MICROBIAL DRUG RESISTANCE Volume 00, Number 00, 2019 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2018.0234

Activity of -Avibactam Alone and in Combination with , , and Tigecycline Against Carbapenemase-Producing

Dominika Ojdana, Anna Gutowska, Paweł Sacha, Piotr Majewski, Piotr Wieczorek, and Elzbieta_ Tryniszewska

The aim of this study was to investigate the synergy between ceftazidime-avibactam, ertapenem, fosfomycin, and tigecycline against carbapenemase-producing Klebsiella pneumoniae using the E test MIC:MIC (minimum inhibitory concentration) ratio synergy method. The results were interpreted using fractional inhibitory con- centration index (FICI) to describe the effects of antimicrobial combinations in vitro. To assess the clinical significance of each combination, the susceptible breakpoint index (SBPI) was calculated for each combination, and within each strain. The FICI method revealed that the most synergistic combinations against carbapenemase-producing K. pneumoniae were ceftazidime-avibactam with ertapenem and ceftazidime- avibactam with fosfomycin. This effect was demonstrated in 47% (9/19) of all tested clinical K. pneumoniae isolates. Considering the effects of all drug combinations in K. pneumoniae harboring blaKPC, blaNDM, and blaOXA-48 genes, we observed that the combination of ceftazidime-avibactam with fosfomycin was the most synergistic in New Delhi metallo-b-lactamase (NDM)-producing K. pneumoniae, and the combination of ceftazidime-avibactam with ertapenem was the most synergistic in K. pneumoniae carbapenemase (KPC)- producing K. pneumoniae. In addition, all tested combinations were synergistic against oxacillinase (OXA)-48- producing K. pneumoniae, except the combination of ceftazidime-avibactam with tigecycline. The SBPI index showed that ceftazidime-avibactam in combination with fosfomycin reduced the MIC to less than the sus- ceptibility breakpoint among all tested carbapenemase-producing K. pneumoniae. Moreover, the combinations of ceftazidime-avibactam with ertapenem, and ceftazidime-avibactam with tigecycline were able to reduce the MIC to less than the susceptibility breakpoint in all KPC- and OXA-48-producing K. pneumoniae.

Keywords: carbapenemase-producing K. pneumoniae, synergy, ceftazidime-avibactam

Downloaded by 151.82.108.227 from www.liebertpub.com at 08/03/19. For personal use only. Introduction avibactam demonstrates inhibitory properties against KPC and OXA-48 producers.6 The U.S. Food and Drug Administration .pneumoniae carbapenemase (KPC)-producing bac- (FDA) has approved ceftazidime-avibactam for the treatment K teria have been reported worldwide.1 The resistance of of intraabdominal infection, , and pneu- K. pneumoniae against is often caused by the monia.7,8 Unfortunately, recent data show that monotherapy production of various carbapenemases, mainly KPCs, New may result in the emergence of ceftazidime-avibactam resis- 9 Delhi metallo-b-lactamases (NDM), and OXA-48-type ox- tance among K. pneumoniae carrying blaKPC genes. acillinases (OXA-48).2 Carbapenemase-producing K. pneu- The lack of ceftazidime-avibactam inhibitory properties moniae often present simultaneous resistance to multiple against NDM-producing K. pneumoniae and reports about antimicrobials, and infections caused by these bacteria are dif- ceftazidime-avibactam resistance among KPC-producing ficult to be cured because of limited therapeutic options, thereby K. pneumoniae enhance the need for the evaluation of carbapenemase-producing K. pneumoniae have become a ceftazidime-avibactam activity in combination with other problem in health care.3,4 antimicrobials. Therefore, in this study, we evaluated the Ceftazidime-avibactam has been proven to be an ef- activity of ceftazidime-avibactam alone and in combina- fective medicine for the treatment of infections caused by tion with ertapenem, fosfomycin, and tigecycline against carbapenemase-producing Enterobacterales.5 Ceftazidime- carbapenemase-producing K. pneumoniae.

Department of Microbiological Diagnostics and Infectious Immunology, Medical University of Bialystok, Bialystok, Poland.

