulbactam

Mouse pneumonia model

No therapeutic difference compared with monotherapy No therapeutic difference compared to monotherapy Synergism Reduced bacterial counts No therapeutic difference compared with monotherapy Synergism

Imipenem and rifampin

Rabbit meningitis model Time-kill studies Mouse pneumonia model Rabbit meningitis model

Imipenem and colistin

Chequerboard assays Time-kill studies Meropenem and colistin Chequerboard assays Time-kill studies Doripenem and colistin Chequerboard assays Time-kill studies Infection of Galleria mellonella larvae Doripenem, colistin and sulbactam Rifampin and sulbactam Colistin and rifampin

Vancomycin and colistin

Teicoplanin and colistin

Doripenem and vancomycin

Telavancin and colistin

Daptomycin and colistin Tigecycline and colistin

Tigecycline and polymyxin B Gallium nitrate and colistin Sulbactam and fosfomycin Colistin and fosfomycin


Time-kill studies Time-kill studies Mouse pneumonia model Time-kill studies In vitro pharmacokinetic/ pharmacodynamic model Mouse pneumonia model Rabbit meningitis model Chequerboard assays Time-kill studies Infection of G. mellonella larvae Chequerboard assays Time-kill studies Infection of G. mellonella larvae Chequerboard assays Time-kill studies Infection of G. mellonella larvae Chequerboard assays Time-kill studies Infection of G. mellonella larvae Etest agar dilution method Chequerboard assays Time-kill studies Rat pneumonia model

Ref. [43,44]

[45–47] [43,44]

[46–48]

Synergism

[48]

Synergism

[48,49]

More active than either drug alone Active against colistin-resistant strains Greater log-kills than two-drug combinations Synergism Synergism Synergism

Reduced bacterial counts No therapeutic difference compared with monotherapy Synergism

[49] [50] [47] [43,44] [42,46,47,51,52]

[43,44,53]

[54]

More active than either drug alone Active against colistin-resistant strains Synergism

[49,54]

More active than either drug alone Synergism

[49,54]

More active than either drug alone against colistinresistant strains Synergism

[54,55]

[49] [49] [56]

In vitro pharmacodynamic model

More active than either drug alone Synergism Additivity Synergism No significant differences between monotherapy and combined therapy Synergistic or additive effects

[60]

Chequerboard assays

Synergism

[61]

Chequerboard assays

Synergism

[62]

Chequerboard assays

Synergism

[62]


[57] [58] [59] [59]

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Review Durante-Mangoni, Utili & Zarrilli carbapenem-resistant A. baumannii strains as assessed using an Etest agar dilution method [58] . The efficacy of tigecycline/colistin combination against an XDR A. baumannii strain resistant to all antimicrobial agents except tigecycline and colistin was recently demonstrated in time-kill studies in vitro but not in an in vivo rat pneumonia model [59] . However, combination therapy with human simulated exposures of polymyxin B 1 mg/kg every 12 h and tigecycline at the aggressive dose of 200 mg every 12 h produced synergistic or additive effects against four carbapenem-resistant A. baumannii isolates in an in vitro PD model [60] . Furthermore, the combination of colistin and gallium nitrate, an iron mimetic compound that has antimicrobial activity against A. baumannii, showed strong in vitro synergism against MDR and XDR A. baumannii strains, suggesting that a colistin–gallium combination holds promise as a last-resort therapy for infections caused by XDR and PDR A. baumannii [61] . Finally, the synergistic effects of fosfomycin in combination with sulbactam or colistin were demonstrated in vitro in six of eight and one of eight carbapenem-resistant A. baumannii strains, respectively [62] . Based on the above overall in vitro and in vivo evidences, it should be concluded that combination therapy against MDR or XDR A. baumannii is more effective than single-agent therapies alone. However, as far as combinations of antimicrobials are concerned, it should be noted that results of synergy testing may be significantly affected by the actual method used and the experimental conditions applied (Table 2) . Moreover, the efficacy of combination therapies in in vitro and in vivo models of A. baumannii infection does not necessarily translate into improved clinical effectiveness of combination treatment in human disease, as we will discuss below. ●●Rationale for combination therapy in XDR

A. baumannii infections

XDR A. baumannii causes hospital-acquired or healthcare-related infections, especially in intensive care unit (ICU) patients. It usually affects fragile, immunocompromised or ventilator-dependent patients, causing pneumonia and other lower respiratory tract infections, bloodstream infections (BSIs), complicated intra-abdominal or urinary tract infections, and wound infections [1] . The morbidity, costs and mortality of nosocomial infections due to

