Abstract
β-Lactam susceptibility of 499 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae, determined by reference broth microdilution, demonstrated that 14.4% of isolates were categorized as cefepime susceptible according to current CLSI breakpoints. Ceftazidime- and meropenem-susceptible isolates were also observed (2.6 and 3.0%, respectively). Cefepime-susceptible KPC-producing isolates may confuse laboratory staff and clinicians in their therapeutic choices.
TEXT
Since their first description in 2001, Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria have been isolated in microbiology laboratories from health institutions worldwide (1, 2). This carbapenem-hydrolyzing β-lactamase is commonly identified in Klebsiella clinical isolates, but it can also be found among other Enterobacteriaceae, Pseudomonas spp., and Acinetobacter spp. (2–4). Several phenotypic methods to detect carbapenemases such as KPC have been proposed (5, 6); however, these methods can generate false-positive results due to concomitant hyperproduction of chromosomal cephalosporinases, antimicrobial inhibitory effects of the enzyme inhibitor alone, or other technical artifacts (6, 7). Furthermore, some of these methodologies are not suitable to identify all carbapenemases, and different tests for detection of serine carbapenemases and metallo-β-lactamases might need to be applied (6).
Evidence that the MICs have a greater significance in predicting clinical outcomes than the presence or absence of β-lactamases and the absence of simple and reliable phenotypic methods to detect carbapenemase-producing Enterobacteriaceae clinical isolates prompted the Clinical and Laboratory Standards Institute (CLSI) to modify the carbapenem breakpoints for interpreting susceptibility test results (8, 9). Lower carbapenem MIC breakpoint values were recently recommended, aiming to categorize the vast majority of carbapenemase-producing Enterobacteriaceae isolates as resistant to these compounds and eliminating the need for additional phenotypic tests, such as the modified Hodge test (8). However, breakpoints for cefepime and some β-lactam/β-lactamase inhibitor combinations were not changed, and KPC producers might be categorized as susceptible to these antimicrobials, generating confusion for the interpretation of isolates now considered resistant to carbapenems but susceptible to other β-lactam agents (10).
In this study, we evaluated the MICs and current CLSI breakpoints for eight β-lactams tested against 499 KPC-producing enterobacterial isolates collected during an 11-year period as part of two international surveillance studies. Nonduplicated clinical isolates originating from medical centers located in Europe, Asia, and North and Latin America and recovered from bloodstream, respiratory tract, or skin/skin structure infections were included according to defined protocols used by the SENTRY Antimicrobial Surveillance Program and the MYSTIC Program USA. All isolates were identified in the participating medical center and forwarded to a central laboratory (JMI Laboratories, North Liberty, IA), where species identification was confirmed by standard biochemical tests and by using the Vitek 2 systems (bioMérieux, Hazelwood, MO) when necessary. PCR experiments targeting blaKPC were conducted for all isolates by using previously described primers and cycling conditions (11). Isolates belonged to nine bacterial species: Citrobacter freundii (n = 13), Enterobacter cloacae (n = 54), Enterobacter gergoviae (n = 2), Escherichia coli (n = 16), Klebsiella oxytoca (n = 18), Klebsiella pneumoniae (n = 389), Raoultella ornithinolytica (n = 1), Raoultella planticola (n = 2), and Serratia marcescens (n = 4).
All isolates were susceptibility tested by the CLSI broth microdilution method (12) using validated panels (ThermoFisher Scientific [formerly TREK Diagnostics], Cleveland, OH). Results were interpreted using CLSI criteria (13). Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were tested for quality assurance.
All KPC-producing organisms were resistant to aztreonam; however, isolates showing susceptibility to piperacillin-tazobactam (0.6%), ceftriaxone (0.2%), ceftazidime (2.6%), ertapenem (0.4%), imipenem (0.6%), and meropenem (3.0%) were observed (Table 1). Of note, 72 (14.4%) KPC producers were categorized as susceptible to cefepime, including K. pneumoniae (n = 31), K. oxytoca (n = 13), C. freundii (n = 10), E. cloacae (n = 8), E. coli (n = 8), and R. planticola (n = 2), in which cefepime MIC results were as low as 0.5 μg/ml. Cefepime-susceptible isolates included five E. coli, four K. oxytoca, and four K. pneumoniae isolates that were also categorized as ceftazidime susceptible. Among these, two E. coli and two K. pneumoniae isolates showed meropenem susceptibility, and one K. pneumoniae strain was also susceptible to imipenem using the revised CLSI breakpoints (13). All strains showing low MICs for the β-lactam agents tested had the presence of blaKPC confirmed by multiple PCR experiments (11).
Table 1.