1 2 OJDANA ET AL.

The obtained results were interpreted with the use of combination reduces the MIC values below their suscepti- fractional inhibitory concentration (FIC) index to describe bility breakpoint.18 the effects of antimicrobial combinations in vitro. Moreover, Summary statistics, including mean, minimum, maxi- to assess clinical significance, the susceptible breakpoint mum, and median, for SFIC and SBPI values, for each index (SBPI) was calculated for each combination within antibiotic combination among NDM-, KPC-, and OXA-48- each strain. producing K. pneumoniae and according to antibiotic com- bination effects described by SFIC were calculated with STATA 13.1 (StataCorp LP). Moreover, the two-sample Materials and Methods Wilcoxon rank-sum (Mann–Whitney) test was used to as- The study was conducted on 19 non-duplicated K. sess the differences in SFIC and SBPI values between pneumoniae clinical strains. Tested strains were isolated NDM- and KPC-producing K. pneumoniae. p < 0.001 was during the period between 2012 and 2016 from various considered significant. materials obtained from patients hospitalized at the Uni- versity Hospital of Bialystok, Poland. Escherichia coli Results ATCC 25922, K. pneumoniae ATCC BAA-2146, K. pneu- moniae ATCC BAA-1705, and K. pneumoniae NCTC Biochemical identification, screening test for carbapene- 13442 were used as a quality control strain. mases, biochemical Carba NP test, and PCR tests allowed us Biochemical identification and screening tests for antimi- to choose the group of NDM-, KPC-, and OXA-48- crobial resistance among selected strains were prepared with producing K. pneumoniae strains for further assays. Tested the VITEK 2 automated system (bioMerieux, Marcy l’Etoile, K. pneumoniae strains were isolated from clinical materials France) according to the manufacturer’s instructions. originated from the digestive tract (21%), respiratory tract The screening detection of carbapenemases was per- (16%), urinary tract (37%), and blood (26%). formed according to European Committee on Antimicrobial In the present work, we investigated the synergy between Susceptibility Testing (EUCAST) guidelines.10 Moreover, ceftazidime-avibactam, ertapenem, fosfomycin, and tigecy- the biochemical Carba NP test was performed according to cline against carbapenemase-producing K. pneumoniae us- the Nordmann–Poirel protocol.11 ing the E test MIC:MIC ratio synergy method. Before The blaKPC, blaNDM, and blaOXA-48 genes were deter- synergy investigation, we determined MIC for each antibi- mined using a PCR technique as described previously.12,13 otic alone. The results of susceptibility testing showed that, Moreover, for the determination of genetic relationships among all tested K. pneumoniae strains, 53% (10/19) were among the different K. pneumoniae isolates, multilocus se- resistant to ceftazidime-avibactam, and 47% (9/19) were quence typing (MLST) was prepared according to Institut susceptible. Moreover, all tested strains, 100% (19/19), Pasteur’s MLST scheme.14 presented insusceptibility for ertapenem. In contrast to er- Determination tests of minimum inhibitory concen- tapenem, all tested strains, 100% (19/19), were susceptible trations (MICs) for each antibiotic were prepared on to tigecycline. Against fosfomycin, 53% (10/19) of the Mueller-Hinton agar (Oxoid, Basingstoke, United King- strains were resistant, and 47% (9/19) were susceptible. The dom) using MIC test strips: ceftazidime-avibactam, fos- summary of MIC values and the analysis of carbapenemase fomycin (Liofilchem, Roseto degli Abruzzi, TE, Italy); genes occurrence among the tested strains are presented in and E tests: ertapenem, tigecycline (bioMerieux) follow- Table 1. ing the manufacturer’s instructions, for all tested isolates, The E test MIC:MIC ratio synergy method revealed in two repetitions. Obtained results were interpreted in synergism among all tested combinations of ceftazidime- accordance with EUCAST clinical breakpoints (2018), avibactam with ertapenem, fosfomycin, and tigecycline. version 8.0.15 A detailed analysis of in vitro synergy results showed that Antibiotic synergy testing was performed with the E test the combination of ceftazidime-avibactam with ertapenem Downloaded by 151.82.108.227 from www.liebertpub.com at 08/03/19. For personal use only. MIC:MIC ratio method for four antimicrobial combinations, was synergistic against 47% (9/19) of all isolates. An ad- including ceftazidime-avibactam with ertapenem, ceftazidime- ditive effect for this combination was observed against 16% avibactam with fosfomycin, and ceftazidime-avibactam with (3/19) of isolates. This combination presented an indifferent tigecycline, for all tested isolates, in two repetitions. For each effect in 32% (6/19), and in the case of one isolate, antag- combination, and within each strain, FICs were calculated ac- onism was observed. A synergistic effect for ceftazidime- cording to the formula: FIC A = MIC antibiotic A in combi- avibactam combined with ertapenem was observed in 1/1 nation with antibiotic B/MIC antibiotic A alone; FIC B = MIC strain with blaOXA-48, and 100% (8/8) of strains with blaKPC. antibiotic B in combination with antibiotic A/MIC antibiotic B The second tested combination of ceftazidime-avibactam alone. The sum of both FICs (SFIC) for each combination, and with fosfomycin was synergistic for 47% (9/19) strains. within each strain (SFIC = FIC antibiotic A + FIC antibiotic B), Against 37% (7/19) of all strains, this combination was was used to describe the antibiotic interaction effects in vitro.16 additive, and for 16% (3/19), this was indifferent. A syner- Synergy was defined as SFIC £0.5, additivity SFIC >0.5–1, gistic effect for this combination was noticed among 50% 17 indifference SFIC >1–4, and antagonism SFIC >4. (5/10) of strains with blaNDM, 37.5% (3/8) of strains with To assess the clinical significance of each antibiotic blaKPC, and 1/1 strain with blaOXA-48 genes. An additive combination, SBPI was calculated for each strain, as fol- effect of this combination was observed against 30% (3/10) lows: (susceptible breakpoint for antibiotic A/MIC antibi- of isolates with blaNDM and 50% (4/8) strains with blaKPC otic A in combination with antibiotic B) + (susceptible genes. However, ceftazidime-avibactam combination with breakpoint for antibiotic B/MIC antibiotic B in combination fosfomycin was indifferent against 20% (2/10) of isolates with antibiotic A). SBPI values >2 mean that antimicrobial with blaNDM, and 12.5% (1/8) of strains with blaKPC genes. Downloaded by 151.82.108.227 from www.liebertpub.com at 08/03/19. For personal use only.

