Pharmaceutical Sciences Asia Pharm Sci Asia 2020; 47 (1), 37-42 DOI:10.29090/psa.2020.01.019.0012 Research Article Comparative in vitro activity of sitafloxacin against multidrug-resistant and carbapenem-resistant Acinetobacter baumannii clinical isolates in Thailand Taniya Paiboonvong1, Vipavee Rodjun2, Jantana Houngsaitong1, Mullika Chomnawang3, Preecha Montakantikul2, Suvatna Chulavatnatol2* 1 Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand 2 Faculty of Pharmacy, Siam University, Bangkok, Thailand 3 Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand *Corresponding author: suvatna.chu@mahidol.ac.th KEYWORDS: Sitafloxacin; Multidrug-resistant A. baumannii; Carbapenem-resistant A. baumannii ABSTRACT The rapid emergence of multidrug-resistant Acinetobacter baumannii (MDRAB) is recognized as a significant health problem worldwide, including Thailand. Due to a limitation of treatment option, a new antimicrobial agent has been challenged. Sitafloxacin, a new fluoroquinolone antimicrobial agent, has shown a good in vitro activity against MDRAB and carbapenemresistant A. baumannii (CRAB). The aim of this study was to evaluate in vitro activity of sitafloxacin and compare that with other antimicrobial agents against A. baumannii clinical isolates, focusing on multidrug-resistant A. baumannii and carbapenemresistant A. baumannii (MDR-CRAB). All 350 A. baumannii clinical isolates were collected from thirteen tertiary care hospitals in all regions of Thailand. The minimum inhibitory concentrations (MICs) were determined by broth microdilution method. To determine rate of susceptibilities, a susceptible isolate with sitafloxacin was considered as MIC values of ≤ 2 µg/mL and ≤ 1 µg/mL. For other antimicrobial agents, MIC breakpoints were considered according to the Clinical and Laboratory Standards Institute (CLSI) 2018. Our study found that 278 clinical isolates were identified as MDR-CRAB. The MIC range, MIC50 and MIC90 of sitafloxacin against MDRCRAB isolates were ≤ 0.0625-8 µg/mL, 1 µg/mL and 2 µg/ mL, respectively. Additionally, almost of the colistin-resistant isolates were susceptible to sitafloxacin (92.86%). Sitafloxacin had a good activity against multidrug-resistant isolates in Thailand. Thus, sitafloxacin can be considered as an alternative choice for treatment of MDRAB and CRAB infections. Further studies are needed to evaluate treatment outcomes. 1. INTRODUCTION https://www.pharmacy.mahidol.ac.th/journal/ © Faculty of Pharmacy, Mahidol University (Thailand) 2020 Acinetobacter baumannii is an important cause of nosocomial infections, and becomes a major problem in healthcare settings in Asian countries, including Thailand. The resistance to numerous different drug classes known as multidrug-resistant A. baumannii (MDRAB) has rapidly emerged, and associated with a high mortality rate, especially in critically ill patients1. Carbapenems have been mainly used for treatment of MDRAB, whereas the resistant rate has been rising over the world. According to the National Antimicrobial Resistance Surveillance T. Paiboonvong et al. Thailand (NARST) data, carbapenem-resistant A. baumannii (CRAB) has rapidly emerged for two decades up to approximately 80% in 20162. Moreover, several studies reported high rates of MDRAB and CRAB from tertiary care hospitals in Thailand3,4. Choosing the effective agent for treatment of infections caused by those resistant pathogens is challenging. The therapeutic option for MDRAB and CRAB is mainly limited to colistin as the firstline treatment. However, the limitations of colistin are the issue of renal toxicity and poor tissue penetration. Therefore, new antimicrobial agents are needed for treating resistant A. baumannii infections Sitafloxacin (DU-6859a) is a new broadspectrum fluoroquinolone antimicrobial agent, showed an active activity against many drug resistant pathogens. Comparing with other