Abstract
The reevaluation of “forgotten” antibiotics can identify new therapeutic options against extensively drug-resistant Gram-negative pathogens. We sought to investigate isepamicin in this regard. We retrospectively evaluated the antimicrobial susceptibility to isepamicin of Enterobacteriaceae sp. isolates from unique patients, collected at the microbiological laboratory of the University Hospital of Heraklion, Crete, Greece, from 2004 to 2009. Susceptibility testing was done with the automated Vitek 2 system. The breakpoints for susceptibility to isepamicin, tigecycline, and other antibiotics were those proposed by the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM), the FDA, and the CLSI, respectively. A total of 6,296 isolates were studied, including primarily 3,401 (54.0%) Escherichia coli, 1,040 (16.5%) Klebsiella pneumoniae, 590 (9.4%) Proteus mirabilis, and 460 (7.3%) Enterobacter sp. isolates. Excluding the species with intrinsic resistance to each antibiotic, antimicrobial susceptibility was highest for colistin (5,275/5,441 isolates [96.9%]) and isepamicin (6,103/6,296 [96.9%]), followed by meropenem (5,890/6,296 [93.6%]), imipenem (5,874/6,296 [93.3%]), and amikacin (5,492/6,296 [87.2%]). The antimicrobial susceptibility of the 1,040 K. pneumoniae isolates was highest for isepamicin (95.3%), followed by colistin (89.3%) and meropenem (63.0%). Regarding resistant K. pneumoniae isolates, susceptibility to isepamicin was observed for 91.1% of the 392, 87.7% of the 375, and 85.6% of the 111 isolates that were nonsusceptible to the carbapenems, all other aminoglycosides, and colistin, respectively. Isepamicin exhibited high in vitro activity against almost all of the Enterobacteriaceae species. It could particularly serve as a last-resort therapeutic option for carbapenem-resistant K. pneumoniae in our region, where it is endemic, as it does not show considerable cross-resistance with other aminoglycosides.
INTRODUCTION
In the era of increasing antimicrobial resistance, multidrug-resistant Enterobacteriaceae, particularly Klebsiella pneumoniae, Escherichia coli, and Enterobacter spp. that produce carbapenemases, extended-spectrum β-lactamases, or AmpC β-lactamases, are of major concern worldwide (4). Carbapenem-resistant K. pneumoniae isolates are typically resistant to all antibiotics except for colistin, tigecycline, gentamicin, and fosfomycin, but resistance to these agents, particularly to colistin, is increasing (16).
Although several antimicrobial agents targeting Gram-positive cocci have rather recently been approved for clinical use, this is not the case for Gram-negative bacilli (2, 23). The reevaluation of older but “forgotten” antimicrobials can lead to the identification of agents that have retained activity against contemporary problem pathogens, according to the example of colistin and fosfomycin. These agents could then serve as a last-resort treatment for infections with extensively resistant Gram-negative pathogens (14, 15).
Members of the aminoglycoside class of antibiotics can have antimicrobial activity against certain multidrug-resistant Gram-negative pathogens (9). Isepamicin, a derivative of gentamicin B, is one of the latest aminoglycosides to have been introduced in clinical practice (i.e., in 1988 in Japan) (31). It has been available in only a limited number of countries (including Japan, South Korea, China, and other Asian countries as well as certain European countries like Belgium and Italy) (27, 38). The susceptibility of Gram-negative bacterial isolates to isepamicin can be higher than that to amikacin (12). In a selection of 65 of carbapenem-resistant Enterobacteriaceae strains, other than NDM-1 producers, 63 were found to be susceptible to isepamicin (27).
We sought to evaluate the in vitro activity of isepamicin against contemporary Enterobacteriaceae clinical isolates in Crete, Greece, a region with a generally high prevalence of antimicrobial resistance among Gram-negative pathogens.
MATERIALS AND METHODS
Study design and setting.
We retrospectively analyzed all the clinical Enterobacteriaceae sp. isolates that were collected at the microbiological laboratory of the University Hospital of Heraklion (Heraklion, Crete, Greece) from February 2004 to September 2009 and for which data for susceptibility to isepamicin were available. When multiple isolates of the same species were identified in the same patient more than once within 6 months, we included only the first isolate per patient. The University Hospital of Heraklion is a 680-bed, tertiary care, general hospital serving a population of around 650,000 persons.
Specimen processing and culture were done according to routine laboratory methods. The identification of bacterial species was performed by the use of standard biochemical methods, the API 20E system and the Vitek 2 automated system (bioMérieux SA, Marcy l'Etoile, France). The Vitek 2 system was also used for antimicrobial susceptibility testing. The Clinical and Laboratory Standards Institute (CLSI) breakpoints (6a) were used for the interpretation of susceptibility to all antimicrobial agents except for isepamicin and tigecycline. For isepamicin, the breakpoints proposed in 2010 by the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM) were used: susceptibility was defined by an MIC less than or equal to 8 mg/liter, and resistance was defined by an MIC higher than 16 mg/liter (3). For tigecycline, the U.S. Food and Drug Administration (FDA) guidelines for Enterobacteriaceae were used. The study isolates were not routinely tested against chloramphenicol, fosfomycin, and tigecycline, because these antibiotics were either not available or not considered for clinical use for most of the study period.