Table 1. Summary of Minimum Inhibitory Concentration Values and Summation of Fractional Inhibitory Concentrations Among Carbapenemase-Producing Klebsiella pneumoniae Isolates

Antimicrobial susceptibility (MIC, lg/mL)

Interpre- Interpre- Interpre- Interpre- Antimicrobial combination (RFIC index) tation tation tation tation Carbapenemase CAZ- S £ 8; S £ 0.5; S £ 32; S £ 1; CAZ- Interpre- CAZ- Interpre- CAZ- Interpre- genes MLST Strains AVI R < 8 ETP R < 1 FOS R < 32 TIG R < 2 AVI+ETP tation AVI+FOS tation AVI+TIG tation

blaNDM ST-11 52 256 R 8 R 32 S 0.5 S 0.5625 Additivity 0.5 Synergy 3 Indifferent blaNDM ST-11 51 256 R 6 R 64 R 0.75 S 1.125 Indifferent 0.3281 Synergy 2.354 Indifferent blaNDM ST-11 50 256 R 6 R 24 S 0.5 S 0.8125 Additivity 1.0416 Indifferent 2.75 Indifferent blaNDM ST-11 49 256 R 6 R 48 R 0.5 S 0.8568 Additivity 0.2187 Synergy 3.2968 Indifferent blaNDM ST-11 45 256 R 8 R 24 S 0.75 S 1.125 Indifferent 0.8125 Additivity 2.1666 Indifferent blaNDM ST-11 41 256 R 6 R 32 S 0.5 S 1.3541 Indifferent 0.5312 Additivity 2.75 Indifferent 3 blaNDM ST-11 48 256 R 6 R 48 R 0.75 S 1.6666 Indifferent 0.2708 Synergy 2.3333 Indifferent blaNDM ST-11 39 256 R 8 R 48 R 0.75 S 1.625 Indifferent 0.3646 Synergy 1.9166 Indifferent blaNDM ST-11 46 256 R 6 R 48 R 0.75 S 3.5208 Indifferent 0.802 Additivity 2.3333 Indifferent blaNDM ST-11 44 256 R 8 R 48 R 0.75 S 5.6666 Antagonism 1.1667 Indifferent 2.6665 Indifferent blaKPC ST-258 29 1.5 S 1.5 R 16 S 0.5 S 0.0259 Synergy 0.7321 Additivity 1.065 Indifferent blaKPC ST-11 42 3 S 32 R 1,024 R 0.75 S 0.086 Synergy 0.5603 Additivity 0.374 Synergy blaKPC ST-258 35 1.5 S 8 R 64 R 0.75 S 0.0175 Synergy 0.6181 Additivity 1.158 Indifferent blaKPC ST-258 38 1 S 1.5 R 24 S 0.5 S 0.0341 Synergy 0.845 Additivity 1.625 Indifferent blaKPC ST-258 32 0.75 S 1 I 12 S 0.5 S 0.0353 Synergy 1.2533 Indifferent 2 Indifferent blaKPC ST-258 27 1.5 S 4 R 1,024 R 0.5 S 0.0258 Synergy 0.0524 Synergy 3.3333 Indifferent blaKPC ST-258 31 0.75 S 1 I 32 S 1 S 0.0388 Synergy 0.2459 Synergy 1.875 Indifferent blaKPC ST-258 28 1.5 S 6 R 32 S 0.75 S 0.0179 Synergy 0.4487 Synergy 1.8333 Indifferent blaOXA-48 ST-15 43 1 S 2 R 64 R 0.25 S 0.0612 Synergy 0.2353 Synergy 1.89 Indifferent CAZ-AVI, ceftazidime-avibactam; ETP, ertapenem; SFIC, summation of fractional inhibitory concentrations; FOS, fosfomycin; I, intermediate; KPC, K. pneumoniae carbapenemase; MIC, minimum inhibitory concentration; MLST, multilocus sequence typing; NDM, New Delhi metallo-b-lactamase; OXA-48, oxacillinase OXA-48; R, resistant; S, susceptible; ST, sequence type; TIG, tigecycline. 4 OJDANA ET AL.