fluoroquinolones, sitafloxacin has the lowest inhibitory concentration (IC50) against DNA gyrase and topoisomerase IV5, 6. It has been approved for oral formulation and used in Thailand for treatment of urinary tract infections (UTIs) and lower respiratory tract infections (LRIs) since 2011. Moreover, several studies reported a potent activity of sitafloxacin against drug-resistant A. baumannii7-12. Nevertheless, the activity of sitafloxacin against both MDRAB and CRAB (MDR-CRAB) isolates collected from all regions of Thailand has not been reported. Therefore, this study was conducted to provide an informative data of sitafloxacin against A. baumannii clinical isolates at tertiary hospitals in Thailand, focusing on MDR-CRAB. 2. MATERIALS AND METHODS 2.1. Hospitals and bacterial isolates All 350 A. baumannii clinical isolates were collected from in-patients of thirteen tertiary care hospitals, Thailand during the period of 2016. Non-duplicated isolates were collected. All isolates were cultured in suitable media and storage before sending to the laboratory of microbiology at Faculty of Pharmacy, Mahidol University. Bacterial glycerol stocks were prepared and stored at -80°C until being tested. 2.2. Antimicrobial susceptibility tests Broth microdilution method was used to determine minimum inhibitory concentration (MIC) 38 Pharm Sci Asia 2020; 47 (1), 37-42 for sitafloxacin and comparators; ciprofloxacin, ceftazidime, imipenem, meropenem, doripenem, sulbactam, and colistin according to recommendation from CLSI 201613. The standard powder of sitafloxacin was provided by Daiichi Sankyo, Thailand. Standard powders of ciprofloxacin, ceftazidime, colistin, and sulbactam were purchased from Tokyo Chemical Industry CO.,LTD. Standard powders of imipenem and meropenem were purchased from Siam Bheasach CO.,LTD, and doripenem was purchased from Shinogal & CO.,LTD. The MIC values were defined as the lowest concentration of an antimicrobial agent to inhibit a visible growth. The MIC50 and the MIC90 were determined as a concentration that inhibited 50% and 90% of isolates, respectively. Since no MIC breakpoint of sitafloxacin has been published by the CLSI, MIC breakpoint ≤ 2 µg/mL and ≤ 1 µg/mL were considered as susceptible to sitafloxacin in previous studies7,8,12. Other antimicrobial susceptibilities were interpreted according to breakpoints criteria from CLSI 201814. MDRAB and CRAB were identified by the results of susceptibility by broth microdilution method. MDRAB in this study was defined as an isolate resistant to at least three antimicrobial classes according to the interim standard definitions for multidrug-resistant organism, which was created by the European Centre for Disease Prevention and Control and the Centers for Disease Control and Prevention15. Escherichia coli ATCC 25922 was used for quality control strain in this study. The bacterial isolates were grown in Luria-Bertani broth (LB, BD) and cation-adjusted Mueller-Hinton Broth (CAMHB, BD) media. Serial two-fold dilutions of antimicrobials tested for MIC were prepared in CAMHB as described in CLSI guidelines. Bacterial isolates were adjusted for a final inoculum density approximately 5 × 105 CFU/mL using 0.5 McFarland turbidity standard. All 96-well plates were incubated at 37°C in ambient air for 20 hours. The method was performed as the recommendations from CLSI 201613. 