In vivo efficacy of phage cocktails against carbapenem resistance Acinetobacter baumannii in the rat pneumonia model.

Acinetobacter baumannii, an opportunistic pathogen, poses a significant threat in intensive care units, leading to severe nosocomial infections. The rise of multi-drug-resistant strains, particularly carbapenem-resistant A. baumannii, has created formidable challenges for effective treatment. Given the prolonged development cycle and high costs associated with antibiotics, phages have garnered clinical attention as an alternative for combating infections caused by drug-resistant bacteria. However, the utilization of phage therapy encounters notable challenges, including the narrow host spectrum, where each phage targets a limited subset of bacteria, increasing the risk of phage resistance development. Additionally, uncertainties in immune system dynamics during treatment hinder tailoring symptomatic interventions based on patient-specific states. In this study, we isolated two A. baumannii phages from wastewater and conducted a comprehensive assessment of their potential applications. This evaluation included sequencing analysis, genome classification, pH and temperature stability assessments, and in vitro bacterial inhibition assays. Further investigations involved analyzing histological and cytokine alterations in rats undergoing phage cocktail treatment for pneumonia. The therapeutic efficacy of the phages was validated, and transcriptomic studies of rat lung tissue during phage treatment revealed crucial changes in the immune system. The findings from our study underscore the potential of phages for future development as a treatment strategy and offer compelling evidence regarding immune system dynamics throughout the treatment process.IMPORTANCEDue to the growing problem of multi-drug-resistant bacteria, the use of phages is being considered as an alternative to antibiotics, and the genetic safety and application stability of phages determine the potential of phage application. The absence of drug resistance genes and virulence genes in the phage genome can ensure the safety of phage application, and the fact that phage can remain active in a wide range of temperatures and pH is also necessary for application. In addition, the effect evaluation of preclinical studies is especially important for clinical application. By simulating the immune response situation during the treatment process through mammalian models, the changes in animal immunity can be observed, and the effect of phage therapy can be further evaluated. Our study provides compelling evidence that phages hold promise for further development as therapeutic agents for Acinetobacter baumannii infections.

Characterization and therapeutic potential of MRABP9, a novel lytic bacteriophage infecting multidrug-resistant Acinetobacter baumannii clinical strains.

Acinetobacter baumannii is one of the most important pathogens of healthcare-associated infections. The rising prevalence of multidrug-resistant A. baumannii (MRAB) strains and biofilm formation impact the outcome of conventional treatment. Phage-related therapy is a promising strategy to tame troublesome multidrug-resistant bacteria. Here, we isolated and evaluated a highly efficient lytic phage called MRABP9 from hospital sewage. The phage was a novel species within the genus Friunavirus and exhibited lytic activity against 2 other identified MRAB strains. Genomic analysis revealed it was a safe virulent phage and a pectate lyase domain was identified within its tail spike protein. MRABP9 showed potent bactericidal and anti-biofilm activity against MRAB, significantly delaying the time point of bacterial regrowth in vitro. Phage administration could rescue the mice from acute lethal MRAB infection. Considering its features, MRABP9 has the potential as an efficient candidate for prophylactic and therapeutic use against acute infections caused by MRAB strains.

Phage therapy combats pan drug-resistant Acinetobacter baumannii infection safely and efficiently.

Phage therapy offers a promising approach to combat the growing threat of antimicrobial resistance. Yet, key questions remain regarding dosage, administration routes, combination therapy, and the causes of therapeutic failure. In this study, we focused on a novel lytic phage, ФAb4B, which specifically targeted the Acinetobacter baumannii strains with KL160 capsular polysaccharide, including the pan-drug resistant A. baumannii YQ4. ФAb4B exhibited the ability to effectively inhibit biofilm formation and eradicate mature biofilms independently of dosage. Additionally, it demonstrated a wide spectrum of antibiotic-phage synergy and did not show any cytotoxic or haemolytic effects. Continuous phage injections, both intraperitoneally and intravenously over 7 d, showed no acute toxicity in vivo. Importantly, phage therapy significantly improved neutrophil counts, outperforming ciprofloxacin. However, excessive phage injections suppressed neutrophil levels. The combinatorial treatment of phage-ciprofloxacin rescued 91% of the mice, a superior outcome compared to phage alone (67%). The efficacy of the combinatorial treatment was independent of phage dosage. Notably, prophylactic administration of the combinatorial regimen provided no protection, but even when combined with a delayed therapeutic regimen, it saved all the mice. Bacterial resistance to the phage was not a contributing factor to treatment failure. Our preclinical study systematically describes the lytic phage's effectiveness in both in vitro and in vivo settings, filling in crucial details about phage treatment against bacteriemia caused by A. baumannii, which will provide a robust foundation for the future of phage therapy.