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XDR A. baumannii are very high. Crude mortality rates up to 60% have been reported in patients with A. baumannii infection [37,63,64] . Notably, mortality rates of patients with XDR A. baumannii infection were significantly higher than those observed in patients with infection due to more susceptible strains [64] . The 30-day mortality attributable to A. baumannii in patients with nosocomial BSIs exceeds 35%, and is significantly lower (∼15%) in matched controls infected by other microorganisms [65] . In a systematic review of matched studies, attributable mortality in the ICU ranged from 10 to 43% [66] . Management of A. baumannii infections may be very difficult for its unique capacity of acquiring several mechanisms of antibiotic resistance, as outlined above. Thus, XDR A. baumannii strains represent a significant threat, particularly in high-dependency units, owing to treatment of these infections being unfeasible with most traditional agents, a scenario that has been described as a return to the preantibiotic era. Colistin is a polypeptide antibiotic that was introduced in clinical use in the 1950s, and abandoned in 1970s when it was replaced by other antibiotics because of concerns about its potential side effects, mainly nephrotoxicity. Owing to its unique mechanism of action, that is, disruption of bacterial outer membrane lipids, colistin activity has not been hampered by the spread of common antimicrobial resistance mechanisms. As a consequence of the increasing prevalence of infections caused by MDR/XDR Gram-negatives that are still susceptible to colistin, there has been a revival of this ‘old’ molecule as the only clinically useful agent under these circumstances [67] . However, colistin use is weakened by several flaws, not the least the essential absence of placebo- or active comparator-controlled studies of treatment in XDR microorganism infections (Box 2) . Two forms of colistin are commercially available: colistin sulfate, used in general topically, and colistimethate sodium (sodium colistin methanesulfonate). The latter, a prodrug of colistin, is better suited for parenteral use since it is less toxic than colistin sulfate. PK and PD data of colistimethate sodium have been recently re-evaluated and its toxicity has been revisited [69] . Indeed, recent clinical reports have demonstrated that earlier concerns about colistin safety were overstated due to inappropriate patient



Review

Box 2. Major flaws of colistin for extensively drug-resistant Acinetobacter baumannii infections. ●●

Today, colistin use is essentially based on the antimicrobial susceptibility patterns of

major MDR/XDR Gram-negative pathogens, where it increasingly remains the only in vitro active agent. It has not shown efficacy in randomized controlled trials against other molecules [68] ●●

There are major uncertainties regarding optimal daily dosage and timing of

administration of colistin, especially in critically ill patients. The correct dose adjustments for the different types of renal replacement therapies are still to be established. Results of different studies are not immediately comparable, and common confusion between colistin base, colistin sulfate, colistimethate sodium or even polymixin B or E adds complexity to the system [69,70] ●●

As XDR A. baumannii commonly affects the lung, suboptimal therapeutic levels of colistin in the epithelial lining fluid may be an issue due to its poor lung diffusion. Administration of colistin via nebulization has been proposed, but results are uncertain [71–73]

●●

In the presence of suboptimal dosing or reduced bioavailability at the infection site, emergence of colistin heteroresistance may become an issue [42]

●●

Colistin use and adequate dosing may be hampered by the fear of nephrotoxicity

or neurotoxicity; however, few studies have assessed these potential adverse events prospectively [74,75] ●●

Clinical use of colistin may induce cross-resistance to host antimicrobial peptides.

In all studies, mortality attributable to A. baumannii infections remains high despite colistin treatment [33,66] MDR: Multidrug-resistant; XDR: Extensively drug resistant.

selection and inadequate monitoring [67,76] . These authors have demonstrated a satisfactory safety profile of colistin when administered intravenously (iv.) at the recommended doses according to renal clearance. Colistimethate sodium, referred from now on as colistin, is available in the EU as vials labeled in international units, containing 1 million IU per vial; since there are 12,500 units per mg, each vial contains 80 mg of colistimethate sodium. The recommended therapeutic dose for this product, in severe infections of an average-weight adult patient showing normal renal function, is 2 million units three-times daily, corresponding to an average daily dose of 6.4 mg/kg body weight [69] . Clinical efficacy and toxicity of colistin, however, have been investigated only in nonrandomized, small sample studies and prospective large sample studies are lacking [76–79] . Furthermore, the clinical efficacy of colistin may be hampered by poor diffusion into lung epithelial lining fluid and emergence of both colistin resistance and colistin heteroresistance in MDR A. baumannii strains following exposure to suboptimal concentrations [29–33,42,67,69] . This latter occurrence, defined as ‘heteroresistance to