E. coli ATCC 25922 was tested as a quality control strain for susceptibility to isepamicin with the Vitek 2 system once weekly throughout the study period. To evaluate the accuracy of isepamicin susceptibility testing with the Vitek 2 system, 10 isepamicin-susceptible and 10 isepamicin-resistant, randomly selected isolates were retested by using the disk diffusion method (3). Thirty-microgram isepamicin disks (Bio-Rad SA, Marnes La Coquette, France) were used, and the disk zone diameters were interpreted according to CA-SFM criteria (3).
Definitions and data analysis.
We assessed the percentages of susceptible, intermediately susceptible, and resistant isolates for each of the major Enterobacteriaceae sp. isolates studied and against all isolates. We did not take into consideration the automated susceptibility testing data for antibiotics against species that are considered to have intrinsic resistance, according to the European Committee on Antimicrobial Susceptibility Testing (26).
We specifically focused on subsets of isolates exhibiting clinically relevant resistance patterns, such as resistance to carbapenems, aminoglycosides, or colistin, and isolates with an extensively drug-resistant (XDR) or pandrug-resistant (PDR) phenotype. XDR isolates were those that were resistant to all but one or two classes of antimicrobial agents among those available during the study period, while PDR isolates were those isolates that were resistant to all classes of antimicrobial agents (11). To adjust for the fact that susceptibility to fosfomycin and tigecycline was not routinely tested, the XDR and PDR isolates that were characterized according to the definitions mentioned above are referred to as possible XDR (p-XDR) and possible PDR (p-PDR) isolates, respectively (28). Differences in susceptibility were compared by using the chi-square test.
RESULTS
Characteristics of the studied isolates.
A total of 12,796 clinical Enterobacteriaceae sp. isolates were collected at the microbiological laboratory of the University Hospital of Heraklion during the 6-year study period. For 7,589 (59.3%) unselected isolates, data for susceptibility to isepamicin were available. After the exclusion of multiple isolates of the same species per patient, 6,296 clinical isolates were finally included in our study. The most common pathogens were E. coli (3,401 isolates [54%]), followed by K. pneumoniae (1,040 [16.5%]), Proteus mirabilis (590 [9.4%]), and Enterobacter spp. (460 [7.3%]). Further data for the frequency of different species among the included Enterobacteriaceae isolates are provided in Table 1.
Table 1.
Main species of the 6,296 Enterobacteriaceae isolates studied
| Organism | No. of isolates (%) |
|---|---|
| E. coli | 3,401 (54.0) |
| K. pneumoniae | 1,040 (16.5) |
| P. mirabilis | 590 (9.4) |
| Enterobacter spp. | 460 (7.3) |
| Citrobacter spp. | 197 (3.1) |
| K. oxytoca | 156 (2.5) |
| Salmonella sp. | 136 (2.2) |
| S. marcescens | 132 (2.1) |
| M. morganii | 62 (1.0) |
| Proteus vulgaris group | 54 (0.9) |
| Providencia spp. | 17 (0.3) |
| Othersa | 51 (0.8) |
Including other Serratia spp. (n = 30), Yersinia enterocolitica (n = 8), Hafnia alvei (n = 5), other Escherichia spp. (n = 4), Leclercia adecarboxylata (n = 1), Pantoea dispersa (n = 1), Shigella flexneri (n = 1), and Klebsiella spp. (n = 1).
Almost two-thirds of the patients who provided the clinical isolates analyzed in this study were inpatients (4,211/6,296 patients [66.9%]), and the remaining one-third were outpatients. Of the inpatients, 1,308 (31%) patients were hospitalized in medical departments, 747 (18%) patients were hospitalized in hematology or oncology departments, 1,045 (25%) patients were hospitalized in surgical departments, 547 (13%) patients were hospitalized in pediatric departments, 494 (12%) patients were hospitalized in intensive care units (ICU), and the remaining 70 (2%) patients were hospitalized in renal replacement units. The types of culture specimens from which the studied pathogens were most frequently isolated included urine (3,608 specimens [57.3%]), abscess (866 [13.8%]), lower respiratory tract specimens (339 [5.4%]), blood (336 [5.3%]), genital specimens (289 [4.6%]), normally sterile fluids (286 [4.5%]), feces (144 [2.3%]), intravenous catheter tips (126 [2.0%]), and other specimens (302 [4.8%]).
Antimicrobial susceptibility.
Detailed data on the susceptibility of the studied Enterobacteriaceae sp. isolates to all tested antibiotics are presented in Table 2. Among all the isolates studied (excluding for each antibiotic those with intrinsic resistance), antimicrobial susceptibility was highest for colistin (5,275/5,441 [96.9%] isolates were susceptible) and isepamicin (6,103/6,296 [96.9%]), followed by meropenem (5,890/6,296 [93.6%]), imipenem (5,874/6,296 [93.3%]), and amikacin (5,492/6,296 [87.2%]). No isolates with intermediate-level resistance to isepamicin were noted. Excluding the 136 Salmonella sp. isolates, 6,090 (98.9%) of the 6,160 remaining Enterobacteriaceae sp. isolates were susceptible to isepamicin.
Table 2.