Animal model testing for efficacy and adverse effects of bacteriophages against Acinetobacter baumannii.

This study aimed to conduct a detailed study on murine model testing of bacteriophage against Acinetobacter baumannii. These bacteriophages were tested not only for their efficacy in healing wound of murine models infected with multidrug resistant A. baumannii but were also studied for any derangement in hematological parameters as well as liver and kidney function. The study also included any histological changes observed in hepatic and renal tissues of the bacteriophage treated murine animals. This experimental study was conducted at Institute of Basic Medical Sciences, Khyber Medical University, Peshawar and Institute of Pure and Applied Biology, Department of Microbiology and Molecular Genetics, Bahauddin Zakariya University, Multan. A. baumannii isolates were obtained from the Microbiology Department, Armed Forces Institute of Pathology, Rawalpindi. Antimicrobial susceptibility was done by using standard procedures and as per Clinical Laboratory Standards Institute guidelines. Bacteriophages were isolated from sewage water samples collected from different hospitals in Multan. These bacteriophages were characterized and finally used for treating the murine model and efficacy of phage as a therapeutic option was determined by using superficial rat wound model. In this study, the isolated lytic bacteriophage was effective in relatively faster wound healing of the infected animals. Moreover, there were no significant hematological or renal and hepatic profile changes in treated animals. Histology of renal and hepatic tissues was also normal as compared to control animals. Our study concluded that the isolated phage could serve as an attractive therapeutic candidate to combat the deadly multidrug resistant A. baumannii.

Therapeutic evaluation of the Acinetobacter baumannii phage Phab24 for clinical use.

Phages have shown to be effective in treating bacterial infections. However, when evaluating the therapeutic potential of novel phage isolates which have the ability to infect and kill a pathogen, it is important to include parameters such as stability (crucial for storage and delivery), infection dynamics in vitro and in vivo (for efficacy and dosing), and an in-depth genome analysis (to exclude the presence of virulence or lysogeny genes), among others. In this study, we characterized bacteriophage Phab24, which infects a colistin-resistant strain of the notorious nosocomial pathogen Acinetobacter baumannii. Our study is crucial for the use of Phab24 in therapy, while also advancing our understanding of phage predation.

Comparative genomics of Acinetobacter baumannii and therapeutic bacteriophages from a patient undergoing phage therapy.

In 2016, a 68-year-old patient with a disseminated multidrug-resistant Acinetobacter baumannii infection was successfully treated using lytic bacteriophages. Here we report the genomes of the nine phages used for treatment and three strains of A. baumannii isolated prior to and during treatment. The phages used in the initial treatment are related, T4-like myophages. Analysis of 19 A. baumannii isolates collected before and during phage treatment shows that resistance to the T4-like phages appeared two days following the start of treatment. We generate complete genomic sequences for three A. baumannii strains (TP1, TP2 and TP3) collected before and during treatment, supporting a clonal relationship. Furthermore, we use strain TP1 to select for increased resistance to five of the phages in vitro, and identify mutations that are also found in phage-insensitive isolates TP2 and TP3 (which evolved in vivo during phage treatment). These results support that in vitro investigations can produce results that are relevant to the in vivo environment.

Isolation of a lytic bacteriophage against extensively drug-resistant Acinetobacter baumannii infections and its dramatic effect in rat model of burn infection.

OBJECTIVES: Acinetobacter Baumannii is an opportunistic nosocomial pathogen belonging to the Moraxellaceae family. The emergence of multidrug resistant strains of this pathogen caused many problems for hospitals and patients. The aim of the current study was to isolate, identify, and morphologically, physiologically, and in vivo analyze a new lytic bacteriophage targeting extensively drug-resistant (XDR) A. baumannii. MATERIALS AND METHODS: Different wastewater samples were tested for isolation of lytic bacteriophage against 19 A. baumannii isolates obtained from patients hospitalized in a hospital in Arak, Iran, from January 2019 to March 2019. The phenotypic and genotypic characteristics of A. baumannii strains (resistance genes including: adeA, adeB, adeC, adeR, adeS, ISAba1, blaOXA-23, blaOXA-24) were analyzed. The isolated phage characteristics including adsorption time, pH and thermal stability, host range, one-step growth rate, electron microscopy examination, and therapeutic efficacy of the phage were also investigated. Therapeutic efficacy of the phage was evaluated in a rat model with burn infection of XDR A. baumannii. The lesion image was taken on different days after burning and infection induction and was compared with phage untreated lesions. RESULTS: The results showed unique characteristics of the isolated phage (vB-AbauM-Arak1) including high specificity for Acinetobacter baumannii, stability at a relatively wide range of temperatures and pH values, short adsorption time, short latent period, and large burst size. In relation to the therapeutic efficacy of the phage, the lesion area decreased in phage-treated groups over 14 days than in those untreated, significantly (p < 0.05). CONCLUSION: Our findings demonstrated that isolated lytic phage was able to eliminate burn infections caused by XDR A. baumannii in a rat model. So, it may be recommended as alternative options toward to developing a treatment for extensively drug resistant Acinetobacter infections.

