ARBEKACIN – A RAY OF HOPE TO FIGHT AGAINST MDR AND XDR GRAM-NEGATIVE BACTERIA IN A SCIENTIFIC AND COST-EFFECTIVE WAY IN INDIAN SCENARIO

Objective: The objective of the study was to see the in vitro activity of arbekacin, a novel aminoglycoside, against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacilli (GNB) so that it can become a good alternative as empirical treatment for severe sepsis. Methods: Identification and antibiotic sensitivity testing of the GNB isolated from the clinical samples were done using the VITEK-II system in a tertiary care hospital, Kolkata. MDR and XDR strains were selected by their definitions and molecular characterization was done by multiplex polymerase chain reaction. The minimum inhibitory concentration (MIC) value of arbekacin was detected by the E-test strip and compared with other aminoglycosides. Results: A total of 140 drug-resistant strains including ESBL- and carbapenemase-producing GNB were selected for the study. Arbekacin showed reduced values of MIC 50 and MIC 90 compared to other aminoglycosides for most of the drug-resistant GNB. Conclusion: Hence, in this drug-resistant era, arbekacin with the advantage of a single daily dose can be used as an empirical choice in severe sepsis as monotherapy or in combination with other antibiotics such as colistin or polymyxin to fight against MDR and XDR bugs.


INTRODUCTION
Antimicrobial resistance poses a major threat to public health and makes therapeutic decisions more challenging. India carries one of the largest burdens of drug-resistant pathogens worldwide [1]. Emergence of their multidrug-resistant (MDR) and extensively drugresistant (XDR) Gram-negative pathogens adds on to the complexity of infection, rendering them difficult to treat [2]. As there are not enough new antibiotics in the pipeline to meet the challenge of antibiotic resistance, the pharmaceutical industry suggests drug repurposing, to find new uses of old drugs [3]. The drug arbekacin had been licensed in Japan for use under the trade name "Habekacin" for the treatment of MRSA caused sepsis and pneumonia since 1990 [4,5]. Arbekacin is an aminoglycoside dideoxykanamycin B (dibekacin) belonging to the kanamycin family [6]. Arbekacin has the structure of 1-N-[(S)-4-amino-2-hydroxybutyryl (AHB)]-DKB. Due to the introduction of AHB to 1-N position of DKB, the resulting arbekacin exhibited activity against drug-resistant bacteria [7]. Arbekacin is stable to the aminoglycoside-inactivating enzymes such as aminoglycoside phosphotransferase (APH), aminoglycoside adenylyltransferase (AAD), and aminoglycoside acetyltransferase, whereas gentamicin, amikacin, tobramycin, and kanamycin were completely inactivated by APH (2″) [8]. Arbekacin is a good bactericidal drug against MRSA, enterococci and shows concentration-dependent and long-lasting postantibiotic effects [9][10][11][12], but its activity against MDR and XDR Gramnegative bacilli (GNB) is not evaluated. Hence, the objective of the study was to see the in vitro activity of arbekacin against MDR and XDR GNB isolated from different clinical samples.

Identification of the isolates from clinical samples
The study was conducted in the Department of Microbiology, NRS Medical College, Kolkata, from May 2019 to August 2019. Isolation of GNB was done from different samples (urine, respiratory samples, pus, and blood) by standard microbiological procedure and identification up to the species level was done using VITEK-GN cards (bioMérieux India Private Limited).

Antimicrobial susceptibility testing
Antibiotic sensitivity testing was performed in the VITEK-2 system using AST-GN280 and AST-GN281 susceptibility cards and interpreted according to the Clinical and Laboratory Standards Institute recommendations. P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were used as quality control strains.

Inclusion and exclusion criteria
Selection for MDR and XDR strains from the isolated GNB was based on the definition of MDR (acquired non-susceptibility to at least one agent in three or more antimicrobial categories) and XDR [non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remain susceptible to only one or two antimicrobial categories)]. Isolated GNB strains sensitive or resistant to one/two antimicrobial categories were excluded from the study.
ESBL production was determined by placing ceftazidime (CAZ 30 µg) disks with or without clavulanic acid (CA 10 µg) on MHA plate. After overnight incubation, if there was an augmentation of ≥5 mm in the inhibitory zone diameter of CAZ-CA in comparison to CAZ alone, ESBL by that strain was phenotypically confirmed.  arbekacin E-test strip was applied onto it. After overnight incubation, the reading of MIC was taken as the value at the point where ellipse intersects the scale (Fig. 2).   ACT TGG CCT TGC TGT CCT T  603 bp  Reverse CAT TAG CCG CTG CAT TGA T  blaVIM  Forward TGT CCG TGA TGG TGA TGA G T  437

The minimum inhibitory concentration (MIC) of arbekacin against MDR and XDR Gram-negative bacterial isolates
To calculate the MIC of arbekacin by E-test strip, a lawn culture is made by the organism to be tested, over a Mueller-Hinton agar plate and the Sarkar et al.