Antimicrobial susceptibilities of the evaluated isolates
| Antibiotic | No. (%) of susceptible isolatesa |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| E. coli (n = 3,401) | K. pneumoniae (n = 1,040) | P. mirabilis (n = 590) | Enterobacter spp. (n = 460) | Citrobacter spp. (n = 197) | K. oxytoca (n = 156) | S. marcescens (n = 132) | Salmonella sp. (n = 136) | M. morganii (n = 62) | All isolates except intrinsically resistantb | All isolates (n = 6,296)c | |
| Ampicillin | 1,920 (56.5) | IR | 304 (51.5) | IR | IR | IR | IR | 123 (90.4) | IR | 2,351/4,198 (56.0)b | 2,351 (37.3)c |
| Amoxicillin-clavulanate | 2,792 (82.1) | 502 (48.3) | 444 (75.3) | IR | IR | 138 (88.5) | IR | 134 (98.5) | IR | 4,156/5,542 (75.0)b | 4,156 (66.0)c |
| Ticarcillin-clavulanate | 2,897 (85.2) | 499 (48.0) | 509 (86.3) | 338 (73.5) | 163 (82.7) | 138 (88.5) | 125 (94.7) | 131 (96.3) | 48 (77.4) | 4,968/6,296 (78.9) | 4,968 (78.9)c |
| Piperacillin-tazobactam | 3,212 (94.4) | 512 (49.2) | 527 (89.3) | 353 (76.7) | 168 (85.3) | 140 (89.7) | 126 (95.5) | 135 (99.3) | 53 (85.5) | 5,346/6,296 (84.9) | 5,346 (84.9)c |
| Cephalothin | 2,177 (64.0) | 473 (45.5) | 391 (66.3) | IR | IR | 136 (87.2) | IR | 12 (8.8) | IR | 3,280/5,488 (59.8)b | 3,280 (52.1)c |
| Cefoxitin | 3,145 (92.5) | 493 (47.4) | 509 (86.3) | IR | IR | 147 (94.2) | 30 (22.7) | 101 (74.3) | 14 (22.6) | 4,616/5,753 (80.2)b | 4,616 (73.3)c |
| Cefuroxime | 3,051 (89.7) | 482 (46.3) | 512 (86.8) | 153 (33.3) | 139 (70.6) | 138 (88.5) | IR | 12 (8.8) | IR | 4,514/6,048 (74.6)b | 4,514 (71.7)c |
| Cefotaxime | 3,235 (95.1) | 516 (49.6) | 543 (92.0) | 352 (76.5) | 166 (84.3) | 148 (94.9) | 127 (96.2) | 134 (98.5) | 50 (80.6) | 5,392/6,296 (85.6) | 5,392 (85.6) |
| Ceftriaxone | 3,232 (95.0) | 516 (49.6) | 536 (90.8) | 351 (76.3) | 165 (83.8) | 140 (89.7) | 127 (96.2) | 134 (98.5) | 51 (82.3) | 5,373/6,296 (85.3) | 5,373 (85.3) |
| Ceftazidime | 3,225 (94.8) | 514 (49.4) | 525 (89.0) | 351 (76.3) | 166 (84.3) | 148 (94.9) | 126 (95.5) | 134 (98.5) | 49 (79.0) | 5,358/6,296 (85.1) | 5,358 (85.1) |
| Cefepime | 3,237 (95.2) | 521 (50.1) | 548 (92.9) | 364 (79.1) | 168 (85.3) | 149 (95.5) | 127 (96.2) | 134 (98.5) | 53 (85.5) | 5,422/6,296 (86.1) | 5,422 (86.1) |
| Aztreonam | 3,235 (95.1) | 516 (49.6) | 549 (93.1) | 353 (76.7) | 165 (83.8) | 140 (89.7) | 127 (96.2) | 134 (98.5) | 51 (82.3) | 5,391/6,296 (85.6) | 5,391 (85.6) |
| Imipenem | 3,394 (99.8) | 648 (62.3) | 580 (98.3) | 450 (97.8) | 197 (100) | 155 (99.4) | 131 (99.2) | 136 (100) | 61 (98.4) | 5,874/6,296 (93.3) | 5,874 (93.3) |
| Meropenem | 3,395 (99.8) | 655 (63.0) | 586 (99.3) | 449 (97.6) | 197 (100) | 156 (100) | 132 (100) | 136 (100) | 62 (100) | 5,890/6,296 (93.6) | 5,890 (93.6) |
| Tobramycin | 3,212 (94.4) | 579 (55.7) | 510 (86.4) | 426 (92.6) | 188 (95.4) | 151 (96.8) | 91 (68.9) | 12 (8.8) | 53 (85.5) | 5,321/6,279 (84.7)b | 5,321 (84.5)c |
| Amikacin | 3,291 (96.8) | 609 (58.6) | 536 (90.8) | 435 (94.6) | 189 (95.9) | 153 (98.1) | 95 (72.0) | 12 (8.8) | 57 (91.9) | 5,492/6,296 (87.2) | 5,492 (87.2) |
| Gentamicin | 3,209 (94.4) | 651 (62.6) | 513 (86.9) | 437 (95.0) | 190 (96.4) | 152 (97.4) | 125 (94.7) | 12 (8.8) | 53 (85.5) | 5,444/6,279 (86.7)b | 5,444 (86.5)c |
| Netilmicin | 3,229 (94.9) | 578 (55.6) | 519 (88.0) | 428 (93.0) | 188 (95.4) | 152 (97.4) | 94 (71.2) | 12 (8.8) | 55 (88.7) | 5,354/6,279 (85.3)b | 5,354 (85.0)c |
| Isepamicin | 3,397 (99.9) | 991 (95.3) | 588 (99.7) | 455 (98.9) | 195 (99.0) | 154 (98.7) | 130 (98.5) | 13 (9.6) | 60 (96.8) | 6,103/6,279 (97.2) | 6,103 (96.9) |
| Tetracycline | 2,421 (71.2) | 555 (53.4) | IR | 317 (68.9) | 173 (87.8) | 149 (95.5) | 50 (37.9) | 120 (88.2) | IR | 3,821/5,590 (68.4)b | 3,821 (60.7)c |
| Colistin | 3,394 (99.8) | 929 (89.3) | IR | 448 (97.4) | 195 (99.0) | 156 (100) | IR | 134 (98.5) | IR | 5,275/5,441 (96.9)b | 5,275 (83.8)c |
| Trimethoprim-sulfamethoxazole | 2,672 (78.6) | 528 (50.8) | 453 (76.8) | 420 (91.3) | 185 (93.9) | 144 (92.3) | 127 (96.2) | 135 (99.3) | 43 (69.4) | 4,814/6,296 (76.5) | 4,814 (76.5) |
| Ciprofloxacin | 3,034 (89.2) | 518 (49.8) | 482 (81.7) | 429 (93.3) | 190 (96.4) | 156 (100) | 130 (98.5) | 136 (100) | 44 (71.0) | 5,236/6,296 (83.2) | 5,236 (83.2) |
Intermediately susceptible isolates were excluded. IR, intrinsic resistance.