Enhanced antibacterial effect of a novel Friunavirus phage vWU2001 in combination with colistin against carbapenem-resistant Acinetobacter baumannii.

The emergence of carbapenem-resistant Acinetobacter baumannii (CRAB) has been increasingly reported, leading to greater challenges in treating infections. With the development of phage therapy and phage-antibiotic combinations, it is promising to improve the treatment of bacterial infections. In the present study, a novel vB_AbaP_WU2001 (vWU2001) phage-specific CRAB with a genome of 40,792 bp was isolated. Genomic analysis disclosed that it belongs to the Autographiviridae family of the order Caudovirales. Phage vWU2001 had a broad host range with a high adsorption rate, short latent period, large burst size and good stability. The phage could reduce preformed biofilms and inhibit biofilm formation. The combination of phage vWU2001 and colistin had significantly higher bacterial growth inhibition activity than that of phage, or colistin alone. The efficacy of the combined treatment was also evaluated in Galleria mellonella. Evaluation of its therapeutic potential showed that the combination of phage and colistin resulted in a significantly greater increase in G. mellonella survival and in bacterial clearance, as compared with that of phage or colistin alone, indicating that the combination was synergistic against CRAB. The results demonstrated that phage vWU2001 has the potential to be developed as an antibacterial agent.

Isolation and Characterization of a Novel Autographiviridae Phage and Its Combined Effect with Tigecycline in Controlling Multidrug-Resistant Acinetobacter baumannii-Associated Skin and Soft Tissue Infections.

Multidrug-resistant Acinetobacter baumannii (MDR A. baumannii) is one of the ESKAPE pathogens that restricts available treatment options. MDR A. baumannii is responsible for a dramatic increase in case numbers of a wide variety of infections, including skin and soft tissue infections (SSTIs), resulting in pyoderma, surgical debridement, and necrotizing fasciitis. To investigate an alternative medical treatment for SSTIs, a broad range lytic Acinetobacter phage, vB _AbP_ABWU2101 (phage vABWU2101), for lysing MDR A. baumannii in associated SSTIs was isolated and the biological aspects of this phage were investigated. Morphological characterization and genomic analysis revealed that phage vABWU2101 was a new species in the Friunavirus, Beijerinckvirinae, family Autographiviridae, and order Caudovirales. Antibiofilm activity of phage vABWU2101 demonstrated good activity against both preformed biofilms and biofilm formation. The combination of phage vABWU2101 and tigecycline showed synergistic antimicrobial activities against planktonic and biofilm cells. Scanning electron microscopy confirmed that the antibacterial efficacy of the combination of phage vABWU2101 and tigecycline was more effective than the phage or antibiotic alone. Hence, our findings could potentially be used to develop a therapeutic option for the treatment of SSTIs caused by MDR A. baumannii.

Bacteriophage therapeutics to confront multidrug-resistant Acinetobacter baumannii - a global health menace.

We have already entered the post-antibiotic era as the outbreaks of numerous multidrug-resistant strains in the community as well as hospital-acquired infections are ringing alarm bells in the health sector. Acinetobacter baumannii is one such pathogen that has been considered a worldwide threat as it acquires multidrug resistance. It is one of the most challenging hospital-acquired pathogens as World Health Organization has listed carbapenem-resistant A. baumannii as a critical priority pathogen with limited therapeutic options. There is an urgent need to develop novel strategies against such pathogens to tackle the global crisis. Bacteriophages (phages), especially the lytic ones have re-emerged as a potential therapeutic approach. This review encompasses vast majority of phages against A. baumannii strains with special references related to single phage or monophage therapy, use of phage cocktails, combination therapy with antibiotics, use of phage-derived enzymes like endolysins and depolymerases to combat the pathogen and explore their therapeutic aspects. The concurrent ecological as well as evolutionary interplay between the phages and host bacteria demands in depth-research and knowledge, so as to utilize the maximum potential of the bacteriophage therapy.