colistin’, has been described in clinical isolates of A. baumannii that were apparently susceptible to colistin on the basis of MIC [42,69] . However, the actual significance of this phenomenon is unknown and there are no data showing its possible influence on the therapeutic efficacy of colistin monotherapy. For all these reasons, combination antibiotic therapy has traditionally been a strategy empirically employed in the treatment of MDR A. baumannii. ●●Uncontrolled clinical studies of

combination antimicrobial therapy in XDR A. baumannii infections

Based on in vitro and experimental animal studies (detailed above), the combination of colistin with rifampicin has been widely clinically employed [80,81] . It should be noted that in routine clinical and microbiological practice, rifampicin is not employed or even tested for its activity towards Gram-negative organisms. However, as already outlined, in vitro and in vivo studies have consistently shown synergistic activity with both β-lactams and colistin against


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Review Durante-Mangoni, Utili & Zarrilli MDR Gram-negative bacteria. Since its use as a monotherapy is generally hampered by the rapid development of resistance [82] , rifampicin is much more suitable for use as a partner drug for other antimicrobial molecules. The first report of the clinical use of rifampicin in combination with colistin against XDR A. baumannii came from Petrosillo et al., who reported on their experience with a consecutive cohort of 14 patients with ventilator-associated pneumonia (VAP) [83] . iv. colistin sulfomethate sodium, at the dose of 2 MU three-times daily or less for reduced creatinine clearance, was administered in combination with iv. rifampicin, 600 mg once daily, for a median of 12 days. Overall, seven of 14 patients (50%) died, and the A. baumannii attributable mortality was 36%, with a 64% rate of eradication. Of note, five of ten patients with sulbactam-susceptible carbapenem-resistant A. baumannii also received iv. ampicillin–sulbactam. In a single-center study in critically ill Moroccan patients with a mean Acute Physiology And Chronic Health Evaluation (APACHE) II score of 6.3 [84] , iv. rifampicin, given at the dose of 10 mg/kg every 12 h, was associated with aerosolized colistin, 1 MU three-times daily, in 16 VAP cases and with iv. colistin, 2 MU three-times daily, in nine BSIs. In this study, the reported clinical evolution was ‘favorable’ for all patients, although the actual study end point was not specified. No renal adverse events or significant liver toxicity was reported. Bassetti et al. treated 19 VAP and ten BSI cases with 2 MU tid of colistin combined with rifampicin (10 mg/kg every 12 h) for an average of 18 days [85] . Most of these patients, including all VAP cases, were on mechanical ventilation and the mean APACHE II score was 17. All strains were ‘susceptible’ to rifampicin. In this study, favourable microbiological and clinical responses were observed in 76% of cases. The overall 30 day in-hospital mortality was 31% (9/29 cases), whilst the infection-related mortality was 21%. No resistance to rifampicin and colistin developed. The rate of nephrotoxicity was low (10%) and no neurotoxicity was noted. Another retrospective study reported on the efficacy and safety of colistin and rifampicin in 10 patients with VAP caused by carbapenemresistant A. baumannii susceptible only to colistin and resistant to rifampicin. Combining colistin methanesulfonate at the dose of 4,500,000 IU every 12 h with rifampicin at 600 mg daily,

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there was a 20% rate of therapeutic failure without significant renal or hepatic toxicity [86] . Interestingly, a higher level of rifampicin resistance (MIC >64 mg/l) was associated with a reduced rate of microbiological eradication. Rifampicin may exert a mild and transient liver dysfunction, usually of limited clinical significance. Incidence of this complication appeared to be low in all mentioned studies. In summary, uncontrolled reports on limited series of patients have shown a high rate of clinical response to colistin and rifampicin combination therapy in severe infections caused by carbapenem-resistant A. baumannii [83–85] . Whether this combination yields better results over colistin monotherapy could not be determined from these studies. The clinical results of combination therapy with colistin plus meropenem compared with colistin alone are reported in a very small number of patients in a single retrospective study [87] . In this study, 37 patients with A. baumannii nosocomial infections were included, together with another 34 patients with infection due to other resistant Gram-negative organisms. Of the 37 former patients, five received colistin alone and 32 received the combination of colistin and meropenen. The rates of clinical response and in-hospital death in these two subgroups were not reported separately, but the overall clinical response rates in the 71 patients included were not significantly different. Based on these data, survival actually worsened with combination therapy. Overall, the studies shown above were stimulated by the results of antibiotic synergy tests. It should however be considered that a direct application of data emerging from in vitro studies in clinical practice may be misleading. Not rarely, antimicrobial combination strategies showing clear synergy in vitro appeared to only marginally improve clinically relevant outcomes when compared with monotherapy in human clinical studies [88,89] . In several settings of bacterial infection, a combination of antimicrobials appeared to improve only surrogate measures of treatment efficacy [89,90] . In human infections, bacterial concentrations may be very high, antimicrobials may poorly diffuse within the infected site reaching suboptimal concentrations, and drug doses actually administered may be inadequate due to toxicity, renal or liver dysfunction. Moreover, interactions in terms of metabolism and clearance may add variability