Isolates of species with intrinsic resistance to an antibiotic were excluded from the total number of isolates.
Isolates of species with intrinsic resistance to an antibiotic were interpreted as being resistant, regardless of the MIC.
With regard to the subset of the 336 blood culture isolates, 324 (96.4%) were susceptible to isepamicin; the susceptibility to isepamicin was similar to that observed for the rest of the isolates. This was also observed for colistin (293/336 [87.2%] blood culture isolates were susceptible) but not for meropenem (264/336 [78.6%]) or cefepime (232/336 [69.0%]), for which the susceptibility of the blood culture isolates was lower than that of the isolates from all other specimens combined (P < 0.001 for both antibiotics). With regard to the subset of the 494 isolates collected from patients hospitalized in the ICU, 478 (96.8%) were susceptible to isepamicin, similarly to what was observed for the rest of the isolates. However, the susceptibility to colistin (364/494 isolates [73.7%]), meropenem (344/494 [69.6%]), and cefepime (279/494 [56.5%]) was significantly lower than that of the isolates collected from all other patients combined (P < 0.001 for each antibiotic).
Susceptibility to isepamicin was highest among all of the antimicrobial agents tested against E. coli (3,397/3,401 [99.9%] isolates were susceptible), K. pneumoniae (991/1,040 [95.3%]), P. mirabilis (588/590 [99.7%]), and Enterobacter spp. (455/460 [98.9%]). Figure 1 shows the temporal trend in the susceptibility of K. pneumoniae isolates to isepamicin and selected other antibiotics during each of the 6 years of the study period; the susceptibility to isepamicin remained relatively constant over the 6 years of the study period, in contrast with most other antibiotics. Furthermore, the susceptibility to isepamicin was the second highest after carbapenems against Citrobacter spp. (195/197 [99.0%] isolates were susceptible to isepamicin), Serratia marcescens (130/132 [98.5%]), and Morganella morganii (60/62 [96.8%]), while it was the fourth highest after carbapenems, ciprofloxacin, and colistin against Klebsiella oxytoca (154/156 [98.7%]). On the contrary, the susceptibility of Salmonella sp. to isepamicin was low (13/136 [9.6%] isolates were susceptible), similarly to what was observed for all other aminoglycosides tested.
Fig 1.
Temporal trend in susceptibilities of the studied Klebsiella pneumoniae isolates to isepamicin, other tested aminoglycosides, and selected other antibiotics during the study period. The horizontal axis refers to the study year, and the vertical axis refers to the percentages of K. pneumoniae isolates that were susceptible to each antibiotic.
Antimicrobial susceptibilities of specific types of resistant isolates.
Of the 6,296 Enterobacteriaceae sp. isolates studied, 423 (6.7%) were nonsusceptible to either imipenem or meropenem. The carbapenem-nonsusceptible Enterobacteriaceae sp. isolates included, specifically, 392 K. pneumoniae isolates, 11 Enterobacter sp. isolates, 10 P. mirabilis isolates, 7 E. coli isolates, 1 K. oxytoca isolate, 1 M. morganii isolate, and 1 S. marcescens isolate. Of the 423 above-mentioned carbapenem-nonsusceptible isolates, 379 (89.6%) were susceptible to isepamicin. Regarding the 392 carbapenem-nonsusceptible K. pneumoniae isolates, 357 (91.1%) were susceptible to isepamicin, 297 (75.8%) were susceptible to colistin, 96 (24.5%) were susceptible to gentamicin, 84 (21.4%) were susceptible to tetracycline, 47 (12.0%) were susceptible to amikacin, 34 (8.7%) were susceptible to tobramycin, 33 (8.4%) were susceptible to netilmicin, 18 (4.6%) were susceptible to trimethoprim-sulfamethoxazole, and 8 (2.0%) were susceptible to ciprofloxacin. With regard to the remaining 31 carbapenem-nonsusceptible Enterobacteriaceae sp. isolates (other than K. pneumoniae), 22 (71.0%) were susceptible to isepamicin.