In addition, a synergy test showed that ceftazidime- ceftazidime-avibactam resistance among KPC-producing avibactam combined with tigecycline was synergistic for isolates, mediated by additional production of NDM-1, or 5% (1/19) of isolates. Moreover, for 95% (18/19) of the VIM-4, and decreased expression of ompK36 among these tested strains, the combination of ceftazidime-avibactam isolates.22 Nonsusceptibility to ceftazidime-avibactam against with tigecycline was indifferent. A synergistic effect of NDM producers was also presented by Sader et al., who ceftazidime-avibactam with tigecycline was observed in showed that only 23 among 36,380 Enterobacteriaceae 23 12.5% (1/8) of strains harboring blaKPC, while 87.5% (7/8) isolates were resistant, and they carried blaNDM genes. of isolates with blaKPC presented an indifferent effect for Despite previous reports of ceftazidime-avibactam activity this combination. Also, an indifferent effect for this com- as an effective medicine for the treatment of infections caused bination was observed among all strains with blaNDM and by carbapenemase-producing Enterobacteriaceae, there are blaOXA-48 genes. recent data, described by Shields et al., regarding the possi- 24 Statistical analysis of SFIC values between NDM- and bility of resistance due to blaKPC-3 mutations. These authors KPC-producing K. pneumoniae strains revealed significant reported the first three cases of ceftazidime-avibactam resis- differences ( p < 0.001) for ceftazidime-avibactam combina- tance among KPC-producing K. pneumoniae during mono- tion with ertapenem and tigecycline, but not for ceftazidime- therapy.9 Moreover, Haidar et al. expect that, with an avibactam and fosfomycin combination. increased use of ceftazidime-avibactam, this resistance will Summary statistics, including range, mean, and median, continue to emerge by plasmid-carrying mutant genes and for SFIC values for each antibiotic combination among may be disseminated by horizontal gene transfer.25 NDM-, KPC-, and OXA-48-producing K. pneumoniae, and In light of new reports about the emergence of ceftazidime- according to antibiotic combination effects described by avibactam resistance, and the lack of ceftazidime-avibactam SFIC, are presented in Table 2. inhibitory properties against NDM-producing K. pneumoniae, On the contrary, to assess the clinical significance of anti- we decided to investigate the interactions of ceftazidime- biotic combinations, SBPI was calculated for each combina- avibactam in combination with other , including tion within each strain, and the synergy was noticed when ertapenem, fosfomycin, and tigecycline. SBPI >2. We observed SBPI values >2 among all KPC- and Because the problem of multidrug-resistant carbapenemase- OXA-48-producing K. pneumoniae in all tested combinations producing K. pneumoniae is becoming more and more common, of antibiotics. Whereas among NDM-producing K. pneumo- we see the need to evaluate the synergy of antibiotics against niae, SBPI values were >2 for ceftazidime-avibactam such strains in everyday routine work of a laboratory. We combination with fosfomycin, which indicates a possible evaluated ceftazidime-avibactam activity in combination synergistic effect in vivo. However, SBPI values <2 were with other antimicrobials against carbapenemase-producing observed in combinations of ceftazidime-avibactam with er- K. pneumoniae with the E test MIC:MIC ratio method. tapenem and ceftazidime-avibactam with tigecycline among Other E test synergy methods have been described, such as NDM-producing K. pneumoniae. Additionally, combinations direct overlay or 90C angle methods.26 According to of ceftazidime-avibactam with ertapenem, ceftazidime- Pankey et al., results obtained with the E test MIC:MIC ratio avibactam with tigecycline, and ceftazidime-avibactam with method had the highest agreement (80.6%) with time-kill fosfomycin showed higher SBPI values among KPC- assayresults,incomparisontootherE test methods.27 producing K. pneumoniae isolates in comparison with NDM- Moreover, Pankey et al. also observed an 83% agreement producing isolates, and these differences were statistically between the E test MIC:MIC ratio method and the time-kill significant ( p < 0.001). A summary of obtained results, in- assay for levofloxacin- combination against cluding mean, minimum, maximum, and median, for SBPI Stenotrophomonas maltophilia.28 values for each antibiotic combination among NDM-, KPC-, Considering the in vitro effectiveness of three ceftazidime- and OXA-48-producing K. pneumoniae, and according to avibactam combinations with ertapenem, fosfomycin, and ti- antibiotic combination effects described by SFIC, are de- gecycline against carbapenemase-producing K. pneumoniae, Downloaded by 151.82.108.227 from www.liebertpub.com at 08/03/19. For personal use only. scribed in Table 3. we calculated SFIC. The results showed ceftazidime- avibactam with ertapenem to be a synergistic combination in 47% (9/19) of all tested K. pneumoniae isolates. In addition, we Discussion noticed the combination of ceftazidime-avibactam with erta- All K. pneumoniae isolates tested in this study produced to be the most synergistic in KPC-producing K. pneu- various types of carbapenemase enzymes, including NDM, moniae. These results are in accordance with the synergistic KPC, and OXA-48. When investigating ceftazidime- activity of ceftazidime-avibactam with ertapenem as described avibactam in vitro activity against these isolates, we noticed by Gaibani et al. among all (n = 13) K. pneumoniae-carrying 29 that all KPC and OXA-48 producers were fully susceptible, blaKPC genes. Moreover, in our study, the synergistic effect of while NDM-producing K. pneumoniae showed resistance. ceftazidime-avibactam and fosfomycin combination was In vitro activity of ceftazidime-avibactam against Ambler demonstrated in 47% (9/19) of strains. We observed that this class A- and class C-producing Enterobacteriaceae was also combination was the most synergistic mainly in NDM- described by Bonnefoy et al. and Berkhout et al.19,20 producing K. pneumoniae, but also in 37% of tested KPC- Moreover, according to Aktasx et al., ceftazidime-avibactam producing K. pneumoniae. The synergistic effect against presented a promising therapeutic strategy to treat infections metallo-b-lactamase-producing Enterobacteriaceae was re- due to K. pneumoniae with OXA-48 enzymes.21 Castanheira vealed by Wenzler et al. for ceftazidime-avibactam and az- et al. evaluated ceftazidime-avibactam activity against 456 treonam combination.30 In addition, in our study, the Enterobacteriaceae isolates carrying blaKPC at a level as synergistic effect of ceftazidime-avibactam with tigecycline high as 99.3%. In addition, they revealed a low frequency of was observed in 12.5% (1/8) of strains harboring blaKPC genes. Downloaded by 151.82.108.227 from www.liebertpub.com at 08/03/19. For personal use only.