Combination therapy in severe A. baumannii infections: an update on the evidence to date to the system. It should also be considered that administration of two or three antimicrobials may have more profound ecologic consequences, alter the intestinal microbiota to a larger extent, favor the emergence of further MDR organisms or fungi, and increase drug-related adverse events and healthcare costs. Thus, to optimize the use of already available drugs in combination schedules against MDR organisms, any effort should be done to pursue adequately powered, well-designed, randomized clinical trials. Recently, several research groups and consortia started to adopt this approach, and a few randomized controlled trials (RCT) have actually been completed and published, as we will detail in the next section of this review. ●●Randomized clinical trials of combination

antimicrobial therapy in XDR A. baumannii infections

Based on the results of the uncontrolled studies, colistin has become the treatment of choice for carbapenem resistant A. baumannii, with tigecycline as an alternative in intra-abdominal infections. Moreover, it was recommended that the addition of rifampicin for susceptible strains be considered on a case-by-case basis [91] . However, it also became clear that literature data on combination regimens including rifampicin were limited. In particular, the actual net clinical benefit of rifampicin addition in the therapeutic approach to XDR A. baumannii was unclear, especially in terms of hard clinical end points, such as survival [92] . A strong call for action was released, acknowledging the urgent need for randomized, controlled studies of treatment of XDR Gram-negative infections, with a specific focus on combinations including colistin versus colistin alone [93] . This call was caught by several research groups and consortia, and randomized controlled studies were started. Some of them have been concluded and published, and their results are summarized as follows. The first randomized comparative trial of A. baumannii infection treatment was published by Aydemir et al. [94] . This was an open-label, randomized, single-center study conducted at Bulent Ecevit University Teaching and Research Hospital in Turkey. This study aimed at comparing the efficacy of colistin with that of the combination of colistin and rifampicin in the treatment of patients with VAP due to carbapenem-resistant A. baumannii. Colistin was given iv. at a dose of 3 MU every 8 h together


Review

with rifampin 600 mg daily orally via nasogastric tube. The primary outcome was clinical response, as defined by resolution of fever or hypothermia, disappearance of tracheal secretions or airway purulent discharge, amelioration of blood gas exchanges or chest objective signs. The secondary outcomes included microbiological, laboratory and radiological responses. In particular, microbiological response was considered to be achieved if on-treatment cultures of bronchial secretions and blood became negative for A. baumannii. Although in the a priori design 88 patients were expected to be enrolled, the study only included 43 patients, thus being highly underpowered. Moreover, patients assigned to colistin monotherapy had a significantly lower baseline Sequential Organ Failure Assessment (SOFA) score than those receiving combination therapy. Despite of this imbalance, in-hospital and VAP-related mortality were lower, although not significantly, in the combination arm. Consistently, clinical as well as microbiological, laboratory and radiological responses were all better in the combination therapy arm. None of the differences, however, were statistically significant. The only outcome measure showing significant differences was the time to microbiological clearance, that appeared to be shorter in the colistin–rifampicin group (3.1 vs 4.5 days in the colistin monotherapy; p = 0.029). In the associated microbiology studies, synergy between colistin and rifampicin was demonstrated for all isolates, despite elevated rifampicin MICs (256 to ≥512 μg/ml) in 11 and intermediate MICs (8–16 μg/ml) in three isolates. The remaining 29 isolates were fully susceptible to rifampicin (MIC ≤4 μg/ml). In this study, no hepatotoxicity was observed. In contrast, ten patients (23%) developed nephrotoxicity during colistin treatment despite none having prior kidney disease. Colistin dose adjustment allowed regression of renal damage and no patient required discontinuation of treatment or renal replacement therapy. These results were essentially confirmed in a much larger study performed by our own group [75] . This was an Italian multicenter, randomized, open-label clinical trial comparing colistin and rifampicin with colistin alone for the treatment of serious infections due to XDR A. baumannii. A total of 210 patients with lifethreatening infections due to XDR A. baumannii from ICUs of five Italian tertiary care hospitals were enrolled. Allocation (with a 1:1 ratio)