3. RESULTS AND DISCUSSION Our study found that 278 clinical isolates were MDR-CRAB. Most of the isolates were from sputum (78.06%) followed by pus (8.99%), urine (5.40%), blood (4.32%), tissue (2.16%), and nasophyrynx (0.72%). Pharmaceutical Sciences Asia Overall, sitafloxacin showed high rate of susceptibility than those of other comparative agents. Sitafloxacin showed the best activity among comparators against MDR-CRAB isolates when using MIC breakpoint ≤ 2 µg/mL; the susceptibility rate was 90.65%. Secondary to colistin, the susceptibility rate was 64.03% when MIC breakpoint ≤ 1 µg/mL. The MIC range, MIC50 and MIC90 of sitafloxacin were ≤ 0.0625-8 µg/mL, 1 µg/mL and 2 µg/mL, respectively. Additionally, 41 out of 278 isolates (14.75%) were resistant to colistin (MIC ≥ 4 µg/mL), and almost of all isolates (92.86%) were susceptible to sitafloxacin with MICs of ≤ 2 µg/mL (MIC values ranged from < 0.0625-2 µg/mL). We found the high MIC50 (2 µg/mL) and MIC90 (4 µg/mL) of sitafloxacin from isolates of the hospitals in the central region. Antimicrobial susceptibility results and MIC distribution of sitafloxacin categorized by regions in Thailand are presented in Table 1, and Table 2, respectively. Table 1. The antimicrobial susceptibility and MIC values of 278 MDR-CRAB isolates Antimicrobial Susceptibility breakpoints (μg/mL) agents Colistin Sitafloxacin Ciprofloxacin Imipenem Meropenem Doripenem Sulbactam Ceftazidime ≤2 ≤ 1, ≤ 2 ≤1 ≤2 ≤2 ≤2 ≤4 ≤8 MIC (µg/mL) MIC50 MIC90 ranges 0.0625 - 512 ≤ 0.0625 - 8 0.5 - > 512 8 - 256 8 - 256 2 - 256 2 - 2048 16 - > 512 0.5 1 64 32 32 16 16 512 Susceptibility % 4 2 > 512 128 64 64 128 > 512 85.25 64.03, 90.65 1.08 0 0 0.72 8.27 0 Table 2. The MIC distribution of sitafloxacin against MDR-CRAB isolates (n=278) Hospital Regions Central* (n=88) North (n=66) South (n=40) East (n=23) Northeast (n=61) Total (278) ≤ 0.0625 0.25 2 2 2 0 0 6 3 0 0 2 2 7 0.5 MIC (µg/mL) 1 2 4 8 MIC50 MIC90 11 24 5 10 17 67 19 26 21 7 25 98 0 0 1 1 0 2 2 1 1 0.5 1 1 4 2 2 2 4 2 39 12 10 3 10 74 14 2 1 0 7 24 * central region included Bangkok Our results suggest that sitafloxacin has a good activity against MDR-CRAB. The results were consistent with previous studies that showed a high susceptibility rate of sitafloxacin against A. baumannii. The susceptibility rates of sitafloxacin against A. baumannii isolated in 2016 and 2012 from Thai patients with UTIs and LRIs were 57.9% and 87.9% and were 66.9% and 94.1% when MIC breakpoint ≤ 1 µg/mL, and ≤ 2 µg/mL, respectively7,8. This indicated that sitafloxacin remains active against A. baumannii isolated from Thai patients over the past 5 years. However, the MIC90 of A. baumannii isolated in 2016 was 4 µg/mL, higher than that in 2012 (the MIC90 was 2 µg/mL)7,8. Similar to the study of Thamlikitkul and Tiengrim, the susceptibility of CRAB to sitafloxacin was 91.4% when MIC breakpoint ≤ 2 µg/mL9. Another report from Thailand showed a similar result that 39 T. Paiboonvong et al. the susceptibility rate of sitafloxacin against CRAB using disk diffusion method was 66.32% in which the zone diameters for susceptibility referred to the MIC breakpoint ≤ 1 µg/mL10. According to Dong et al, the susceptibility rate of sitafloxacin against 24 extensively drug-resistant (XDR) A. baumannii isolates was 91.67% when MIC breakpoint ≤ 2 µg/mL11. By contrast from Huang et al, the susceptibility rate of sitafloxacin against bacteremic isolates of CRAB was 58.9% that lower than the rate of our study, and the MIC50 and MIC90 were high as 2 µg/mL and 8 µg/mL, respectively12. This may result from those isolates were collected from only single center of teaching hospital, but our study collected the isolates from both teaching and provincial hospitals in all regions of Thailand. Comparing to other agents, sitafloxacin was more active than ceftazidime, ciprofloxacin, and sulbactam in which the resistant rate was 100%, 98.92%, and 91.73%, respectively. Our results were consistent with previous reports that overall susceptibility rate of those agents was lower than 25%7,8,10,12. Multiple acquired resistance determinants have been found in MDRAB and CRAB, including transfer of plasmids, transposons, and integrons, encoding a resistant gene to several antimicrobial classes. A mutation in quinolone resistance-determining region (QRDR) of target genes encoding DNA gyrase (gyrA) and topoisomerase IV (parC) has been reported as the