In Vitro Antibacterial Interaction of Doripenem and Amikacin against Multidrug-Resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae Isolates.
The wide overuse or misuse of antibiotics has been followed by the emergence of resistant bacteria causing healthcare-associated and community-acquired infections worldwide . Many of the pathogens related to the epidemic infectious diseases in humans have evolved into multidrug-resistant (MDR) bacteria . Some isolates change further and become extended drug-resistant (XDR) or pandrug-resistant (PDR) pathogens. Antibiotic resistance pattern in Cipto Mangunkusumo Hospital (CMH), Jakarta, showed that the most prevalent PDR bacteria last year were Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa [3, 4].
Empirical treatment with antibiotics combination has been recommended in severe sepsis and septic shock to reduce mortality such as meropenem and amikacin, meropenem and levofloxacin, and carbapenem (meropenem, imipenem, and doripenem) and polymyxin [5, 6]. In vitro studies showed that combining antimicrobial agents could be more effective against resistant pathogens than the single antibiotic. It can be used to evaluate the efficacy of an antibiotic combination to treat severe infection caused by MDR Gram-negative bacteria . A study reported that the combination of carbapenem and polymyxin resulted in high synergy against Acinetobacter baumannii, among which doripenem was superior to meropenem and imipenem . Other study found that the combination of cefepime and amikacin or meropenem and amikacin resulted in high synergy against MDR P. aeruginosa isolates from intensive care unit patients . Higher synergy can be achieved by combining 2 or 3 antibiotics of aminoglycosides, aztreonam, carbapenem, colistin, rifampin, tigecycline, and fosfomycin .
Doripenem is a relatively new carbapenem and has not been widely studied as meropenem . Therefore, it is interesting to know how doripenem will interact with other class of antibiotic in combination. Aminoglycosides has been a preferable agent for combining antibiotics due to its wide spectrum and good interaction with antibiotics that act on the bacterial cell wall, such as penicillin, cephalosporins, monobactam, carbapenems, and glycopeptides [6, 7]. This study was aimed to evaluate the interaction of doripenem and amikacin combination against three resistant Gram-negative bacteria, that is, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa.
2.1. Study Design and Specimens. This study was a laboratory experimental test in the Department of Clinical Pathology, Cipto Mangunkusumo National Central General Hospital (CMNCGH), Jakarta, between April and October 2016. Study materials were bacterial isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Specimens were collected from hospitalized or ambulatory patients in CMNCGH, which were sent to the Microbiology Laboratory of Clinical Pathology Department.
2.2. Bacterial Identification and Antibiotic Susceptibility Test. Bacteria were identified by using colony morphology identification, Gram staining, and biochemical testing. Isolates were included if the antibiotic susceptibility testing using Kirby-Bauer disk diffusion method showed resistant or sensitive to doripenem and amikacin. Isolates with intermediate resistant were excluded from analyses. The quality of susceptibility testing was controlled by using Escherichia coli ATCC 25922 against Enterobacteriaceae and Pseudomonas aeruginosa ATCC 27853 against nonfermenter Gram-negative rod as recommended by the Clinical and Laboratory Standards Institute (CLSI) guidelines . When the results were within the allowed concentration range, the same isolates underwent a second antibiotic susceptibility test using Kirby-Bauer disk difussion method to confirm their susceptibility against doripenem and amikacin. Antibiotic interaction test was done only if the second antibiotic susceptibility test confirmed the results of the first test. Resistance was defined based on the inhibitory zone diameter on the diffusion disk method by CLSI 2016. Resistant bacteria were grouped as MDR, if nonsusceptible to [greater than or equal to] 1 agent in [greater than or equal to] 3 antimicrobial categories; XDR, if nonsusceptible to [greater than or equal to] 1 agent in all but [less than or equal to] 2 categories; PDR, if nonsusceptible to all antimicrobial agents listed .