Table 2. Summary of Summation of Fractional Inhibitory Concentrations Among Carbapenemase-Producing Klebsiella pneumoniae Isolates

Antimicrobial combination (RFIC index) CAZ-AVI+ETP CAZ-AVI+FOS CAZ-AVI+TIG Strains Range Mean Median Range Mean Median Range Mean Median According to the presence of carbapenemase genes NDM 0.5625–5.6666 1.8315 1.2395 0.2187–1.1667 0.6036 0.5156 1.9166–3.2968 2.5567 2.5102 KPC 0.0175–0.086 0.0352 0.03 0.0524–1.2533 0.5945 0.5892 0.374–3.3333 1.6579 1.7291 OXA-48 0.0612 0.0612 0.0612 0.2353 0.2353 0.2353 1.89 1.89 1.89 According to SFIC among all tested isolates S 0.0175–0.086 0.038 0.0341 0.0524–0.5 0.296 0.2708 0.374 0.374 0.374 5 A 0.5625–0.8568 0.7454 0.8125 0.5312–0.845 0.7002 0.7321 — — — I 1.125–3.5208 1.7361 1.4895 1.0416–1.2533 1.1538 1.1667 1.065–3.3333 2.2415 2.2499 AN 5.6666 5.6666 5.6666 — — — — — — According to SFIC among NDM-producing K. pneumoniae S — — — 0.2187–0.5 0.3364 0.3281 — — — A 0.5625–0.8568 0.7454 0.8125 0.5312–0.8125 0.7152 0.802 — — — I 1.125–3.5208 1.7361 1.4895 1.0416–1.1667 1.1041 1.1041 1.9166–3.2968 2.5567 2.5102 AN 5.6666 5.6666 5.6666 — — — — — — According to SFIC among KPC-producing K. pneumoniae S 0.0175–0.086 0.0352 0.03 0.0524–0.4487 0.249 0.2459 0.374 0.374 0.374 A — — — 0.5603–0.845 0.6888 0.6751 — — — I — — — 1.2533 1.2533 1.2533 1.065–3.3333 1.8414 1.8333 According to SFIC among OXA-48-producing K. pneumoniae S 0.0612 0.0612 0.0612 0.2353 0.2353 0.2353 — — — I — — — — — — 1.89 1.89 1.89

A, additivity; AN, antagonism; I, indifferent; S, synergy. Downloaded by 151.82.108.227 from www.liebertpub.com at 08/03/19. For personal use only.

Table 3. Summary of Susceptible Breakpoint Index Among Carbapenemase-Producing Klebsiella pneumoniae Isolates Susceptible breakpoint index CAZ-AVI+ETP CAZ-AVI+FOS CAZ-AVI+TIG Strains Range Mean Median Range Mean Median Range Mean Median According to the presence of NDM 0.0469–0.6562 0.2718 0.2447 2.5468–8.3333 4.7395 4.7812 0.7174–5.0156 1.4679 1.1484 carbapenemase genes KPC 130–536.4583 440.2899 483.6674 21.1629–237.913 80.9113 37.5775 5–62.129 18.1238 12.0833 OXA-48 332.8226 332.8226 332.8226 1,293 1,293 1,293 14.614 14.614 14.614 According to S;FIC among all S 130–536.4583 428.3491 452.5589 3.9791–1,293 189.7567 8.3333 62.129 62.129 62.129