Of the 683 isolates that were nonsusceptible to all of the other aminoglycosides, 500 (73.2%) were susceptible to isepamicin, specifically 329 (87.7%) of 375 K. pneumoniae isolates, 1 (0.8%) of 124 Salmonella sp. isolates, and 170 (92.4%) of 184 other Enterobacteriaceae sp. isolates. Further data on the antimicrobial susceptibilities of isolates that were nonsusceptible to any of the aminoglycosides or to colistin (excluding the intrinsically resistant ones) as well as of the p-XDR and p-PDR isolates are shown in Table 3.
Table 3.
Susceptibilities to isepamicin of Enterobacteriaceae isolates with specific resistance phenotypesg
| Resistance phenotype |
K. pneumoniae |
Other Enterobacteriaceae |
||||
|---|---|---|---|---|---|---|
| Total no. of isolates | No. (%) of isolates susceptible to: |
Total no. of isolates | No. (%) of isolates susceptible to isepamicin | |||
| Isepamicin | Meropenem | Colistin | ||||
| Any AG-R | 464 | 415 (89.4) | 111 (23.9) | 382 (82.3) | 554 | 410 (74) |
| Tobramycin resistant | 461 | 412 (89.4) | 109 (23.6) | 379 (82.2) | 514 | 371 (72.2)a |
| Amikacin resistant | 431 | 382 (88.6) | 88 (20.4) | 351 (81.4) | 373 | 234 (62.7)a |
| Gentamicin resistance | 389 | 343 (88.2) | 95 (24.4) | 315 (81) | 463 | 324 (70)a |
| All AG-R | 375 | 329 (87.7) | 81 (21.6) | 301 (80.3) | 308f | 171 (55.5) |
| Carbapenem resistant | 392 | 357 (91.1) | NA | 297 (75.6) | 31 | 22 (71) |
| Colistin resistantb | 111 | 95 (85.6) | 18 (16.2) | NA | 55c | 52 (94.5)c |
| p-XDRd | 423 | 386 (91.3) | 54 (12.8) | 317 (74.9) | ND | ND |
| Susceptible to colistin onlyd | 220 | 209 (95.0) | NA | NA | ND | ND |
| p-PDRd,e | 54 | 49 (90.7) | NA | NA | ND | ND |
P = 0.003 for amikacin-resistant versus tobramycin-resistant isolates, P = 0.027 for amikacin-resistant versus gentamicin-resistant isolates, and P = 0.45 for tobramycin-resistant versus gentamicin-resistant isolates.
A total of 22/111 (20%) colistin-resistant K. pneumoniae isolates were tested against tigecycline and fosfomycin: all 22 isolates were susceptible to tigecycline, 21/22 (96%) were susceptible to fosfomycin, and 16/22 (73%) were susceptible to isepamicin.
Refers only to species that are not nonintrinsically resistant to colistin.
Isolates were not tested against tigecycline, fosfomycin, and chloramphenicol.
A total of 11/54 (20%) p-PDR isolates were tested against tigecycline and fosfomycin. All were susceptible to tigecycline, 10/11 (91%) were susceptible to fosfomycin, and 10/11 (91%) were susceptible to isepamicin.
The 308 isolates included 6 Citrobacter sp. isolates, 19 Enterobacter sp. isolates, 95 Escherichia coli isolates, 3 Klebsiella oxytoca isolates, 5 Morganella morganii isolates, 47 Proteus sp. isolates, 1 Providencia rettgeri isolate, 124 Salmonella sp. isolates, 7 Serratia marcescens isolates, and 1 Shigella flexneri isolate.
AG, aminoglycoside(s); All AG-R, nonsusceptible to all aminoglycosides (excluding isepamicin); Any AG-R, nonsusceptible to at least one of the aminoglycosides (excluding isepamicin); NA, not applicable; ND, not determined; p-XDR, possibly extensively drug resistant; p-PDR, possibly pandrug resistant. Resistant isolates include intermediately resistant isolates.
Quality control testing.
The isepamicin MICs for E. coli ATCC 25922 were always ≤1 mg/liter, similarly to what was described previously (27). There was categorical agreement in the interpretations of susceptibility to isepamicin between the disk diffusion method and the Vitek 2 system for all of the 20 randomly selected isolates that were tested with the latter method.
DISCUSSION
In our study, isepamicin exhibited very high in vitro activity against the 6,296 unselected clinical Enterobacteriaceae sp. isolates that were collected in a tertiary care university general hospital in Crete, Greece, from 2004 to 2009 from unique patients. Isepamicin was the most active in vitro against all isolates among all of the antibiotics tested, followed by colistin, meropenem, imipenem, and amikacin. High susceptibility to isepamicin was also observed for the subsets of the blood culture isolates and the isolates collected from patients hospitalized in the ICU, in contrast to other antibiotics, such as meropenem. High susceptibility to isepamicin was observed for the carbapenem-nonsusceptible Enterobacteriaceae sp. isolates, the great majority of which were K. pneumoniae isolates. Very high susceptibility to isepamicin was also observed for the Enterobacteriaceae sp. isolates that were nonsusceptible to all of the other aminoglycosides tested. The only exception was Salmonella sp., for which the aminoglycosides are not considered appropriate therapy, even if they appear to be active in vitro, and should not be reported as susceptible according to CLSI guidelines. Resistance to isepamicin did not appear to correlate with resistance to the other aminoglycosides in the K. pneumoniae isolates but was higher for the amikacin-resistant than for the gentamicin- or tobramycin-resistant isolates in, cumulatively, the remaining Enterobacteriaceae spp.