781

Review Durante-Mangoni, Utili & Zarrilli to either colistin alone or colistin plus rifampicin iv. was based on severity of underlying illness and was stratified by clinical site. Colistin was given at the dose of 2 MU (or 160 mg of colistimethate sodium) every 8 h and rifampicin at 600 mg every 12 h iv. Patients enrolled included those with ventilator-associated or hospital-acquired pneumonia (VAP/HAP), BSI and complicated intrabdominal infections. The study was designed to identify an absolute mortality reduction of 20% with combination therapy. Indeed, the hard end point of 30-day all-cause mortality was set as the primary study outcome. All patients were followed-up for at least 30 days after randomization, and whenever this occurred later up to the end of the hospitalization. The study secondary end points were infection-related death, microbiological eradication, hospitalization length and on treatment emergence of resistance to colistin. Death within 30 days from randomization occurred in 43.3% of patients in the combination arm and in 42.9% in the monotherapy arm. After taking into account all possible interfering clinical variables, the difference was not statistically significant (adjusted odds ratio: 0.88; 95% CI: 0.46–1.69; p = 0.71). Similarly, no difference was observed for infection-related death and length of hospitalization. However, a significant increase of microbiological eradication rate was observed in the colistin plus rifampicin arm (60.6 vs 44.8%; p = 0.034). Neither emergence of colistin resistance nor colistin MIC increase were observed. Factors significantly associated with 30-day mortality in the multivariable analysis were the type of initial admission (medical/surgical vs emergency/trauma), the severity of illness measured with the Simplified Acute Physiology Score (SAPS) II score and the number of comorbidities assessed by means of the Charlson index. Adverse events were observed in 34.6% of patients, without differences between the experimental and control arm. Renal impairment according to the RIFLE criteria occurred in 53 patients (26.2%) and led to colistin dose reduction or discontinuation in 17% of patients and to renal replacement therapy in two (1%). Liver dysfunction associated with hyperbilirubinemia was observed in 16% of patients and was more frequent, although not significantly, in the rifampicin arm. The results of this large RCT suggest that in serious XDR A. baumannii infections, 30-day mortality is not reduced by addition

782


of rifampicin to colistin. Moreover, combination treatment does not reduce infection-related death and does not shorten the length of hospitalization or ICU stay. Interestingly, however, and in line with the findings of Aydemir et al., A. baumannii eradication from the primary source of infection is more often observed with combination treatment. This is also consistent with all previous experimental findings [43,44,53] . These findings have been interpreted conservatively. It can be hypothesized that the beneficial effect of rifampicin addition to colistin may not become evident due to the inherently high severity of underlying illnesses of index patients. Indeed, 30-day mortality was related to the SAPS II score and the comorbid conditions, that is, the overall patient clinical status. These results indicate that, currently, rifampicin combined with colistin should not be used in routine clinical practice. However, an increased rate of A. baumannii eradication with combination treatment could still infer a benefit with regard to infection relapse in individual subjects and prevention of further pathogen spread. The major weakness of this trial was represented by the use of a low dose of colistin (2 million units every 8 h), which could not translate into adequate tissue concentrations and leave rifampicin functionally as a monotherapy. Thus, it would be interesting to assess whether the currently used schedule (including a loading dose), based on novel PK data [70] , would disclose a superiority of rifampicin combination over colistin monotherapy. On the other hand, by possibly improving colistin efficacy, a higher colistin dose could have driven the difference even more towards the absence of superiority of combination, although as of yet, no study has shown clinical superiority of a higher colistin dose. Growing interest concerns the possible activity of fosfomycin, another old and long underused antibiotic, against XDR A. baumannii [62] . Also, the possibility of a synergistic effect of fosfomycin in combination with colistin against carbapenem-resistant A. baumannii strains has been recently suggested [95] . Based on this still uncertain rationale, Sirijatuphat and coworkers have recently conducted a prospective, randomized, open-label clinical trial to compare efficacy and safety of colistin plus fosfomycin compared with colistin alone for the treatment of infections caused by carbapenem-resistant A. baumannii. Ninety-four patients were