Evaluation of bacteriophage cocktail on septicaemia caused by colistin-resistant Acinetobacter baumannii in immunocompromised mice model.

Background & objectives: Since the bacterium, Acinetobacter baumannii (AB) has acquired resistance to almost all commercially available antibiotics, the search for alternative treatment options continues to be need of the hour. Bacteriophage therapy seems to be the most promising amongst various proposed alternatives (e.g. antimicrobial peptides, bacteriocin, probiotics, etc.). The present study, therefore, aimed to evaluate the effect of different dosages of specific phages in immunocompromised rodents in a septicaemia model caused by AB mimicking real clinical situations. Methods: The three most active and unique phages (ɸAb4, ɸAb7 and ɸAb14) were selected for this study. A constant dose (100 µl of 108 pfu/ml) of AB was given in all the experiments. Five different sets of experiments were designed: prophylactic administration of phage cocktail in the volume of 100 µl (109 pfu/ml) before and simultaneous with the bacterial challenge; and therapeutic i.e. administration of phage cocktail six, 12 and 24 h after bacterial challenge. Since there were deaths in mice when phage was given 24 h after bacterial challenge, the reduced dosage i.e. 100 µl of 107, 10[6], 105 pfu/ml of phage cocktail was also evaluated. Results: The administration of 100 µl (109 pfu/ml) of phage cocktail after six, 12 and 24 h of the bacterial challenge resulted in the mortality ranging between 20 to 60 per cent. However, no mortality could be observed with simultaneous or prophylactic administration of phages with the bacterial challenge. No mortality was observed with reduced doses of the cocktail (10[6] and10[5] pfu/ml). Interpretation & conclusions: As per the results of this study, it may be concluded that even if patients with acute infections report late to the hospital, a relatively low dose of the phage cocktail may be therapeutically beneficial.

Colistin-phage combinations decrease antibiotic resistance in Acinetobacter baumannii via changes in envelope architecture.

Multidrug-resistant bacterial infections are becoming increasingly common, with only few last-resort antibiotics such as colistin available for clinical therapy. An alternative therapeutic strategy gaining momentum is phage therapy, which has the advantage of not being affected by bacterial resistance to antibiotics. However, a major challenge in phage therapy is the rapid emergence of phage-resistant bacteria. In this work, our main aim was to understand the mechanisms of phage-resistance used by the top priority pathogen Acinetobacter baumannii. We isolated the novel phage Phab24, capable of infecting colistin-sensitive and -resistant strains of A. baumannii. After co-incubating Phab24 with its hosts, we obtained phage-resistant mutants which were characterized on both genotypic and phenotypic levels. Using whole genome sequencing, we identified phage-resistant strains that displayed mutations in genes that alter the architecture of the bacterial envelope at two levels: the capsule and the outer membrane. Using an adsorption assay, we confirmed that phage Phab24 uses the bacterial capsule as its primary receptor, with the outer membrane possibly serving as the secondary receptor. Interestingly, the phage-resistant isolates were less virulent compared to the parental strains in a Galleria mellonella infection model. Most importantly, we observed that phage-resistant bacteria that evolved in the absence of antibiotics exhibited an increased sensitivity to colistin, even though the antibiotic resistance mechanism per se remained unaltered. This increase in antibiotic sensitivity is a direct consequence of the phage-resistance mechanism, and could potentially be exploited in the clinical setting.

Hydrogen peroxide-producing electrochemical bandage controlled by a wearable potentiostat for treatment of wound infections.

Chronic wound infections caused by biofilm-forming microorganisms represent a major burden to healthcare systems. Treatment of chronic wound infections using conventional antibiotics is often ineffective due to the presence of bacteria with acquired antibiotic resistance and biofilm-associated antibiotic tolerance. We previously developed an electrochemical scaffold that generates hydrogen peroxide (H2 O2 ) at low concentrations in the vicinity of biofilms. The goal of this study was to transition our electrochemical scaffold into an H2 O2 -generating electrochemical bandage (e-bandage) that can be used in vivo. The developed e-bandage uses a xanthan gum-based hydrogel to maintain electrolytic conductivity between e-bandage electrodes and biofilms. The e-bandage is controlled using a lightweight, battery-powered wearable potentiostat suitable for use in animal experiments. We show that e-bandage treatment reduced colony-forming units of Acinetobacter buamannii biofilms (treatment vs. control) in 12 h (7.32 ± 1.70 vs. 9.73 ± 0.09 log10 [CFU/cm2 ]) and 24 h (4.10 ± 12.64 vs. 9.78 ± 0.08 log10 [CFU/cm2 ]) treatments, with 48 h treatment reducing viable cells below the limit of detection of quantitative and broth cultures. The developed H2 O2 -generating e-bandage was effective against in vitro A. baumannii biofilms and should be further evaluated and developed as a potential alternative to topical antibiotic treatment of wound infections.