2.3. In Vitro Antibiotic Interaction Test. Antibiotic interaction test was done using the Etest method (Liofilchem[R] MIC Test Strip, Italy) of doripenem and amikacin on each bacterial isolates. Strips of both antibiotics were placed on the surface of preinoculated Mueller-Hinton (MH) agar with the scale upside. Strips were gently pressed on the agar surface and were left there for 15 minutes. Then, the strips and agar were incubated for 18-24 h at 35[degrees]C. The minimal inhibitory concentration (MIC) of each antibiotic was the value at which the inhibition zone intersected the scale on E strip . The concentration range was 0.002-32 [micro]g/mL for doripenem ([MIC.sub.DOR]) and 0.016-256 [micro]g/mL for amikacin ([MIC.sub.AMK]).
The MIC for combined antibiotics was then performed using both strips placed perpendicular and intersect at each MIC as shown in Figure 1. First, the doripenem MIC strip was placed on the MH agar surface, and then, the amikacin MIC strip was put on it at 90[degrees] and intersected at each MIC of the isolate against doripenem alone and amikacin alone. Then, both strips were pressed gently, left for 15 minutes, and then incubated for 18-24 h at 35[degrees]C. The MIC of each antibiotic and combined antibiotics was read at the tip of inhibitory zone intersected on the E strips [13, 14].
To evaluate the effect of the combinations of doripenem (DOR) and amikacin (AMK), the fractional inhibitory concentration (FIC) index was calculated as ([MIC.sub.DOR-AMK]/[MIC.sub.DOR]) + ([MIC.sub.AMK-DOR]/[MIC.sub.AMK]). Results were interpreted as synergistic (FIC index [less than or equal to] 0.5), additive (FIC index >0.5-1.0), indifferent (FIC index >1.0-4.0), and antagonistic (FIC index >4.0) interaction as shown in Figure 2 .
2.4. Statistical Analyses. Results were analyzed and presented descriptively. Mean MIC differences between single and combined antibiotics were analyzed using paired t-test or Wilcoxon's signed-rank test. A p value of less than 0.05 was considered significant. Statistical analyses were done using the SPSS software version 17.0 (SPSS Inc., Chicago, Illinois, USA).
During the study period, 80 isolates were collected, but only 68 isolates met the study criteria, consisted of 24 A. baumannii, 20 P. aeruginosa, and 24 K. pneumoniae isolates. There were only 2 isolates of P. aeruginosa that were doripenem sensitive and amikacin resistant. Patients' diagnoses varied greatly, that is, 5 (7%) burn wounds, 4 (6%) sepsis, 4 (6%) hospital-acquired pneumonia, 3 (4%) tuberculosis, 2 (3%) chronic kidney disease, 2 (3%) healthcare-associated pneumonia, 2 (3%) postlaparatomy wound, 2 (3%) liver abscess, 2 (3%) ulcers, and many other diagnosis. The most common specimen was sputum (44%), followed by tissue or pus or wound (24%), bronchoalveolar lavage (7%), urine (6%), blood (4%), feces (4%), drain (3%), CVC tip (3%), liver abscess (2%), bronchial fluid (2%), and vitreous fluid (2%). Distribution of each pathogen according to the resistant pattern and source of specimens is given in Table 1.
Sensitivity pattern of the isolates was 5 (7.5%) non-MDR, 24 (35.3%) MDR, 34 (50%) XDR, and 5 (7.5%) PDR. The XDR K. pneumoniae has the highest proportion. Within the XDR bacteria group, the XDR carbapenem-resistant isolate was found in 9/24 (37.5%) of A. baumannii, 10/20 (50.0%) of P. aeruginosa, and 9/24 (37.5%) of K. pneumoniae isolates. Only 1 MDR carbapenem-resistant isolate was found in each of the three bacterial isolates.
3.1. In Vitro Antibiotic Interaction Test. Interaction test was done between doripenem and amikacin. The results showed that most isolates were indifferent (63.2%). Except for P. aeruginosa, there were 6 isolates allocated in each group of doripenem-amikacin interaction. Only two P. aeruginosa isolates were available in the DOR-S/AMK-R group. Synergistic interaction occurred in only 1 (1.5%) isolate, that is, A. baumannii within the DOR-S/AMK-S group. The most common result was the indifferent interaction, which occurred in 43 (63.2%) isolates. Additive interaction occurred in 24 (35.3%) isolates. No antagonistic interaction was found (Table 2). Based on drug-resistant class, synergy was only found in 1 non-MDR isolate. Additive interaction was found almost similar between MDR and XDR isolates, but lower in PDR group (Figure 3).
