In vitro comparison of colistin-versus tigecycline-based combinations against carbapenem resistant Acinetobacter baumannii intensive care unit clinical isolates

Background: Acinetobacter baumannii (A. baumannii) has emerged as a nosocomial pathogen especially in the intensive care units (ICUs). It’s enlisted at the top of urgent threat level organisms in centers for disease control and prevention (CDC’s) antibiotic resistance threats report. Objectives: To assess prevalence, risk factors of health care associated infection by A. baumannii , and to compare the in-vitro efficacy of colistin sulfate-tigecycline combinations versus their individual combination with levofloxacin and meropenem against carbapenem resistant A. baumannii clinical isolates from an Egyptian tertiary care hospital ICUs. Methods: The study included 250 ICU patients, samples were collected according to the site of infection. Acinetobacter baumannii was isolated, identified and tested for antibiotic susceptibility by disc diffusion. Broth microdilution method was used for assessment of colistin, tigecycline, levofloxacin, and meropenem. Thirty isolates resistant to all carbapenems were tested by the checkerboard method to assess effect of antibiotic combinations. Results: forty-six A. baumannii were isolated, with highest prevalence in respiratory secretions. Prior antibiotic administration and failure of empirical antibiotic therapy were found to be a major risk factors of infections by A. baumannii . Colistin combination with meropenem showed the highest synergy (50%). Tigecycline-meropenem combination had the highest antagonistic effect (66.7%). Conclusion: No antagonistic effect of colistin combination with meropenem was confirmed in this study. Only colistin-based combinations, particularly those with meropenem may confer therapeutic benefits against carbapenem-resistant A. baumannii .


Multidrug-resistant
Acinetobacter baumannii (A.baumannii) is a considerable pathogen causing health care associated infection (HAIs), especially in critically ill patients admitted to intensive care units (ICUs) [1].Acinetobacter baumannii is characterized by its great intrinsic and acquired resistance to many antibiotics leading to the frightening reality of its treatment failure [2].
antimicrobial resistance [4].Even the promising agents such as cefiderocol with activity towards carbapenem resistant microorganisms including carbapenem-resistant A. baumannii are quiet in early clinical development stages and will be eventually available only in the coming years [5].Besides, the new βlactam-β-lactamase inhibitor combinations are not active against carbapenemresistant A. baumannii [6].All of these reasons have led to the reliance on polymyxins as salvage therapy.
Colistin use has many constraints including toxicity, hetero-resistant isolates development, and finally the clinical and laboratory and standard inistitute (CLSI) warning of the limited clinical efficacy even if intermediate in vitro susceptibility results are obtained that drives clinicians to try other treatment strategies including antimicrobial combinations [7].Tigecycline, a semi-synthetic tetracycline product, proved to have in vitro antimicrobial activity against carbapenem-resistant A. baumannii isolates.Unfortunately increasing resistance has been reported in different localities, in addition to reports of tigecycline monotherapy failure suggest a need for tigecycline combination with other antimicrobials [8,9].Synergistic combination therapy using available antibiotics offers a promising and tangible option to treat infections by MDR bacteria.For carbapenem-resistant A. baumannii, there is no consensus on optimal antimicrobial treatments for such strains [10].
This study aimed to assess prevalence, risk factors of HAI by A. baumannii, and to compare the in vitro efficacy of colistin sulfate-tigecycline combinations versus their individual combination with levofloxacin and meropenem against carbapenem resistant A. baumannii clinical isolates from an Egyptian tertiary care hospital ICUs.

Study design and participants
This cross sectional study was executed in Anesthesia and surgical ICU, emergency ICU, Tropical Medicine ICU, and Medical Microbiology and Immunology Department, Faculty of Medicine, Zagazig University throughout 12 months from April 2019 to April 2020.This comprehensive study included all patients admitted to ICUs during study period if they developed fever, leukocytosis and other evidences of infection after 48 hours of hospital admission.
Patients were excluded if there was evidence of infection prior to hospital admission.

Ethical approval
The study was approved by Zagazig University Institution Review Board (ZU-IRB) (Approval code 6353).This study was carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).Informed consent was obtained from patients or their relatives.

Microbiological work up
Clinical samples collection and A. baumannii identification Clinical samples were aseptically collected according to the site of infection from ICU admitted patients with evidence of HAI.Samples included endotracheal aspirates, urine sample, pus, and blood.Detailed history of patients included age, sex, length of hospital stay, use of invasive medical devices, problem obliging ICU admission and previous administration of empirical antibiotics were stated.Samples were transported and processed for isolation and identification of the causative organism.
Acinetobacter baumannii initial identification was done via conventional biochemical methods [11] and confirmed to the species level by API 20NE (Bio-Mérieux, France).Only one A. baumannii isolate per patient was included.
Carbapenem resistance was defined as A. baumannii that test resistant to imipenem, meropenem or doripenem based on current CLSI M100 standards [12].Isolates that were resistant to all tested carbapenems (n=30) were used for subsequent steps in the study.

