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Antimicrobial Susceptibility Among Colistin, Sulbactam, And Hindawi Journal of Pathogens Volume 2018, Article ID 3893492, 5 pages https://doi.org/10.1155/2018/3893492 Research Article Antimicrobial Susceptibility among Colistin, Sulbactam, and Fosfomycin and a Synergism Study of Colistin in Combination with Sulbactam or Fosfomycin against Clinical Isolates of Carbapenem-Resistant Acinetobacter baumannii Sombat Leelasupasri,1 Wichai Santimaleeworagun ,2,3 and Tossawan Jitwasinkul3,4 1 Internal Medicine Unit, Phyathai II International Hospital, Bangkok, Tailand 2Department of Pharmacy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Tailand 3Antibiotic Optimization and Patient Care Project by Pharmaceutical Initiative for Resistant Bacteria and Infectious Diseases Working Group (PIRBIG), Silpakorn University, Nakhon Pathom, Tailand 4Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Tailand Correspondence should be addressed to Wichai Santimaleeworagun; [email protected] Received 28 August 2017; Revised 9 December 2017; Accepted 25 December 2017; Published 18 January 2018 Academic Editor: Jose Yuste Copyright © 2018 Sombat Leelasupasri et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Tis in vitro study aimed to determine the activity of colistin plus sulbactam and colistin plus fosfomycin against carbapenem- resistant A. baumannii (CRAB). Fifeen clinical isolates were obtained from patients admitted to Phyathai II International Hospital, Bangkok, Tailand, from August 2014 to April 2015. Te antimicrobial susceptibilities of colistin, sulbactam, and fosfomycin were evaluated using the E-test or broth microdilution and the synergistic activity of the antibacterial combinations (colistin plus sulbactam or fosfomycin) was determined using the chequerboard method. Clonal relationships were explored using repetitive element palindromic- (REP-) PCR. Te CRAB isolates were categorized by REP-PCR in 8 groups [A-H]. All CRAB isolates were universally susceptible to colistin but only 20.0% were susceptible to sulbactam. Te MIC ranges for colistin, sulbactam, and fosfomycin were 0.75–2 mg/L, 2–96 mg/L, and 64–256 mg/L, respectively. A chequerboard assay revealed that the rates of synergistic and additive efect rates of colistin plus sulbactam and colistin plus fosfomycin were 53.3% and 73.3% of isolates, respectively. No antagonistic efect in any colistin-based combination was observed. However, almost CRAB strains in clone A showed the synergy or additive efects of colistin-sulbactam combination, whereas the other clone (B-H) mostly showed indiferent efects. In conclusion, colistin plus sulbactam and colistin plus fosfomycin against CRAB seem to be interesting option but the efcacy in clinical use has to be evaluated. 1. Introduction meropenem (National Antimicrobial Resistant Surveillance, Tailand, 2015); thus, CRAB infections are difcult to treat in Carbapenem-resistant A. baumannii (CRAB) is the most an increasingly antimicrobial resistant era. One of the various important pathogen in nosocomial infections worldwide strategies to improve clinical outcomes and to prevent emerg- causing high morbidity and mortality and increasing medical ing resistance during treatment is antimicrobial combination expenditure [1, 2]. Data from the National Antimicrobial therapy [3, 4]. Resistant Surveillance, Tailand, in 2015, showed that A. Todate,colistin(polymyxinE)orpolymyxinB-based baumannii wasrankedthethirdandsecondofpathogens combinations are recommended as treatments for CRAB isolated from all specimens and sputum, respectively. Con- because almost all strains of CRAB remain sensitive to sidering the various CRAB strains, the report showed that polymyxins [5], while sulbactam and fosfomycin have better A. baumannii was up to 70% resistant to imipenem or pharmacokinetic profles with moderate and low protein 2 Journal of Pathogens binding. Sulbactam and fosfomycin are currently used at Te quality controlled strain constituted E. coli ATCC 25922 maximum dosage to cover some resistant strains [6, 7]. [Department of Medical Sciences Type (DMST) culture Certain studies have focused on determining the synergist collection, Bangkok, Tailand], which was used to monitor efect of colistin plus sulbactam and colistin plus fosfomycin theaccuracyofMICdeterminationbasedonCLSI2017. against A. baumannii. Tose previous studies of in vitro Te MIC fndings of sulbactam, colistin, and fosfomycin synergismshowedthatcolistinplussulbactammightbe were interpreted using standard breakpoint criteria. Col- a combined therapeutic option for CRAB treatment [3]. istin and sulbactam susceptible breakpoints were defned as Moreover,colistincombinedwithfosfomycinrevealeda ≤2mg/Land≤4mg/L, respectively, according to CLSI 2017. synergistic efect or additive efects against CRAB strains Due to the lack of a standard MIC breakpoint for fosfomycin or extensively drug-resistant A. baumannii, but only two against A. baumannii in CLSI, an MIC resistance breakpoint studies addressed the colistin-fosfomycin combination with of ≤32 mg/L was established for fosfomycin according to the discordant results [8, 9]. European Committee on Antimicrobial Susceptibility Testing As previously described, owing to the inconsistent fnd- 2017 (EUCAST). ings of colistin-based combinations due to the variety of bacterial strains used in related studies from university 2.4. Synergistic Testing. Te synergistic efects of antimicro- medical schools, it is hard to refect on or to generalize the bial combinations (colistin plus sulbactam and colistin plus benefcial results of such synergistic efects. We, therefore, fosfomycin) were determined by the chequerboard tech- performed this study to determine the in vitro synergistic nique. Tis technique was performed with Mueller-Hinton efects of colistin plus sulbactam and colistin plus fosfomycin broth II (Difco), supplemented with 25 �g/mL G-6-P in a ∘ against clinical strains of CRAB. fosfomycin combination. All samples were incubated at 37 C for 18 hours. Te fractional inhibitory concentration indices 2. Materials and Methods (FICI) were calculated for each of the paired antibiotics and were interpreted as ≤0.5 = synergistic, 0.5–≤1.0 = additive 2.1. Bacterial Strains. Clinical CRAB strains were isolated efect, >1–4 = no interaction, and ≥4antagonisticefect. from inpatients admitted to Phyathai II International Hospi- tal, a 550-bed private hospital for inpatient cases in Bangkok, 3. Results Tailand, from August 2014 to April 2015. CRAB was defned as resistance to either imipenem, meropenem, or doripenem Fifeen CRAB isolates were obtained from various sites. No according to the Clinical and Laboratory Standards Institute particular strains were isolated repeatedly within the same screening criteria of carbapenem-resistant strains [10]. All patient (Table 1). According to the clonal relationship study, clinical isolates of CRAB were frstly obtained from various the15samplescouldbeclassifedinto8clonesincludingA-H. specimens in each patient. All CRAB strains were kept at Most strains were in clone A (�=8), whereas the remaining ∘ −20 C until tested. Te institutional review board approved 7 strains were in each member of B-H. the research protocol with a waiver for informed consent Among 15 CRAB isolates, the MIC ranges for colistin, [number ID0013/59]. sulbactam, and fosfomycin were 0.75–2 mg/L, 2–96 mg/L, and 64–256 mg/L,respectively.Tepercentageofsusceptible 2.2. Clonal Relationships. Te clonal relationships of CRAB strains was 100 and 20 for colistin and sulbactam, respectively, were evaluated using the REP-PCR method. Te PCR reac- according to CLSI breakpoint, but all CRAB strains showed tion mixture had been described elsewhere [11]. Briefy, a resistance to fosfomycin according to EUCAST breakpoint. � couple primer (REP-forward: 5 -IIIGCGCCGICATCAGGC- Te antibiotic combination fndings from the chequer- � � � 3 and REP-reverse: 5 -ACGTCTTATCAGGCCTAC-3 )was board study are shown in Table 2. Both synergistic and addi- used to amplify the REP region under the following condi- tive efects were found for colistin plus sulbactam in 2 of 15 ∘ tions, starting with heating at 94 Cfor10minutes,followed isolates (13.3%) and 6 of 15 isolates (40.0%), respectively. Both ∘ ∘ ∘ by 30 cycles of 94 Cfor1minute,45Cfor1minute,72C synergistic and additive efects were also seen for the colistin- ∘ for 2 minutes, and fnally 72 C for 16 minutes. Te REP-PCR fosfomycin combination in 4 of 15 isolates (26.7%) and 7 of 15 products were performed using agarose gel electrophoresis isolates (46.7%), respectively. Indiferent efect was seen for and were stained with ethidium bromide. Te criterion for colistin-sulbactam and colistin-fosfomycin combination in 7 classifying the diferent clones was a pattern that difered of 15 isolates (46.7%) and 4 of 15 isolates (26.7%), respectively. from the at least three bands or more of REP-PCR [12]. Likewise,noantagonisticefectswereobservedwithanyof the combinations of antimicrobial agents studied. 2.3. Determination of Minimum Inhibitory Concentration. Te MICs of sulbactam and fosfomycin were determined by 4. Discussion the Epsilometer test (E-test; Liochem) method using Mueller- Hinton agar (Difco, Detroit, MI) plates, specifcally, the E- According to these results, all CRAB isolates were universally test of fosfomycin supplemented with glucose-6-phosphate susceptible to colistin. Our fndings were
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