Pre-optimized phage therapy on secondary Acinetobacter baumannii infection in four critical COVID-19 patients.

Phage therapy is recognized as a promising alternative to antibiotics in treating pulmonary bacterial infections, however, its use has not been reported for treating secondary bacterial infections during virus pandemics such as coronavirus disease 2019 (COVID-19). We enrolled 4 patients hospitalized with critical COVID-19 and pulmonary carbapenem-resistant Acinetobacter baumannii (CRAB) infections to compassionate phage therapy (at 2 successive doses of 109 plaque-forming unit phages). All patients in our COVID-19-specific intensive care unit (ICU) with CRAB positive in bronchoalveolar lavage fluid or sputum samples were eligible for study inclusion if antibiotic treatment failed to eradicate their CRAB infections. While phage susceptibility testing revealed an identical profile of CRAB strains from these patients, treatment with a pre-optimized 2-phage cocktail was associated with reduced CRAB burdens. Our results suggest the potential of phages on rapid responses to secondary CRAB outbreak in COVID-19 patients.

Bacteriophage-resistant Acinetobacter baumannii are resensitized to antimicrobials.

We characterized two bacteriophages, ΦFG02 and ΦCO01, against clinical isolates of Acinetobacter baumannii and established that the bacterial capsule is the receptor for these phages. Phage-resistant mutants harboured loss-of-function mutations in genes responsible for capsule biosynthesis, resulting in capsule loss and disruption of phage adsorption. The phage-resistant strains were resensitized to human complement, beta-lactam antibiotics and alternative phages and exhibited diminished fitness in vivo. Using a mouse model of A. baumannii infection, we showed that phage therapy was effective.

Preclinical Assessment of Bacteriophage Therapy against Experimental Acinetobacter baumannii Lung Infection.

Respiratory infections caused by multidrug-resistant Acinetobacter baumannii are difficult to treat and associated with high mortality among critically ill hospitalized patients. Bacteriophages (phages) eliminate pathogens with high host specificity and efficacy. However, the lack of appropriate preclinical experimental models hampers the progress of clinical development of phages as therapeutic agents. Therefore, we tested the efficacy of a purified lytic phage, vB_AbaM_Acibel004, against multidrug-resistant A. baumannii clinical isolate RUH 2037 infection in immunocompetent mice and a human lung tissue model. Sham- and A. baumannii-infected mice received a single-dose of phage or buffer via intratracheal aerosolization. Group-specific differences in bacterial burden, immune and clinical responses were compared. Phage-treated mice not only recovered faster from infection-associated hypothermia but also had lower pulmonary bacterial burden, lower lung permeability, and cytokine release. Histopathological examination revealed less inflammation with unaffected inflammatory cellular recruitment. No phage-specific adverse events were noted. Additionally, the bactericidal effect of the purified phage on A. baumannii was confirmed after single-dose treatment in an ex vivo human lung infection model. Taken together, our data suggest that the investigated phage has significant potential to treat multidrug-resistant A. baumannii infections and further support the development of appropriate methods for preclinical evaluation of antibacterial efficacy of phages.

Risk factors and outcomes of bloodstream infections caused by Acinetobacter baumannii: a case-control study.

The risk factors and outcomes of patients with bloodstream infections (BSIs) caused by Acinetobacter baumannii are major concerns in clinical therapy. Multicenter case-control studies were performed to compare the clinical characteristics of 47 A. baumannii BSI patients and 124 matched controls with nonbloodstream A. baumannii infections and the clinical and molecular characteristics of BSI survivors and nonsurvivors. Additionally, the mortality of BSIs was assessed. The clinical characteristics, including neutropenia, ICU admission prior to positive culture, primary infection in the central nervous system, and carbapenem use prior to positive culture, were independently associated with BSI caused by A. baumannii. The mortality of the BSI patients was significantly higher than that of the controls. A high Pitt bacteremia score was found to be an independent predictor of mortality in the BSI patients. The healthcare-associated factors, disease severity level, or antibiotic usage increased the risks of A. baumannii BSI and related mortality.