6 tested isolates A 0.2916–0.6562 0.4756 0.4791 2.7708–146.4016 36.2023 22.5526 — — — I 0.1328–0.278 0.2074 0.207 2.5468–21.1629 8.8199 2.75 0.7174–24.1646 6.2308 3.1822 AN 0.0469 0.0469 0.0469 — — — — — According to SFIC among S — — — 3.9791–8.3333 5.8916 4.8125 — — — NDM-producing K. pneumoniae A 0.2916–0.4791 0.4756 0.4791 2.7708–5.0989 4.7395 4.7812 — — — I 0.1328–0.2708 0.2074 0.207 2.5468–2.75 2.6484 2.6484 0.7174–5.0156 1.4679 1.1484 AN 0.0469 0.0469 0.0469 — — — — — — According to SFIC among S 130–536.4583 440.2899 483.6674 34.3859–237.913 128.4507 113.0532 62.129 62.129 62.129 KPC-producing K. pneumoniae A — — — 22.5526–146.4016 60.194 35.9109 — — — I — — — 21.1629 21.1629 21.1629 5–24.1646 11.8374 11.833 According to SFIC among S 332.8226 332.8226 332.8226 1,293 1,293 1,293 — — — OXA-48-producing K. pneumoniae I — — — — — — 14.614 14.614 14.614