Determination of minimal inhibitory concentration (MIC), MIC50, MIC90 of antibiotics to be used in combination study
The antimicrobial agents used in combination assessment were selected based on its empirical combination use for treatment of carbapenemresistant A. baumannii and the previous in vivo and in vitro studies suggesting their efficacy in combination [13][14][15].The broth microdilution method was used for the estimation of minimum inhibitory concentration (MIC) for colistin sulfate (Hebei Shengxue Dacheng Pharmaceutical, china), Tigecycline (Suzhou greenway Biotech / china), levofloxacin and meropenem (Sigma-Aldrich, USA).CLSI standards regarding solvents for stock solution preparation, dilutions, timing of preparation, and broth media were followed.
Interpretation of results were done according to CLSI standards except for tigecycline as no breakpoints were available regarding A. baumannii.MIC50 and MIC90 were estimated and reported for each individual antibiotic.

Synergism testing by checkerboard method
The selected antimicrobials were tested noncombined and in combination by microdilution checkerboard method.The checkerboard method was performed according to the method described [16,17].Briefly, in a sterile 96 well microtiter plates doubling dilutions of one antibiotic were done in the horizontal wells and the other antibiotic dilutions in the vertical wells.Bacterial suspension (1/100 dilution of 0.5 McFarland bacterial suspension) was added to each well.The final antibiotic concentration ranged from 1/8 up to 4 folds of predetermined MIC for the tested isolate.Serially diluted antibiotics without combination, growth control and sterility control wells were included.The plates were covered and incubated at 35˚C for 18-24 h.The wells with no visible growth were identified visually against a dark background, the MICs for antibiotics in combination were recorded.

Patients' outcomes assessment
The ICU consultants were informed about the results of synergism testing.The outcomes of surviving patients who received combination therapy assessed clinically (fever regression-signs of infection resolution -laboratory investigations) and microbiologically by recollection and testing of samples from previously proved infected sites 3-5 days after onset of antibiotic combination administration.

Statistical analysis
All data were analyzed using SPSS 22.0 for windows (SPSS Inc., Chicago, IL, USA) & MedCalc 13 for windows (MedCalc Software bvba, Ostend, Belgium).Categorical variables were expressed as a number (percentage).Chi square test was used to compare percentage.All tests were two sided.p<0.05 was considered statistically significant (S), p<0.001 was considered highly statistically significant (HS) and p≥0.05 was considered non-significant (NS).