The high susceptibility of the Enterobacteriaceae sp. isolates examined in this study to isepamicin could be explained by the stability of this drug to the action of the most common aminoglycoside-modifying enzymes present in these pathogens. Isepamicin has a profile of resistance to the aminoglycoside-modifying enzymes that is similar to that of amikacin, with the advantage that it is not inactivated by the AAC(6′)-I subfamily of enzymes (32). The latter enzymes inactivate all of the commonly used aminoglycosides, except gentamicin (37). The prevalence of AAC(6′)-I enzymes appears to differ by geographical region and can be higher in countries where amikacin is commonly used in clinical practice; Greece appears to be such an example (31). Other aminoglycoside-modifying enzymes that can be found in Enterobacteriaceae and that inactivate isepamicin include ANT(4′)-II, APH(3′)-VI, and AAC(6′)-III, although their prevalences are rather low (32). Of these, the ANT(4′)-II enzyme was first found in Pseudomonas aeruginosa but has also been found in Enterobacteriaceae, and the APH(3′)-IV enzyme is found primarily in Acinetobacter baumannii, while the AAC(6′)-III enzyme has been described in specific geographical regions such as Turkey (21, 35, 37).
In our study, isepamicin exhibited very good in vitro activity specifically against the carbapenem-nonsusceptible K. pneumoniae isolates. The remaining aminoglycosides had poor activity against these pathogens, with gentamicin being the most active, with a susceptibility of 21.4%. In Greece, carbapenem resistance in K. pneumoniae has been related primarily to the production of specific carbapenemases (17, 39). Since 2002, there has been a polyclonal outbreak of VIM-1 metallo-β-lactamase-producing K. pneumoniae, which has largely been replaced by KPC-2-producing K. pneumoniae, belonging more often to ST258 (17, 36). The latter strains first appeared in Crete by late 2007 (18, 30). ST258 KPC-producing K. pneumoniae strains (7 out of 7) showed susceptibility to isepamicin in another study (27). Despite the above-described change in the epidemiology of carbapenem-resistant K. pneumoniae in our region, the susceptibility to isepamicin did not substantially change during the 6 years of our study.
Gentamicin has been the most active aminoglycoside against the most of the prevalent carbapenem-resistant and, particularly, the KPC-producing K. pneumoniae clones (10, 25). This resistance profile is associated with the production of AAC(6′)-I aminoglycoside-modifying enzymes (20, 24). An aminoglycoside, when active in vitro, could be an effective treatment option for infections with Gram-negative pathogens that are resistant to other aminoglycosides (7). However, isepamicin was inferior to gentamicin when they were both used in combination with β-lactams for the treatment of experimental endocarditis caused by AAC(6′)-Ib-producing Enterobacteriaceae (6, 29). This could be attributed to the low-level resistance to isepamicin conferred by this enzyme (33). Genes for additional aminoglycoside-modifying enzymes are commonly carried by the resistance plasmids encoding VIM-1 or KPC-2 in K. pneumoniae (10, 20). This might be an explanation for the low degree of susceptibility to gentamicin observed in our study in comparison with other relevant studies.
Whether isepamicin could constitute an effective treatment option against carbapenemase-producing K. pneumoniae remains to be determined. This might be important considering that the effectiveness and safety profile of the currently available therapeutic options for these pathogens appear to be suboptimal (13). It should be mentioned that isepamicin has formally been evaluated for clinical use and was approved in this respect in many European countries in the mid-1990s (22, 32). Several clinical studies agreed that there is no important difference in the clinical effectiveness and safety profile of isepamicin compared with those of amikacin (5).
The very high susceptibility to isepamicin of the Enterobacteriaceae sp. isolates collected in our study hospital might relate to the fact that isepamicin was not available for clinical use during the study period. There are differences in the patterns of resistance to aminoglycosides between different regions and in different time periods, which relate mainly to the selection pressure from the use of different members of this class (31). Nevertheless, the rather limited relevant published data available from different countries (mainly those in which isepamicin was available for clinical use) support the idea that isepamicin has very good in vitro activity against Gram-negative pathogens compared to other commonly used aminoglycosides (12). The prevalence of resistance to a new aminoglycoside can increase rather quickly, depending on the consumption of the drug (19). The selection of resistance to isepamicin in Gram-negative bacteria was observed in two Belgian hospitals soon after the institution of isepamicin in the market (38). Resistance to isepamicin can result from the combination of more than one aminoglycoside-modifying enzyme or of such an enzyme along with decreased permeability or efflux mechanisms (33, 35). Enterobacteriaceae that carry the NDM metallo-β-lactamase typically produce 16S rRNA methylases, which inactivate all of the aminoglycosides available for clinical use, including isepamicin (8, 27, 34).
The findings of our study should be interpreted in the context of potential limitations. The susceptibilities of the included isolates to the antibiotics tested, including isepamicin, were determined by using an automated susceptibility testing system, owing to the retrospective nature of our study. Moreover, interpretative MIC breakpoints of susceptibility of Enterobacteriaceae to isepamicin are not available through the CLSI. Relevant studies, though, have commonly used an MIC breakpoint of susceptibility to isepamicin that matches that to amikacin (1). According to the CLSI, susceptibility to amikacin is defined by an MIC of ≤16 mg/liter. We opted for a more conservative breakpoint of susceptibility to isepamicin according to what was proposed previously by Barry et al. and has been endorsed by CA-SFM (susceptibility defined as an MIC of ≤8 mg/liter) (1).