Significant MIC reduction was observed in DOR-S/AMK-S in all bacterial isolates compared to single MIC of each antibiotic. However, most of the significant amount of MIC reduction was not enough to reach synergistic effect and could only reach additive or indifferent effect. On the contrary, combination of doripenem-resistant and amikacin-resistant isolates did not change the MIC. Significant MIC reduction was shown by K. pneumoniae isolates when combining either doripenem- or amikacin-resistant isolates with the sensitive counterpart (Table 3).
The antibiotic interaction test using MIC strips is a relatively easy procedure to find the best combination of available antibiotics against MDR pathogens. This study is the first to look for synergy between doripenem and amikacin against the 3 most common resistant Gram-negative bacteria in Cipto Mangunkusumo National Central General Hospital, Jakarta. In this study, specimens were obtained from all wards or polyclinics in the hospital, which were taken from various kinds of underlying diseases. Some studies had used resistant isolates from the intensive care unit only [16, 17], while others used specimens from hospital surveillance without further detail [15, 18].
Our study showed that despite high resistance to doripenem and amikacin, some interactions did occur if both agents were combined resulting in lower MICs than the single antibiotic alone. Lower MIC means the combined antibiotics become more powerful to kill the bacteria. This was more obvious with doripenem sensitive rather than amikacin sensitive. However, antibiotics synergy was only found in one isolate with non-MDR pathogen which showed a synergy with combined doripenem sensitive and amikacin sensitive.
Other study using the checkerboard method showed synergy in 4 of 22 (18.2%) doripenem-resistant A. baumannii isolates when combined with amikacin. Further test conducted by another study using the time-kill curve method showed that only 1 of 8 isolates showed synergy while the rest showed indifferent interaction . Previously, a study using the time-kill curve method on 25 isolates of MDR A. baumannii showed maximum synergistic effect at 24 hours of incubation period, among which 24 (96%) isolates showed synergistic interaction . In addition, lower synergy interaction has been reported among XDR A. baumannii (4% of 48 isolates) .
Synergistic interaction in doripenem sensitive and amikacin sensitive can be due to the doripenem effect in destroying the bacterial cell wall that facilitates amikacin influx. Reduced synergy can be due to enzyme inactivation of the antibiotic, such as beta-lactamase (e.g., carbapenemase) or aminoglycoside-modifying enzyme (AME) produced by the bacteria, modification of antibiotic target, decrease in outer membrane permeability, or efflux pump activation .
Combined doripenem and amikacin showed different results against P. aeruginosa. A study among 100 isolates of carbapenem-resistant P. aeruginosa (89 doripenem resistant and 30 amikacin resistant) found 20% synergistic, 47% additive, and 33% indifferent interactions by using the Etest method . No synergistic effect was found when the bacteria produced metallo-[beta]-lactamase. Another study using the timekill curve method on 25 P. aeruginosa isolates (4 isolates were doripenem resistant) showed synergy in 22 (88%) isolates treated with combined doripenem and amikacin .
In the current study, interaction of doripenem-sensitive isolates was similar with doripenem resistant if the pathogen was also resistant to amikacin. Better interaction was showed by amikacin-sensitive P. aeruginosa in doripenem-sensitive compared to doripenem-resistant isolates (Table 3). Additionally, the MIC of doripenem-sensitive P. aeruginosa was much higher in the amikacin-resistant group when compared to the amikacin-sensitive group (Table 3). This could be due to the cumulative effects of nonenzymatic mechanism of resistance, which develop gradually after several treatments with aminoglycosides .