Combination therapy in severe A. baumannii infections: an update on the evidence to date randomized at a University Hospital in Thailand to receive colistin (5 mg/kg/day) or colistin plus fosfomycin (4 g every 12 h) for 7–14 days. The study outcomes were clinical response, microbiological response, 28-day mortality and adverse events. Patients were well balanced in terms of baseline characteristics, most of them had nosocomial pneumonia (76.6%). A favorable clinical response at both day 3 and treatment completion was observed more often in the combination arm (72.3 and 59.6% vs 66.0 and 55.3%, respectively). All-cause and infection-related 28-day mortality were both lower in the fosfomycin group. However, none of these differences was statistically significant. Similar to what has been observed in studies of colistin–rifampicin combination, a favorable microbiological response was observed significantly more often with fosfomycin and colistin at both day 3 and treatment end (90.7 and 100% vs 58.1 and 82.1%, respectively; p < 0.01). Thus, once again, the use of two antibiotics against XDR A. baumannii was more successful in achieving microbiological eradication but could not significantly improve the clinical outcomes. Of note, no significant increase in renal toxicity was observed with the concomitant use of fosfomycin. Ongoing studies A handful of studies are currently underway in the field of nosocomial infections due to XDR Gram-negative bacteria including A. baumannii. Some of them are approaching the issue with the optimal design of a randomized controlled study. One European and one US trial are currently assessing the efficacy of a colistin–carbapenem combination against XDR A. baumannii. Both studies enroll patients with infections due to other resistant Gram-negatives, including carbapenem-resistant Pseudomonas aeruginosa and KPC-producing Klebsiella pneumoniae. The EU-funded trial (ClinicalTrials.gov identifier: NCT01732250 [96]), nested within the Seventh Framework Program ‘AIDA’ project, is currently enrolling patients with all types of infections due to XDR A. baumannii, P. aeruginosa and K. pneumoniae. This Phase IV, multicenter, multinational, randomized and open-label study has the objective to determine whether the addition of meropenem to colistin is superior than colistin alone in the treatment of clinically significant infections caused by these MDR bacteria. Adult inpatients with clinically


Review

significant BSIs, hospital-acquired pneumonia, VAP and urinary tract infections due to carbapenem-resistant and colistin-susceptible Gramnegative bacteria are eligible. The experimental arm is receiving iv. meropenem, 2 g every 8 h, adjusted for renal function plus iv. colistin with a loading dose of 9 MIU units, followed by a maintenance dose of 4.5 MIU every 12 h or adjusted for renal function. The active comparator arm is receiving colistin alone. Treatment duration is up to 10 days. The primary study outcome is clinical success defined as a composite of all of the following, all measured at 14 days: patient alive, systolic blood pressure >90 mmHg without need for vasopressor support, stable or improved SOFA score, stable or improved PaO2 :FiO2 ratio, clearance of blood cultures (for details see ClinicalTrials.gov [96]). This study also has a number of secondary outcome measures, including 14- and 28-day all-cause mortality, clinical success with modification of therapeutic regimen, time to defervescence or weaning from mechanical ventilation or hospital discharge, microbiological failure and superinfections. The overall number of patients to be enrolled in this trial is 360, but the rate of XDR A. baumannii is currently unknown, and the estimated study completion date is mid 2016. The US trial is a NIH-sponsored project (ClinicalTrials.gov identifier: NCT01597973 [96]) that is conducting a Phase IV, multicenter, randomized, placebo-controlled trial of treatment of XDR Gram-negative bacilli. More than 400 inpatients with BSI or pneumonia due to XDR Gram-negative organisms, including A. baumannii, are expected to be included. The primary study objective is to determine whether the treatment regimen of colistin combined with imipenem–cilistatin is related to a reduced risk of 28-day mortality compared with colistin alone. The secondary objective is to determine which treatment regimen (colistin monotherapy or colistin combined with imipenem) is more likely to reduce colistin resistance emergence during therapy. This study is estimated to be completed by mid 2016. In summary, by the end of 2017, we will likely have enough evidence to decide whether a carbapenem should be added to colistin for the treatment of XDR A. baumannii. Interestingly, it is expected that the results of these two large ongoing trials will be pooled to increase overall statistical power.