Results
A total of 250 clinical samples were included in the study from ICU admitted patients (144 males and 106 females) with clinical and laboratory evidence of HAI.Their age ranged from 18 to 80 years (mean ± SD = 56 ± 19).Of the tested 250 clinical samples, 46 A. baumannii isolates were recovered.Acinetobacter baumannii accounted for 18.4 % of HAI in ICUs.Acinetobacter baumannii showed higher prevalence in endotracheal aspirate samples (22.1 %) with higher percent among patients with late onset ventillator associated pneumonia than early onset ones (27.5% versus 11.8%) respectively with no statistically significance difference between early and late onset VAP.Lower prevalence 14% and 8.3 % in pus and blood culture were detected respectively with no statistical significant difference (P >0.05) (Table 1).
Admission to other hospital wards before ICU and failure of empirical antibiotic were statistically highly significance risk factors for increased prevalence (%) of A. baumannii isolates [(p-value=0.001**for both, Odds (95% CI=3.9(2.03-7.7)& 3.1(1.6-6.21)]respectively.Intensive care unit admission for more than a week showed higher prevalence of A. baumannii isolation but no statistical significant difference from ICU stay for less than a week (p-value=0.08) .In addition the reason for ICU admission wasn't statistically significance risk factor for increased prevalence (%) of A. baumannii isolates (p-value=0.7)(Table 2).
Assessment of antibiotic susceptibility of 9 FDA suggested group A antimicrobials against A. baumannii isolates showed that all isolates were resistant to ampicillin-sulbactam, ceftazidime, and ciprofloxacin.The least detected resistance was to meropenem and doripenem (65.2%) (Table 3).
When effects of antibiotic combinations were assessed on A. baumannii isolates resistant to all tested carbapenems, the highest synergy (50%) was found for combinations of colistin with meropenem, followed by combination of colistin with levofloxacin (16.7%).Tigecycline-meropenem combination had the highest antagonistic effect (66.7%).Tigecycline exerted synergistic activity only with colistin in only 3 isolates (10%) (Isolates with tigecycline MIC>2 µg/mL) while this combination showed no antagonistic effect in any of tested isolates.Neither levofloxacin nor meropenem exerted synergistic activity when combined with tigecycline in any of the tested isolates (Table 5).
Colistin-meropenem combination was prescribed for 15 patients.Tigecycline was not available for use during study period.Among patients who received colistin-meropenem combination (n=15) the infection was cured in 10 patients (the surviving patients).On the other hand in USA according to the fact sheet published as a part of CDCs 2019 antibiotic resistant threat reports, there was reduction in carbapenem-resistance in Acinetobacter isolates from hospitalized patients over time thanks to continued infection control practices and appropriate antibiotic use [28].
All isolates in our study were of intermediate susceptibility for colistin according to CLSI breakpoints declared in 2020.In other Egyptian study colistin resistance was reported to be 6.25% among A. baumannii isolates [29].MIC90 for both colistin and tigecycline were 1µg/ml.This suggests that both colistin and tigecycline are available therapeutic choices for carbapenemresistant A. baumannii infections based on in vitro susceptibility testing.
Three out of 30 isolates (10%) in our study had tigecycline MIC > 2 µg/ml (The US FDA breakpoints of tigecycline approved for Enterobacteriaceae).Similar results were reported by another Egyptian study that reported 9.37% of A. baumannii isolates as tigecycline non susceptible [29].On the other hand 100% of A. baumannii isolates from ventilator associated pneumonia in emergency ICU of Zagazig University hospitals were tigecycline susceptible [24].
On speaking about checkerboard method, our results showed that colistin with meropenem is the most active in vitro combination on carbapenem resistant A. baumannii with 50% synergy and no antagonism was observed.These results matched with results reported by Meliani and coworkers who reported synergy between colistin and imipenem in 57.14% of tested carbapenem resistant isolates [30] and higher synergism (80%) was reported in other studies [27].
Slightly lower synergism between colistin and meropenem (30%) was reported by Bae and coworkers [13].This difference may be caused by difference in colistin resistance between the two studies as they tested colistin resistant isolates while all isolates in our study were of intermediate susceptibility to colistin.
Our results are the reverse of that reported by Kheshti et al. who found that colistin-imipenem combination exhibited the highest (40%) antagonistic effect [31].Sertcelik et al. reported the synergy of colistin with meropenem was only 4.3% and additive effect "partial synergism" in 95.7% of isolates [32].This marked discrepancy between studies' findings could be due to using different in vitro methods for synergism assessment [33].Also, involved mechanism of carbapenem resistance or even MIC values of carbapenem in different isolates may explain this marked discrepancy between different studies that was confirmed by the study done by Zhu and coworkers who studied colistin imipenem combination and reported synergy in 93.3% of isolates with imipenem MICs of 16 μg/ml versus 16.7% of isolates with imipenem MIC of 64 μg/ml and suggested paying more attention to the MICs of single drugs for combination therapy choice [34].Tigecycline-colistin combination showed no antagonistic effect in any of tested isolates in our study.Very limited synergistic effect (10%) of tigecycline combination with colistin was detected only in the 3 strains with MIC > 2 µg/ml.This synergism may be explained by the permeating action of colistin on bacterial outer membrane leading to better access of tigecycline into bacterial cell's target site.Lack of tested bacterial resistance to colistin may explain this synergism.Tigecycline-colistin combination showed addition and indifference in 23.3% and 66.7% of isolates respectively.These results were comparable with a study done by Bae and coworkers who observed indifference of tigecycline combination with colistin in 77.8% of isolates [13].
Tigecycline-imipenem combination had the highest antagonistic effect in 66.7% of isolates, this finding greatly matched with Güçkan et al.
[27].This can be explained by the interference between the bacteriostatic effect of tigecycline with the bactericidal action of meropenem on bacterial cells.
Although tigecycline didn't have a good in vitro evidence for use in a combination to treat infections by carbapenem resistant A. baumannii in our study, Previous clinical study done by Kofteridis and coworkers have evaluated the efficacy of empirical tigecycline-carbapenem combination therapy in the treatment of pan-drug resistant bacteria to be clinically effective in 37.5% of A. baumannii infections [35].Such discrepancy between in vitro and in vivo studies may be due to tigecycline pharmacodynamics properties, as tigecycline is rapidly concentrated into tissues ensuing higher tissue concentrations up to 78-fold its plasma concentration.Another explanation of such discrepancy is the labile nature of tigecycline solution due to oxidative degradation in vitro [36].A rationalization of tigecycline recommendation in treatment is thus essential, it should be limited to treatment of extensive drug resistant organisms.

Conclusion
No undesirable antagonistic effects of colistin combination with meropenem were confirmed in this study.These results are fortunate as colistin / meropenem combination therapy is often given in our ICUs for treatment of carbapenem resistant Gram-negative bacteria.Only colistinbased combinations, particularly those with meropenem may confer therapeutic benefits against carbapenem-resistant A. baumannii.

Recommendation
Application of antibiotic stewardship is mandatory as well as strict infection control policies for prevention of development of pan-drug resistant bacteria.

FICI
Fractional inhibitory concentration index (FICI) was used to describe different combination effects [16].The FIC and FICI was calculated using the following equations: The FIC drug A = MIC of drug A in combination / MIC of drug A alone The FIC drug B = MIC of drug B in combination / MIC of drug B alone FICI = FIC of drug A + FIC of drug B The results of the FICI were interpreted as shown in table (I).Table I. interpretation of FICI of antibiotic combination.

Table 2 .
Risk factors for A. baumannii infections in the ICU.

Table 5 .
Drug combination effect determined by the checkerboard method on A. baumannii isolates resistant to all tested carbapenems (n=30).