main mechanism of quinolone resistance in A. baumannii. In addition, the plasmid-mediated quinolone resistance (PMQR) genes have also been found and conferred high level resistance to ciprofloxacin16,17. Our study found that almost of all isolates were resistant to ciprofloxacin; MIC values ranged from 4 to > 512 µg/mL, however, all of ciprofloxacin-resistant isolates were susceptible to sitafloxacin. This advantage of sitafloxacin may be a potent activity to inhibit the target enzymes of Gram-negative pathogens; it exhibited the lower IC50 than ciprofloxacin approximately 4-fold against the mutations in type II topoisomerases of Pseudomonas aeruginosa6. Colistin-resistant A. baumannii (CoR-AB) has been reported from various countries over the world, including Thailand18-23. Similar to our study, CoR-AB isolates were found in 14.75%. Interestingly, almost of all CoR-AB were susceptible 40 Pharm Sci Asia 2020; 47 (1), 37-42 to sitafloxacin (92.86%) probably involved the difference of resistant mechanism. The main mechanism of CoR-AB has been proposed as alteration of lipopolysaccharide (LPS); lipid A modification or loss of LPS19-21. Overexpression of efflux pump system has been reported for the mechanism of resistance, and recently found in clinical isolates from Thailand22,23. However, sitafloxacin resistance to A. baumannii has not been reported as an overexpression by efflux pump system. Although the mobile colistin resistance gene mcr-1 has been found in gram negative pathogens, especially E. coli, the mcr-1 has not yet been reported on A. baumannii clinical isolates20. However, mcr-1-carrying plasmid could be introduced into A. baumannii, and co-localized with multiple plasmid replicon types, which may be involved in resistance to quinolones24,25. The limitation of this study is that there is no standard MIC breakpoint criteria for susceptibility established for sitafloxacin. Although synergistic effects of sitafloxacin in combination with colistin, sulbactam, rifampicin, and tigycycline were reported against XDR-A. baumannii11, combinations against MDRAB and CRAB should be investigated to confirm a result of different strains in Thailand. 4. CONCLUSION Sitafloxacin showed a good in vitro activity against MDR-CRAB, including colistin-resistant isolates. Thus, sitafloxacin can be considered as an alternative choice for treatment of infections caused by resistant strains. Further studies are needed to evaluate treatment outcomes. 5. ACKNOWLEDGEMENTS We would like to thank Daiichi-Sankyo (Thailand) Ltd. for providing sitafloxacin powder. We also thank microbiology staffs from all hospitals for specimrns collection; Taksin Hospital, Klang Hospital, Nakhon Pathom Hospital, Samut Sakhon Hospital, Maharaj Nakorn Chiang Mai Hospital, Buddhachinaraj Hospital, Sawanpracharak Hospital, Surat Thani Hospital, Maharaj Nakhon Si Thammarat Hospital. Chonburi Hospital, Sunpasitthiprasong Hospital, Surin Hospital, and Buriram Hospital. Pharmaceutical Sciences Asia Conflict of interests The authors declare no conflicts of interests in this research. Funding No funding to declare 7. Ethical approval The study protocol was approved by the Institutional Review Board of the Faculty of Dentistry and Faculty of Pharmacy, Mahidol University (MUDT/PY-IRB 2017/040.2607). 8. Article info: Received February 6, 2019 Received in revised form March 25, 2019 Accepted April 19, 2019 REFERENCES 1. Chung DR, Song JH, Kim SH, Thamlikitkul V, Huang SG, Wang H, et al. High prevalence of multidrug-resistant nonfermenters in hospitalacquired pneumonia in Asia. Am J Respir Crit Care Med. 2011;184(12):1409-17. 2. National Antimicrobial Resistance Surveillance Center (NARST). Antimicrobial Resistance Surveillance 2000-2016. Available from: http:// narst.dmsc.moph.go.th/data/AMR%2020002016.pdf.[Last cited on 2017 May 2]. 3. 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