Interaction tests on K. pneumoniae showed varying results. A study using broth microdilution against XDR K. pneumoniae isolates from patients with hospital-associated infections (all were doripenem resistant) showed synergy in 10% isolates when combined with amikacin. Doripenem can reduce the amikacin MIC 4-32 times lower and amikacin can decrease doripenem MIC 8-16 times lower to reach sensitive breakpoints . Synergistic effect was also observed when amikacin was combined with meropenem or imipenem against Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae .
Overall, our study showed that the antibiotic interaction depends on the resistance pattern of the pathogen tested. Combined antibiotics did lower the MIC to some level although synergy can only be achieved when both agents are still sensitive. Higher resistance pattern results in lower interaction of combined antibiotics. However, in this study, we did not check the mechanism of resistance and thus may limit further assessment on its association with the interaction test results.
In vitro antibiotic interaction test is a useful method to know whether combination of two different antibiotics will be effective to kill multidrug-resistant bacteria. Combination of doripenem and amikacin against Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae showed different interaction effect, which depends on their resistant pattern. Synergistic effect may be difficult to reach, but additive interaction can still be obtained with doripenem sensitive/amikacin resistant or doripenem resistant/amikacin sensitive. When both agents are resistant, nearly all tests showed indifferent interaction. When
combined with amikacin, doripenem has better killing effect on Acinetobacter baumannii and Pseudomonas aeruginosa (nonfermenter Gram-negative bacteria) when compared with Klebsiella pneumoniae (fermenter Gram-negative bacteria).
The data used to support the findings of this study are available from the corresponding author upon request.
Conflicts of Interest
The authors declare that they have no conflicts of interest regarding this study.
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Tonny Loho, Ninik Sukartini, Dalima A. W. Astrawinata, Suzanna Immanuel, Diana Aulia, and Ika Priatni
Infectious Diseases Division, Department of Clinical Pathology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
Correspondence should be addressed to Tonny Loho; firstname.lastname@example.org
Received 23 February 2018; Revised 21 May 2018; Accepted 29 May 2018; Published 3 July 2018
Academic Editor: Jorge Garbino
Caption: Figure 1: Position of the MIC strips for the antibiotic interaction test: (A) doripenem and (B) amikacin.
Caption: Figure 2: Results of the antibiotic interaction test: (a) synergistic (FIC [less than or equal to] 0.5); (b) additive (FIC >0.5-1.0); (c) indifferent (FIC >1.0-4.0); (d) antagonistic (FIC >4.0) interaction.