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Review Durante-Mangoni, Utili & Zarrilli Two other studies have been or are going to be completed soon. The first is a prospective observational study of patients with drugresistant A. baumannii bacteremia (ANTI-AB; ClinicalTrials.gov Identifier: NCT01511224 [96]) sponsored by the National Taiwan University Hospital (Taiwan). In this nonrandomized study, treatment is set at the discretion of the attending clinical team and data are subsequently prospectively collected, including the interval from study enrollment to negative blood A. baumannii PCR and blood sterilization as the study primary end point and survival at 14 and 30 days after enrollment, adverse drug reactions and risk factors for 30-day mortality as secondary end points. Treatment regimens evaluated and compared are variable and include colistin monotherapy and colistin combinations with tigecycline, carbapenems, rifampin, high-dose sulbactam or glycopeptides. The results with colistin-sparing regimens and colistin loading doses will also be provided. Two hundred patients should be included. The second study is a randomized trial of colistin plus rifampicin versus colistin alone in MDR P. aeruginosa and A. baumannii infections. Enrollment of approximately 130 patients is projected. Doses are 2.5–5 mg of colistin base activity/kg/day and 10 mg/kg/day of rifampin. The primary outcome measure is the number of subjects with cure or improvement at day 28 and the secondary outcome is the number of subjects with microbiological eradication. Although not being powered or optimally designed as the two multicenter trials previously described, these latter important clinical studied will almost certainly provide additional evidence to guide the care of patients with infections due to XDR A. baumanni. Their results are therefore eagerly awaited. Conclusion Several areas of uncertainty remain on the optimal treatment of severe infections due to XDR A. baumannii. In our own view, the following major clinical questions should be addressed in the near future: first, the optimal dose, duration and route of administration of colistin for XDR A. baumannii infection should be established in clinical studies and not rely solely on PK/PD modeling; second, efficacy and safety of each combination treatment regimen including colistin should be tested against colistin monotherapy; study outcomes have to be clinically relevant, and should include all-cause mortality and

784


A. baumannii-attributable mortality, as well as microbiological eradication and hospitalization length. XDR pathogen infections represent a major area of clinical investigation where the need for high-quality data, coming out from carefully conducted, sufficiently sized, prospective clinical trials, is vital. Future perspective Treatment of XDR A. baumannii will likely remain an active area of investigation for the foreseeable future. As the pharmacological development of novel antibiotics active against XDR organisms progresses slowly, the master route remains to study available drugs in various combinations based on a wise interpretation of in vitro studies. This should allow us to optimize the use the existing armamentarium. Future options that seem logical to evaluate comprise the new carbapenem doripenem-based combinations, tigecycline-containing regimens, novel colistin-based unorthodox combinations and sitafloxacin [97] . No clinical data have been formally published yet regarding these combinations, except for a single report on colistin–glycopeptide combination [98] . In this retrospective analysis of pooled cases from different centers, use of colistin in combination with a glycopeptide for at least 5 days turned out to be associated with a lower mortality without added nephrotoxicity. The value of colistin as a partner drug for other antimicrobials certainly deserves attention. Colistin may disrupt Gram-negative outer membrane integrity, causing on the one hand bacterial cell death via osmotic rupture of the inner membrane and on the other hand permeabilizing the organism to hydrophobic molecules that are usually unable to penetrate. This activity may result in the acquisition of antimicrobial activity of several molecules that could be the base for the evaluation of clinical effectiveness of unorthodox antimicrobial combinations. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.



Review

Executive summary Antimicrobial resistance in Acinetobacter baumannii ●●

Generation of carbapenem-hydrolizing β-lactamases is the most frequent mechanism of carbapenem resistance in A. baumannii.

●●

A. baumannii can develop resistance to polymyxin antibiotics through total loss of the lipid component of lipopolysaccharide or its phosphorylation.

●●

Decreased susceptibility to rifampicin results from mutations in the rpoB target gene, altered permeability or drug modification by rifampin ADP-ribosyltransferase.

Antimicrobial combinations against A. baumannii: in vitro & in vivo experimental evidences ●●

In vitro synergism against A. baumannii has been demonstrated for imipenem and rifampin.

●●

The combination of a carbapenem with colistin against colistin-resistant A. baumannii strains was synergistic in vitro and in vivo.

●●

Rifampicin showed additive bactericidal activity and synergy when combined with colistin in vitro. An additive effect and/or synergism was observed in strains with low-intermediate rifampicin MIC but not in strains with elevated rifampin MIC (>256 mg/l) and mutations in rpoB. Combined therapy of rifampin and colistin reduced bacterial cell counts in the mouse pneumonia model, without increasing survival of the animals compared with monotherapies.

●●

The efficacy of tigecycline/colistin combination in a pneumonia model caused by extensively drug-resistant (XDR) A. baumannii resistant to all antimicrobial agents except tigecycline and colistin was recently reported.

●●

The combination of fosfomycin and sulbactam was synergic in vitro against carbapenem-resistant A. baumannii strains.