I, indifferent; S, synergy. CEFTAZIDIME-AVIBACTAM ACTIVITY ON K. PNEUMONIAE 7

These results are in agreement with the 8% (1/13) synergistic nosocomial dominance. Antimicrob. Agents Chemother. 59: interaction of ceftazidime-avibactam with tigecycline as ob- 5873–5884. served by Gaibani et al.29 3. Morrill, H.J., J.M. Pogue, K.S. Kaye, and K.L. LaPlante. It should be emphasized that synergistic activities ob- 2015. Treatment options for -resistant En- served in in vitro conditions may not exactly correlate with terobacteriaceae infections. Open Forum Infect. Dis. 2:1–15. clinical efficacy. The reason for this can be connected with 4. Meletis, G. 2016. Carbapenem resistance: overview of the the different pharmacokinetic properties of antibiotics. problem and future perspectives. Ther. Adv. Infect. Dis. 3: In vitro discordance with in vivo activity was described by 15–21. Monogue et al. on ceftazidime-avibactam alone and in 5. Castanheira, M., S.E. Farrell, K.M. Krause, R.N. Jones, and combination with and tigecycline.31 They noticed H.S. Sader. 2014. Contemporary diversity of b-lactamases Enterobacteriaceae the synergistic effect in vivo of ceftazidime-avibactam with among in the nine U.S. census regions and ceftazidime-avibactam activity tested against isolates aztreonam against NDM-producing K. pneumoniae, while producing the most prevalent b-lactamase groups. Anti- no synergy was observed in vivo. This effect may have been microb. Agents Chemother. 58:833–838. caused by too low bacterial reduction by humanized doses 6. Zasowski, E.J., J.M. Rybak, and M.J. Rybak. 2015. The b- of ceftazidime-avibactam in vivo. lactams strike back: ceftazidime-avibactam. Pharma- To establish the clinical efficacy of the revealed in vitro cotherapy 35:755–770. synergistic effects in our study, we used SBPI. According to 7. Sharma, R., T.E. Park, and S. Moy. 2016. Ceftazidime- Milne and Gould, this parameter evaluates the value of in- avibactam: a novel /b-lactamase inhibitor teraction to pharmacodynamic breakpoints using determi- combination for the treatment of resistant gram-negative nation susceptibility in vivo.18 organisms. Clin. Ther. 38:431–444. In our study, by SBPI values, combinations of ceftazidime- 8. Avycaz (ceftazidime-avibactam) for injections (2.5g). avibactam with ertapenem, and ceftazidime-avibactam with 2018. Indication and usage. Available at www.avycaz.com/ tigecycline, were able to reduce MIC to less than the sus- #indications. ceptibility breakpoint in all KPC- and OXA-48-producing K. 9. Shields, R.K., L. Chen, S. Cheng, K.D. Chavda, E.G. Press, pneumoniae. However, ceftazidime-avibactam in combina- A. Snyder, R. Pandey, Y. Doi, B.N. Kreiswirth, M.H. tion with fosfomycin reduced MIC to less than the suscepti- Nguyen, and C.J. Clancya. 2017. Emergence of ceftazidime- bility breakpoint among all tested carbapenemase-producing avibactam resistance due to plasmid-borne blaKPC-3 mu- K. pneumoniae. The observed synergy can present clinical tations during treatment of carbapenem-resistant Klebsiella significance in vivo. pneumoniae infections. Antimicrob. Agents Chemother. 