MIC distributions for eight broad-spectrum β-lactams tested against 499 KPC-producing Enterobacteriaceae isolates collected in the SENTRY Antimicrobial Surveillance Program and MYSTIC Program USA
| Antimicrobial agent and organism (no. tested) | No. (cumulative %) inhibited at MICa (μg/ml): |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ≤0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | >64b | |
| Aztreonam | ||||||||||
| All species (408) | 1 | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 3 (0.7) | 3 (1.5) | —c | — | 402 (100.0) |
| K. pneumoniae (306) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (0.3) | 2 (1.0) | — | — | 303 (100.0) |
| Non-K. pneumoniae (102) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 2 (2.0) | 1 (2.9) | — | — | 99 (100.0) |
| Cefepime | ||||||||||
| All species (499) | 0 (0.0) | 1 (0.2) | 3 (0.8) | 10 (2.8) | 18 (6.4) | 40 (14.4) | 337 (82.0) | — | — | 90 (100.0) |
| K. pneumoniae (389) | 0 (0.0) | 1 (0.3) | 0 (0.0) | 1 (0.5) | 9 (2.8) | 20 (8.0) | 275 (78.7) | — | — | 83 (100.0) |
| Non-K. pneumoniae (110) | 0 (0.0) | 0 (0.0) | 3 (2.7) | 9 (10.9) | 9 (19.1) | 8.0 (37.3) | 62 (93.6) | — | — | 7 (100.0) |
| Ceftazidime | ||||||||||
| All species (499) | 0 (0.0) | 0 (0.0) | 2 (0.4) | 3 (1.0) | 8 (2.6) | 19 (6.4) | 28 (12.0) | — | — | 439 (100.0) |
| K. pneumoniae (389) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (0.3) | 3 (1.0) | 11 (3.9) | 12 (6.9) | — | — | 362 (100.0) |
| Non-K. pneumoniae (110) | 0 (0.0) | 0 (0.0) | 2 (1.8) | 2 (3.6) | 5 (8.2) | 8 (15.5) | 16 (30.0) | — | — | 77 (100.0) |
| Ceftriaxone | ||||||||||
| All species (499) | 0 (0.0) | 0 (0.0) | 1 (0.2) | 0 (0.0) | 5 (1.2) | 8 (2.8) | — | — | — | 485 (100.0) |
| K. pneumoniae (389) | 0 (0.0) | 0 (0.0) | 1 (0.3) | 0 (0.0) | 0 (0.0) | 1 (0.5) | — | — | — | 387 (100.0) |
| Non-K. pneumoniae (110) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 5 (4.5) | 7 (10.9) | — | — | — | 98 (100.0) |
| Ertapenem | ||||||||||
| All species (481) | 0 (0.0) | 2 (0.4) | 8 (2.1) | 12 (4.6) | 34 (11.6) | 58 (23.7) | — | — | — | 367 (100.0) |
| K. pneumoniae (381) | 0 (0.0) | 1 (0.5) | 4 (1.6) | 4 (2.6) | 12 (5.8) | 27 (12.9) | — | — | — | 332 (100.0) |
| Non-K. pneumoniae (100) | 0 (0.0) | 0 (0.0) | 4 (4.0) | 8 (12.0) | 22 (34.0) | 31 (65.0) | — | — | — | 35 (100.0) |
| Imipenem | ||||||||||
| All species (497) | 0 (0.0) | 2 (0.4) | 1 (0.6) | 0 (0.0) | 54 (11.5) | 124 (36.4) | — | — | — | 316 (100.0) |
| K. pneumoniae (387) | 0 (0.0) | 2 (0.5) | 0 (0.0) | 0 (0.0) | 22 (6.2) | 89 (29.2) | — | — | — | 274 (100.0) |
| Non-K. pneumoniae (110) | 0 (0.0) | 0 (0.0) | 1 (0.9) | 0 (0.0) | 32 (30.0) | 35 (61.8) | — | — | — | 42 (100.0) |
| Meropenem | ||||||||||
| All species (499) | 1 (0.2) | 8 (1.8) | 6 (3.0) | 29 (8.8) | 56 (20.0) | 76 (35.3) | — | — | — | 323 (100.0) |
| K. pneumoniae (389) | 1 (0.3) | 5 (1.5) | 3 (2.3) | 11 (5.1) | 20 (10.3) | 51 (23.4) | — | — | — | 298 (100.0) |
| Non-K. pneumoniae (110) | 0 (0.0) | 3 (2.7) | 3 (5.5) | 18 (21.8) | 36 (54.5) | 25 (77.3) | — | — | — | 25 (100.0) |
| Piperacillin-tazobactam | ||||||||||
| All species (499) | 0 (0.0) | 0 (0.0) | 0 (0.0) | (0.0) | 0 (0.0) | 3 (0.6) | 0 (0.0) | 3 (1.2) | 13 (3.8) | 480 (100.0) |
| K. pneumoniae (389) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 3 (0.8) | 0 (0.0) | 2 (1.3) | 3 (2.1) | 381 (100.0) |
| Non-K. pneumoniae (110) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (0.9) | 10 (10.0) | 99 (100.0) |
Susceptible MIC ranges by CLSI criteria are highlighted in gray; EUCAST susceptibility MIC ranges are in boldface.