Effect of nurse-led, goal-directed lung physiotherapy on prognosis of patients with sepsis caused by Acinetobacter baumannii pulmonary infection.

OBJECTIVES: To investigate the role of nurse-led, goal-directed lung physiotherapy on the prognosis of patients with sepsis caused by Acinetobacter baumannii pulmonary infection. METHODS: Patients with sepsis caused by A. baumannii pulmonary infection were recruited and divided into a control group (phase 1) and a treatment group (phase 2). Both groups received standard therapy for sepsis, and patients in phase 2 also received nurse-led, goal-directed lung physiotherapy. The primary outcome measure was 28-day mortality. RESULTS: Among 742 patients with sepsis, 201 were diagnosed with A. baumannii pulmonary infection. Compared with patients in phase 1, patients in phase 2 had a significantly shorter duration of mechanical ventilation {median 4 (interquartile range (IQR) 3-5] vs 5 (IQR 3-12) days; P = 0.004}, lower intensive care unit (ICU) mortality [13.6% (18/132) vs 27.5% (19/69); P = 0.016] and lower 28-day mortality [21.2% (28/132) vs 37.7% (26/69); P = 0.012]. As a protective factor, nurse-led, goal-directed lung physiotherapy (odds ratio 0.341, 95% confidence interval 0.155-0.751; P = 0.008) was an independent risk factor for 28-day mortality. CONCLUSIONS: Nurse-led, goal-directed lung physiotherapy shortened the duration of mechanical ventilation and ICU stay, and decreased ICU mortality and 28-day mortality in patients with sepsis caused by A. baumannii pulmonary infection.

Thermo-responsive chitosan hydrogel for healing of full-thickness wounds infected with XDR bacteria isolated from burn patients: In vitro and in vivo animal model.

Treatment of non-healing skin wounds infected with extensively drug-resistant (XDR) bacteria remains as a big challenge. To date, different biomaterials have been applied for treatment of post-wound infections, nevertheless their efficacy for treatment of the wounds infected with XDR isolates has not been determined yet. In this study, the potential of the thermo-responsive chitosan (TCTS) hydrogel for protection of full-thickness wounds XDR bacteria isolated from burn patients was evaluated both in vitro and in vivo in a rat model. Antibacterial activity of the TCTS hydrogel against standard strain and clinical isolates of Acinetobacter baumannii, cytobiocompatibility for Hu02 fibroblast cells, degradation rate and swelling ratio were determined in vitro. MTT assay and disk diffusion test indicated no detectable cytotoxicity and antibacterial activity in vitro, respectively. In vivo study showed significant acceleration of wound healing, re-epithelialization, wound closure, and decreased colony count in the TCTS hydrogel group compared with control. This study suggests TCTS hydrogel as an excellent wound dressing for management of the wounds infected with XDR bacteria, and now promises to proceed with clinical investigations.

In vitro synergistic activity of the sulbactam/avibactam combination against extensively drug-resistant Acinetobacter baumannii.

Introduction. The therapeutic options to treat Acinetobacter baumannii infections are very limited.Aim. Our aim was to evaluate the activity of sulbactam combined directly with avibactam or the ampicillin-sulbactam/ceftazidime-avibactam combination against extensively drug-resistant A. baumannii isolates.Methodology. Extensively drug-resistant A. baumannii isolates (n=127) collected at several South American hospitals were studied. Synergy with the sulbactam/avibactam combination was assessed in all isolates using the agar dilution method. Avibactam was used at a fixed concentration of 4 mg l-1. A disc diffusion synergy test was also performed. Synergy by a time-kill experiment was performed in a selected isolate.Results. Synergy with sulbactam/avibactam was demonstrated in 124 isolates and it showed MIC values ≤4 mg l-1. This synergy was not detected in the three New Delhi metallo-ß-lactamase-harbouring isolates. Similar results were observed with the disc diffusion synergy test of ampicillin-sulbactam/ceftazidime-avibactam. In the time-kill experiments, sulbactam/avibactam showed a rapid synergistic and bactericidal activity in ampicillin-sulbactam-resistant isolates.Conclusions. This study demonstrated that the sulbactam/avibactam combination displayed synergistic activity against A. baumannii isolates. This synergy was observed when both inhibitors were also used as part of the commercially available combinations: ampicillin-sulbactam and ceftazidime-avibactam.