Rationale for combination therapy in XDR A. baumannii infections ●●

The 30-day mortality attributable to A. baumannii in patients with nosocomial bloodstream infections is 25–35%. Management is difficult due to antibiotic resistance.

●●

Colistin is usually active but often deemed little effective, thus combination antibiotic therapy has traditionally been a strategy empirically employed in the treatment of MDR A. baumannii.

Uncontrolled clinical studies of combination antimicrobial therapy in XDR A. baumannii infections ●●

In limited series of patients, the combination of colistin and rifampicin showed high rates of clinical response in infections caused by carbapenem-resistant A. baumannii.

●●

The results of colistin plus meropenem combination compared with colistin alone are reported in a very small number of patients, where survival worsened with combination therapy.

Randomized clinical trials of combination antimicrobial therapy in XDR A. baumannii infections ●●

In an open-label, randomized, single-center study of treatment of ventilator associated pneumonia, the time to microbiological clearance was shorter in the colistin–rifampicin combination arm.

●●

In a multicenter, randomized, open-label clinical trial comparing colistin and rifampicin with colistin alone for

serious infections due to XDR A. baumannii, 30-day mortality was equal, but a significant increase of microbiological eradication rate was observed in the colistin plus rifampicin arm. ●●

A prospective, randomized, open-label clinical trial comparing efficacy and safety of colistin plus fosfomycin with colistin alone observed a favorable clinical and microbiological response more often in the combination arm.

Ongoing studies & future perspective ●●

Two randomized controlled studies are currently assessing the efficacy of colistin–carbapenem combination against XDR A. baumannii.

●●

Treatment of XDR A. baumannii will likely remain an active area of investigation for the foreseeable future.

The master route remains to study available drugs in various combinations to optimize the use of the existing armamentarium.



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Review Durante-Mangoni, Utili & Zarrilli 12 Nagano N, Nagano Y, Cordevant C, Shibata

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Important investigation demonstrating that the iron mimetic compound gallium nitrate is active against multidrug-resistant Acinetobacter baumannii strains in vitro and in vivo.

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Therapeutic efficacy of lysophosphatidylcholine in severe infections caused by Acinetobacter baumannii.

In Vivo and In Vitro Efficacy of Minocycline-Based Combination Therapy for Minocycline-Resistant Acinetobacter baumannii.

formoterol fixed-dose combination therapy for COPD: the evidence to date.

The Immune Response against Acinetobacter baumannii, an Emerging Pathogen in Nosocomial Infections.

An update on the arsenal for multidrug-resistant Acinetobacter infections: polymyxin antibiotics.

Effects of silver nanoparticles in combination with antibiotics on the resistant bacteria Acinetobacter baumannii.

Combination therapy with polymyxin B and netropsin against clinical isolates of multidrug-resistant Acinetobacter baumannii.

Nosocomial Acinetobacter baumannii Infections and Changing Antibiotic Resistance.

Clinical efficacy and safety of the combination of colistin plus vancomycin for the treatment of severe infections caused by carbapenem-resistant Acinetobacter baumannii.

Update on the evidence regarding maintenance therapy.

Spotlight on valsartan-sacubitril fixed-dose combination for heart failure: the evidence to date.

Monotherapy versus combination therapy for sepsis due to multidrug-resistant Acinetobacter baumannii: analysis of a multicentre prospective cohort.

Monotherapy versus combination therapy for sepsis due to multidrug-resistant Acinetobacter baumannii: analysis of a multicentre prospective cohort--authors' response.

Nintedanib in NSCLC: evidence to date and place in therapy.

Rifampicin as adjunct to colistin therapy in the treatment of multidrug-resistant Acinetobacter baumannii.

Colistin and Fusidic Acid, a Novel Potent Synergistic Combination for Treatment of Multidrug-Resistant Acinetobacter baumannii Infections.

Update on the Epidemiology, Treatment, and Outcomes of Carbapenem-resistant Acinetobacter infections.

Nosocomial Infections Caused by Acinetobacter baumannii: Are We Losing the Battle?

The Response of Acinetobacter baumannii to Zinc Starvation.

Community-acquired Acinetobacter baumannii pneumonia.

Genetic tools for manipulating Acinetobacter baumannii genome: an overview.

The Acinetobacter baumannii group: a systemic review.

Sensitivity of levofloxacin in combination with ampicillin-sulbactam and tigecycline against multidrug-resistant Acinetobacter baumannii.

Polymyxin B in combination with doripenem against heteroresistant Acinetobacter baumannii: pharmacodynamics of new dosing strategies.