In conclusion, isepamicin was highly active in vitro against recent clinical Enterobacteriaceae sp. isolates collected at a tertiary care hospital in Greece. Isepamicin was highly active against isolates with resistance to all of the other aminoglycosides tested and against problem pathogens, particularly carbapenem-resistant K. pneumoniae. These findings appear to be promising for a potential role of isepamicin as a last-resort therapeutic option against infections with Enterobacteriaceae that have acquired resistance to commonly used agents. However, the profile of susceptibility of Enterobacteriaceae to isepamicin that was observed in our study needs to be corroborated by additional relevant studies.
Footnotes
Published ahead of print 5 March 2012
REFERENCES
- 1. Barry AL, Thornsberry C, Jones RN, Gerlach EH. 1980. Interpretive standards for disk susceptibility tests with Sch 21420 and amikacin. Antimicrob. Agents Chemother. 18:616–621 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Bassetti M, Ginocchio F, Giacobbe DR, Mikulska M. 2011. Development of antibiotics for Gram-negatives: where now? Clin. Invest. 1:211–227 [Google Scholar]
- 3. Bonnet R, et al. 2010. Comité de l'Antibiogramme de la Société Française de Microbiologie. Recommandations 2010. Edition de Janvier 2010. Société Française de Microbiologie, Paris, France: www.sfm-microbiologie.org/ [Google Scholar]
- 4. Boucher HW, et al. 2009. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin. Infect. Dis. 48:1–12 [DOI] [PubMed] [Google Scholar]
- 5. Carbon C. 1995. Overview of the efficacy of isepamicin in the adult core clinical trial programme. J. Chemother. 7(Suppl 2):79–85 [PubMed] [Google Scholar]
- 6. Caulin E, Coutrot A, Carbon C, Collatz E. 1996. Resistance to amikacin and isepamicin in rabbits with experimental endocarditis of an aac(6′)-Ib-bearing strain of Klebsiella pneumoniae susceptible in vitro. Antimicrob. Agents Chemother. 40:2848–2853 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6a. Clinical and Laboratory Standards Institute 2010. Performance standards for antimicrobial susceptibility testing; 20th informational supplement. CLSI document M100-S20. CLSI, Wayne, PA [Google Scholar]
- 7. Daikos GK, Kosmidis JC, Hamilton-Miller JM, Brumfitt W. 1976. Amikacin in treatment of infections caused by gram-negative bacteria resistant to gentamicin and other aminoglycosides: clinical and bacteriologic results. J. Infect. Dis. 134(Suppl):S286–S290 [DOI] [PubMed] [Google Scholar]
- 8. Doi Y, Arakawa Y. 2007. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides. Clin. Infect. Dis. 45:88–94 [DOI] [PubMed] [Google Scholar]
- 9. Durante-Mangoni E, Grammatikos A, Utili R, Falagas ME. 2009. Do we still need the aminoglycosides? Int. J. Antimicrob. Agents 33:201–205 [DOI] [PubMed] [Google Scholar]
- 10. Endimiani A, et al. 2009. ACHN-490, a neoglycoside with potent in vitro activity against multidrug-resistant Klebsiella pneumoniae isolates. Antimicrob. Agents Chemother. 53:4504–4507 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Falagas ME, Karageorgopoulos DE. 2008. Pandrug resistance (PDR), extensive drug resistance (XDR), and multidrug resistance (MDR) among Gram-negative bacilli: need for international harmonization in terminology. Clin. Infect. Dis. 46:1121–1122 [DOI] [PubMed] [Google Scholar]
- 12. Falagas ME, et al. 2012. Susceptibility of Gram-negative bacteria to isepamicin: a systematic review. Expert Rev. Anti Infect. Ther. 10:207–218 [DOI] [PubMed] [Google Scholar]
- 13. Falagas ME, Karageorgopoulos DE, Nordmann P. 2011. Therapeutic options for infections with Enterobacteriaceae producing carbapenem-hydrolyzing enzymes. Future Microbiol. 6:653–666 [DOI] [PubMed] [Google Scholar]
- 14. Falagas ME, Kasiakou SK. 2005. Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin. Infect. Dis. 40:1333–1341 [DOI] [PubMed] [Google Scholar]
- 15. Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. 2010. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum beta-lactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect. Dis. 10:43–50 [DOI] [PubMed] [Google Scholar]
- 16. Falagas ME, et al. 2010. Antimicrobial susceptibility of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Enterobacteriaceae isolates to fosfomycin. Int. J. Antimicrob. Agents 35:240–243 [DOI] [PubMed] [Google Scholar]
- 17. Giakkoupi P, et al. 2011. An update of the evolving epidemic of blaKPC-2-carrying Klebsiella pneumoniae in Greece (2009–10). J. Antimicrob. Chemother. 66:1510–1513 [DOI] [PubMed] [Google Scholar]
- 18. Giakoupi P, et al. 2009. KPC-2-producing Klebsiella pneumoniae infections in Greek hospitals are mainly due to a hyperepidemic clone. Euro Surveill. 14(21):pii=19218. http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19218 [DOI] [PubMed] [Google Scholar]