DISCUSSION
The increased prevalence of MDR and XDR GNB along with their rapid spread is a matter of concern in modern medicine. In our study period over 4 months, we have selected 140 drug-resistant strains including ESBL and carbapenemase-producing GNB which were representative of MDR and XDR strains among the total of 1276 isolates. We compared the resistance pattern of arbekacin with other aminoglycosides (amikacin and gentamicin) and other potent antimicrobial drugs with these selected isolates. Arbekacin showed a better sensitivity profile than penicillin, cephalosporin, other aminoglycosides, fluoroquinolones, β-lactam-β -lactamase inhibitors, and carbapenems. Polymyxin B and tigecycline showed better sensitivity than arbekacin. In Proteus spp., the sensitivity of arbekacin was comparable with other aminoglycosides.
This study showed that MIC 50 of arbekacin was 10.67 times lower than that of amikacin and gentamicin for E. coli, 4 times lower than that of amikacin, and 2 times lower than gentamicin in Klebsiella spp., 1.33 times lower than that of amikacin, and 2.67 times lower than that of gentamicin for Enterobacter spp., 1.33 times lower than that of amikacin but MIC 50 of gentamicin was 3 times lower than that of arbekacin in Proteus spp. In the case of Pseudomonas spp., MIC 50 of arbekacin is 5.33 times lower than that of amikacin and gentamicin and in Acinetobacter spp., arbekacin MIC 50 was 8 times lower than amikacin and 4 times lower than gentamicin. However, the value of MIC of arbekacin was high in Klebsiella (MIC 50 = 16, MIC 90 ≥ 64) and Proteus (MIC 50 = 24, MIC 90 ≥ 128) ( Table 5). In these cases, we might consider the combination therapy with polymyxins/tigecycline or carbapenem (if carbapenem was sensitive) [14]. Antibiotic combination therapy study groups suggested the effective combination of arbekacin plus aztreonam against MDR Pseudomonas [15].
Hence, the MIC value of arbekacin is lower than other aminoglycosides in drug-resistant GNB which was similar to the study by Watanabe et al., 2012, Kazuno et al., 1986. Arbekacin is a unique aminoglycoside because of its ability to concentrate in pulmonary epithelial lining fluid [18], unlike other aminoglycosides which will help us to use it against pneumonia and other respiratory diseases. Again, simultaneous Gram-positive and Gram-negative coverage with a single daily dose are the advantage of arbekacin and it can be used as an empirical choice in severe sepsis. To interpret its in vitro activity, the sensitivity of other aminoglycosides (amikacin and gentamicin) can guide arbekacin sensitivity for treating physicians in a resource-limited setting. Although the sensitivity of tigecycline is better than arbekacin, the favorable pharmacodynamics of arbekacin make the drug superior to tigecycline.
A higher dose of arbekacin can improve clinical efficacy. In a study by Matsumoto et al., 2014 [8], it was shown that in patients with normal renal function, the target peak concentration (Cpeak) was not achieved with once-daily administration of 150-200 mg and a higher dosing regimen is required to improve clinical efficacy, and the Japanese Society of Chemotherapy and the Japanese Society of Therapeutic Drug Monitoring Experts recommend 5.5-6.0 mg/kg body weight to reach the target concentration [19]. The pharmacokinetics in healthy volunteers with normal renal function did not change on 400 and 600 mg single dose and the total clearance does not decrease at a high dose [8].

Sarkar et al.
If the dose of arbekacin is formulated as 4 times the normal dose with dose spacing of 72 h, it will have the following benefits: (i) The dose will cross 4 times the normal MIC which is well above minimum bactericidal concentration (MBC) and for that the chance of crossresistance or resistant mutant will be nil. (ii) If the dose is 4 times increased, it will be far less than the toxic dose and therefore safe. The renal-related adverse drug reactions of arbekacin are increased with a higher Ctrough [11]. The incidence of arbekacin induced nephrotoxicity was observed when it was administrated at a total dose of over 5000 mg [20]. Moreover, with a dose spacing of 72 h, the Ctrough will be lower. (iii) Outpatient antimicrobial therapy is possible in resource-limited settings. (iv) It can be cost effective and can be used in remote places where a basic sensitivity pattern is available.
Hence, in case of severe sepsis, we can plan the treatment in the following way -first, send the culture from all relevant sites and give arbekacin 800 mg (4 times the normal dose to attend the MBC). No antibiotics are needed for the next 72 h until the culture sensitivity report comes and plan the antibiotics accordingly. All other supportive measures are to be continued as per the sepsis protocol. The total cost of therapy will be less with a fair chance of patient survival.

CONCLUSION
Hence, in this drug-resistant era, arbekacin can be used as an empirical choice in severe sepsis as monotherapy or in combination with other antibiotics such as colistin or polymyxin to fight against MDR and XDR bugs. The favorable pharmacokinetics, pharmacodynamics, and spectrum (simultaneous Gram-positive and Gram-negative coverage) of the drug with the advantage of a single daily dose will make the antibiotic as a handy choice for the management of sepsis with least time to the thermometer to needle even in resource-limited health-care settings in India.

AUTHORS' CONTRIBUTIONS
Dr. Soma Sarkar -Data collection and manuscript writing, Dr. Dipankar Sarkar -Design the research study, Dr. Anjum Namhata -Preparation of manuscript, and Dr. Manideepa Sengupta -Editing of the manuscript as per journal guidelines.

CONFLICTS OF INTEREST
The authors declare that they have no conflicts of interest.