Table 1: Distribution of each pathogen based on the drug-resistant patterns and sources (n = 68). Source Non-MDR MDR XDR PDR n (%) n (%) n (%) n (%) A. baumannii Sputum (n = 15) 1 (50.0) 6 (66.7) 7 (63.6) 1 (50.0) Tissue (n = 3) -- 1 (11.1) 2 (18.2) -- Blood (n = 2) -- 1 (11.1) 1 (9.1) -- BAL (n = 2) 1 (50.0) -- -- 1 (50.0) Urine (n = 1) -- -- 1 (9.1) -- Stool (n = 1) -- 1 (11.1) -- -- P. aeruginosa Sputum (n = 7) -- 5 (55.6) 2 (20.0) -- Tissue (n = 2) -- -- 1 (10.0) 1 (100) BAL (n = 1) -- -- 1 (10.0) -- Urine (n = 1) -- 1 (11.1) -- -- Wound (n = 3) -- -- 3 (30.0) -- Pus (n = 1) -- -- 1 (10.0) -- Others (n = 5) -- 3 (33.3) 2 (20.0) -- K. pneumoniae Sputum (n = 8) 2 (66.7) 2 (33.3) 3 (23.1) 1 (50.0) Tissue (n = 1) -- -- 1 (7.7) -- Blood (n = 1) -- -- 1 (7.7) -- BAL (n = 2) -- 1 (16.7) 1 (7.7) -- Urine (n = 2) 1 (33.3) -- 1 (7.7) -- Stool (n = 2) -- 2 (33.3) -- -- Wound (n = 2) -- -- 1 (7.7) 1 (50.0) Pus (n = 2) -- -- 2 (15.4) -- Others (n = 4) -- 1 (16.7) 3 (23.1) -- BAL: bronchoalveolar lavage. Table 2: Results of doripenem and amikacin interaction test. Classification A. baumannii P. aeruginosa K. pneumoniae DOR-S/AMK-S (n = 18) Synergy 1 0 0 Additive 5 5 2 Indifferent 0 1 4 Antagonist 0 0 0 DOR-S/AMK-R (n=14) Synergy 0 0 0 Additive 3 0 1 Indifferent 3 2 5 Antagonist 0 0 0 DOR-R/AMK-S (n=18) Synergy 0 0 0 Additive 1 1 4 Indifferent 5 5 2 Antagonist 0 0 0 DOR-R/AMK-R (n=18) Synergy 0 0 0 Additive 0 1 1 Indifferent 6 5 5 Antagonist 0 0 0 Classification Total DOR-S/AMK-S (n = 18) Synergy 1 Additive 12 Indifferent 5 Antagonist 0 DOR-S/AMK-R (n=14) Synergy 0 Additive 4 Indifferent 10 Antagonist 0 DOR-R/AMK-S (n=18) Synergy 0 Additive 6 Indifferent 12 Antagonist 0 DOR-R/AMK-R (n=18) Synergy 0 Additive 2 Indifferent 16 Antagonist 0 Table 3: Mean MIC changes after combining doripenem and amikacin. Bacteria [MIC.sub.DOR] MIC.sub.DOR+AMK] Mean difference DOR-S/AMK-S A. baumannii 0.37 0.13 0.24 P. aeruginosa 0.66 0.32 0.34 K. pneumoniae 0.29 0.09 0.20 DOR-S/AMK-R A. baumannii 4.08 2.33 1.75 P. aeruginosa 4.00 3.00 1.00 K. pneumoniae 0.09 0.06 0.03 DOR-R/AMK-S A. baumannii 32.00 29.33 2.67 P. aeruginosa 32.00 27.67 4.33 K. pneumoniae 26.67 9.12 17.50 DOR-R/AMK-R A. baumannii 32.00 32.00 0.00 P. aeruginosa 32.00 27.33 4.67 K. pneumoniae 23.33 21.33 2.00 Bacteria p value * [MIC.sub.AMK] DOR-S/AMK-S A. baumannii 0.001 2.33 P. aeruginosa 0.006 4.00 K. pneumoniae 0.027 ([conjunction]) 3.25 DOR-S/AMK-R A. baumannii 0.073 121.33 P. aeruginosa 0.157 ([conjunction]) 256.00 K. pneumoniae 0.033 217.33 DOR-R/AMK-S A. baumannii 0.363 3.58 P. aeruginosa 0.363 9.00 K. pneumoniae 0.001 2.50 DOR-R/AMK-R A. baumannii 0.317 ([conjunction]) 256.00 P. aeruginosa 0.180 141.33 K. pneumoniae 0.203 256.00 Bacteria [MIC.sub.AMK+DOR] Mean p value * difference DOR-S/AMK-S A. baumannii 0.88 1.45 0.008 P. aeruginosa 1.69 2.31 0.003 K. pneumoniae 2.42 0.83 0.041 ([conjunction]) DOR-S/AMK-R A. baumannii 92.67 28.67 0.221 P. aeruginosa 192.00 64.00 0.157 K. pneumoniae 162.67 54.66 0.035 DOR-R/AMK-S A. baumannii 2.75 0.83 0.129 P. aeruginosa 7.67 1.33 0.363 K. pneumoniae 1.50 1.00 0.018 DOR-R/AMK-R A. baumannii 256.00 0.00 0.109 ([conjunction]) P. aeruginosa 128.00 13.33 0.180 K. pneumoniae 220.33 26.67 0.363 * Paired t-test; ([conjunction]) Wilcoxon's signed-rank test. Figure 3: Results of the doripenem-amikacin interaction test based on the drug-resistant class. Synergy Additive Indifferent Non-MDR 1 2.4 2 MDR 0 5 9 XDR 0 6 8 PDR 0 2.8 4 Note: Table made from bar graph.