Greater than the highest dilution tested.
—, untested concentration.
Susceptibility data interpreted using the EUCAST criteria revealed susceptibility rates to cefepime (0.8%) and ceftazidime (0.4%) that were substantially lower than those obtained with CLSI criteria (Table 1), while the meropenem susceptibility rate increased considerably (8.8%) (Table 1).
Temporal analysis of susceptibility rates showed that isolates recovered between years 2000 and 2005 were significantly more susceptible to cefepime and ceftazidime than those recovered during the 2006-2010 period (P < 0.05) (Table 2). In addition, more recent isolates showed meropenem and piperacillin-tazobactam resistance rates that were significantly higher than those of isolates recovered between years 2000 and 2005 (P < 0.05) (Table 2). These results indicate increased extended-spectrum β-lactamase (ESBL) production among isolates recovered during the 2006-2010 period, likely due to blaCTX-M dissemination in more recent years (14).
Table 2.
Number tested and percentages of KPC-producing isolates according to susceptibility categories assigned using the current CLSI breakpoints
| Antimicrobial agent | No. tested (all species/K. pneumoniae only) |
Comparison of percentages by categorya (all species/K. pneumoniae only) |
||||
|---|---|---|---|---|---|---|
| Susceptible |
Resistant |
|||||
| 2000–2005 | 2006–2010 | 2000–2005 | 2006–2010 | 2000–2005 | 2006–2010 | |
| Aztreonam | 80/42 | 328/264 | 0.0/0.0 | 0.0/0.0 | 97.5/100.0 | 99.7/99.6 |
| Cefepime | 80/42 | 419/347 | 32.5/14.3 | 11.0/7.2 | 0.0/0.0 | 21.5/31.1 |
| Ceftazidime | 80/42 | 419/347 | 7.5/0.0 | 1.7/1.2 | 88.7/95.2 | 94.5/96.3 |
| Ceftriaxone | 80/42 | 419/347 | 0.0/0.0 | 0.2/0.3 | 100.0/100.0 | 99.8/99.7 |
| Ertapenem | 62/34 | 419/347 | 0.0/0.0 | 0.5/0.6 | 96.8/100.0 | 98.1/98.3 |
| Imipenem | 80/42 | 417/345 | 1.3/0.0 | 0.5/0.6 | 98.7/100.0 | 99.5/99.4 |
| Meropenem | 80/42 | 419/347 | 2.5/0.0 | 3.1/2.6 | 81.2/97.6 | 93.1/94.5 |
| Piperacillin-tazobactam | 80/42 | 419/347 | 0.0/0.0 | 0.7/0.9 | 91.2/100.0 | 97.1/97.7 |
Results in boldface were considered significant (P < 0.05); statistical significance of percentages between groups was assessed using the chi-square test.
The CLSI decision to maintain current cefepime breakpoints was based on reports showing that current standard (or usual) doses of intravenous cefepime (≥3 to 4 g/day) have a high likelihood of achieving optimal exposure against Enterobacteriaceae categorized as susceptible to this drug (15, 16). Additionally, it has been reported recently that KPC-2 expression in Enterobacteriaceae is not enough to confer cefepime resistance (17). On the other hand, cefepime therapy of infections due to ESBL-producing Enterobacteriaceae was recently associated with lower survival rates among patients infected with isolates showing cefepime MICs of >1 μg/ml, within the CLSI susceptibility range (18). Therefore, more studies in animal models, pharmacokinetic/pharmacodynamic modeling, and/or focused clinical trials appear necessary to provide conclusive evidence that cefepime is a useful treatment option for infections due to KPC-producing isolates that demonstrate in vitro susceptibility to this agent under current CLSI breakpoints.
ACKNOWLEDGMENTS
We thank L. M. Deshpande and L. N. Woosley for their excellent technical assistance in screening the KPC strains used in this study.
JMI Laboratories, Inc., has received research and educational grants in 2009 to 2012 from the American Proficiency Institute (API), Anacor, Astellas, AstraZeneca, Bayer, Cempra, Cerexa, Contrafect, Cubist, Daiichi, Dipexium, Enanta, Furiex, GlaxoSmithKline, Johnson & Johnson (Ortho McNeil), LegoChem Biosciences Inc., Meiji Seika Kaisha, Merck, Nabriva, Novartis, Pfizer (Wyeth), Rempex, Rib-X Pharmaceuticals, Seachaid, Shionogi, The Medicines Co., Theravance, ThermoFisher, and some other corporations. Some JMI employees are advisors/consultants for Astellas, Cubist, Pfizer, Cempra, Cerexa-Forest, Johnson & Johnson, and Theravance.
We have no speaker bureaus or stock options to declare.
Footnotes
Published ahead of print 24 April 2013
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