61: This study presents results regarding in vitro enhanced ac- e02097-16. tivity for the combination of ceftazidime-avibactam with erta- 10. The European Committee on Antimicrobial Susceptibility penem, and fosfomycin, in comparison to this antibiotic alone, Testing. 2017. The EUCAST Guideline on Detection of Resistance Mechanisms Version 2.0. Available at www against carbapenemase-producing K. pneumoniae. While there .eucast.org. are limited options for the treatment of carbapenemase- 11. Nordmann, P., L. Poirel, and L. Dortet. 2012. Rapid de- producing K. pneumoniae, the results described here can be tection of carbapenemase-producing Enterobacteriaceae. considered promising. However, a limitation of our study is that Emerg. Infect. Dis. 18:1503–1507. a more sensitive mathematical model could have been used for 12. Ojdana, D., P. Sacha, D. Olszan´ska, P. Majewski, calculating the effect of antibiotic combinations. In the future, P. Wieczorek, J. Jaworowska, A. Sien´ko, A. Jurczak, and E. we plan to expand our research by testing on a larger number of Tryniszewska. 2015. First report of Klebsiella pneumoniae- isolates and subjecting the obtained results to other quantitative carbapenemase-3-producing Escherichia coli ST479 in methods, for example, Greco model or isobolographic analysis, Poland. Biomed. Res. Int. 2015:1–3. for assessing drug synergism.32 Moreover, further clinical trials 13. Rasheed, J.K., B. Kitchel, W. Zhu, K.F. Anderson, N.C. are necessary for the evaluation of safety and efficacy in the Clark, M.J. Ferraro, P. Savard, R.M. Humphries, A.J. Downloaded by 151.82.108.227 from www.liebertpub.com at 08/03/19. For personal use only. treatment of infections due to carbapenemase-producing K. Kallen, and B.M. Limbago. 2013. New Delhi metallo-b- pneumoniae. lactamase-producing Enterobacteriaceae, United States. Emerg. Infect. Dis. 19: 870–878. Acknowledgments 14. Diancourt,L.,V.Passet,J.Verhoef, P.A. Grimont,andS.Brisse. 2005. Multilocus sequence typing of Klebsiella pneumoniae We thank Steven J. Snodgrass for editorial assistance. nosocomial isolates. J. Clin. Microbiol. 43:4178–4182. This work was funded by the Medical University of Bia- 15. The European Committee on Antimicrobial Susceptibility lystok, Poland. Testing. 2018. Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 8.0, 2018. Available at Disclosure Statement www.eucast.org No competing financial interests exist. 16. The European Committee of Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical References Microbiology and Infection Diseases (ESCMID). 2000. Terminology relating to methods for the determination of 1. Nordmann, P., T. Naas, and L. Poirel. 2011. Global spread susceptibility of bacteria to antimicrobial agents. Clin. of carbapenemase-producing Enterobacteriaceae. Emerg. Microbiol. Infect. 6:503–508. Infect. Dis. 17:1791–1798. 17. Doern, C.D. 2014. When does 2 plus 2 equal 5? A review 2. Pitout, J.D., P. Nordmann, and L. Poirel. 2015. Carbapenemase- of antimicrobial synergy testing. J. Clin. Microbiol. 52: producing Klebsiella pneumoniae, a key pathogen set for global 4124–4128. 8 OJDANA ET AL.

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