- 19. Giamarellou H, et al. 1986. Nosocomial consequences of antibiotic usage. Scand. J. Infect. Dis. Suppl. 49:182–188 [PubMed] [Google Scholar]
- 20. Gootz TD, et al. 2009. Genetic organization of transposase regions surrounding blaKPC carbapenemase genes on plasmids from Klebsiella strains isolated in a New York City hospital. Antimicrob. Agents Chemother. 53:1998–2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Jacoby GA, et al. 1990. Appearance of amikacin and tobramycin resistance due to 4′-aminoglycoside nucleotidyltransferase [ANT(4′)-II] in gram-negative pathogens. Antimicrob. Agents Chemother. 34:2381–2386 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Jones RN. 1995. Isepamicin (SCH 21420, 1-N-HAPA gentamicin B): microbiological characteristics including antimicrobial potency of spectrum of activity. J. Chemother. 7(Suppl 2):7–16 [PubMed] [Google Scholar]
- 23. Karageorgopoulos DE, Falagas ME. 2009. New antibiotics: optimal use in current clinical practice. Int. J. Antimicrob. Agents 34(Suppl 4):S55–S62 [DOI] [PubMed] [Google Scholar]
- 24. Kassis-Chikhani N, et al. 2006. First outbreak of multidrug-resistant Klebsiella pneumoniae carrying blaVIM-1 and blaSHV-5 in a French university hospital. J. Antimicrob. Chemother. 57:142–145 [DOI] [PubMed] [Google Scholar]
- 25. Leavitt A, et al. 2010. Molecular epidemiology, sequence types, and plasmid analyses of KPC-producing Klebsiella pneumoniae strains in Israel. Antimicrob. Agents Chemother. 54:3002–3006 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Leclercq R, et al. 2008. Expert rules in antimicrobial susceptibility testing. European Committee on Antimicrobial Susceptibility Testing. http://www.eucast.org/expert_rules/ [DOI] [PubMed]
- 27. Livermore DM, et al. 2011. Activity of aminoglycosides, including ACHN-490, against carbapenem-resistant Enterobacteriaceae isolates. J. Antimicrob. Chemother. 66:48–53 [DOI] [PubMed] [Google Scholar]
- 28. Magiorakos AP, et al. 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18:268–281 [DOI] [PubMed] [Google Scholar]
- 29. Mainardi JL, et al. 1994. Activity of isepamicin and selection of permeability mutants to beta-lactams during aminoglycoside therapy of experimental endocarditis due to Klebsiella pneumoniae CF104 producing an aminoglycoside acetyltransferase 6′ modifying enzyme and a TEM-3 beta-lactamase. J. Infect. Dis. 169:1318–1324 [DOI] [PubMed] [Google Scholar]
- 30. Maltezou HC, et al. 2009. Outbreak of infections due to KPC-2-producing Klebsiella pneumoniae in a hospital in Crete (Greece). J. Infect. 58:213–219 [DOI] [PubMed] [Google Scholar]
- 31. Miller GH, et al. 1997. The most frequent aminoglycoside resistance mechanisms—changes with time and geographic area: a reflection of aminoglycoside usage patterns? Aminoglycoside Resistance Study Groups. Clin. Infect. Dis. 24(Suppl 1):S46–S62 [DOI] [PubMed] [Google Scholar]
- 32. Miller GH, Sabatelli FJ, Naples L, Hare RS, Shaw KJ. 1995. The changing nature of aminoglycoside resistance mechanisms and the role of isepamicin—a new broad-spectrum aminoglycoside. The Aminoglycoside Resistance Study Groups. J. Chemother. 7(Suppl 2):31–44 [PubMed] [Google Scholar]
- 33. Mingeot-Leclercq MP, Glupczynski Y, Tulkens PM. 1999. Aminoglycosides: activity and resistance. Antimicrob. Agents Chemother. 43:727–737 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Nordmann P, Poirel L, Walsh TR, Livermore DM. 2011. The emerging NDM carbapenemases. Trends Microbiol. 19:588–595 [DOI] [PubMed] [Google Scholar]
- 35. Over U, Gur D, Unal S, Miller GH. 2001. The changing nature of aminoglycoside resistance mechanisms and prevalence of newly recognized resistance mechanisms in Turkey. Clin. Microbiol. Infect. 7:470–478 [DOI] [PubMed] [Google Scholar]
- 36. Psichogiou M, et al. 2008. Ongoing epidemic of blaVIM-1-positive Klebsiella pneumoniae in Athens, Greece: a prospective survey. J. Antimicrob. Chemother. 61:59–63 [DOI] [PubMed] [Google Scholar]
- 37. Shaw KJ, Rather PN, Hare RS, Miller GH. 1993. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol. Rev. 57:138–163 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38. Vanhoof R, Nyssen HJ, Van Bossuyt E, Hannecart-Pokorni E. 1999. Aminoglycoside resistance in Gram-negative blood isolates from various hospitals in Belgium and the Grand Duchy of Luxembourg. Aminoglycoside Resistance Study Group. J. Antimicrob. Chemother. 44:483–488 [DOI] [PubMed] [Google Scholar]
- 39. Vatopoulos A. 2008. High rates of metallo-beta-lactamase-producing Klebsiella pneumoniae in Greece—a review of the current evidence. Euro Surveill. 13(4):pii=8023. http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=8023 [PubMed] [Google Scholar]