Department of Medicine, SUNY Downstate Medical Center, 450 Clarkson Avenue, Box 77, Brooklyn, NY 11203, USA
Received 16 February 2005; returned 24 March 2005; revised 31 March 2005; accepted 26 April 2005
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Abstract |
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Methods: Clinical isolates with suspected carbapenem resistance were referred to the central research laboratory from August 2003 to June 2004. Isolates underwent MIC testing, ribotyping, and were analysed for the presence of KPC carbapenemases. Timekill studies using various antibiotic(s) were performed on selected isolates.
Results: Ninety-six isolates were referred from 10 Brooklyn hospitals. All isolates were resistant to the carbapenems with most having MICs >32 mg/L. Few were susceptible to fluoroquinolones and cephalosporins; approximately half were susceptible to aminoglycosides, and 90% to polymyxin B. Two-thirds were susceptible to doxycycline, and all were considered susceptible to the investigational glycylcycline antibiotic tigecycline. Virtually all possessed blaKPC, and over 80% belonged to one ribotype. In timekill studies involving 16 isolates, tigecycline demonstrated bacteriostatic activity and polymyxin B concentration-dependent bactericidal activity. The combination of polymyxin B at 0.5 x MIC plus rifampicin had synergic activity against 15/16 isolates, including two polymyxin-resistant strains. The combination of polymyxin B plus imipenem had synergic bactericidal activity against 10/16 isolates, but was antagonistic for three isolates.
Conclusions: Multiresistant K. pneumoniae with blaKPC are present in multiple hospitals in New York City. The most consistently active agents in vitro were tigecycline and polymyxin B, particularly when the latter was combined with rifampicin. The clinical efficacy of these agents remains to be determined.
Keywords: antibiotic resistance , ß-lactamases , imipenem , tigecycline
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Introduction |
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Over the past few years, a progressive increase in carbapenem-resistant Gram-negative bacteria, particularly Acinetobacter baumannii and Pseudomonas aeruginosa, has been observed in some areas.1,3 In the United States, carbapenem resistance has been largely attributed to expression of a class C cephalosporinase and loss of outer membrane porins in isolates of A. baumannii, P. aeruginosa, and rarely, K. pneumoniae.46 Carbapenem-hydrolysing ß-lactamases have been rarely recovered in K. pneumoniae. However, isolates possessing KPC-1, KPC-2, and KPC-3, class A enzymes, have been recently identified in the northeastern United States.711 These isolates are often resistant to multiple antibiotic classes, presenting clinicians with very limited therapeutic options. In this report, the molecular epidemiology of a large number of referred isolates of carbapenem-resistant K. pneumoniae is described, and the in vitro activity of several antibiotic agents is evaluated.
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Methods |
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All isolates underwent ribotyping with the RiboprinterTM Microbial Characterization System (Qualicon, Wilmington, DE, USA) using the restriction endonuclease EcoRI. A subset of isolates were also fingerprinted by pulsed-field gel electrophoresis (PFGE) using restriction endonuclease XbaI as previously described.3 The PFGE patterns were interpreted according to the recommendations of Tenover et al.14 The presence of KPC-type ß-lactamases was tested by PCR as previously described.15 The following primers were used: KPC forward: 5'-ATGTCACTGTATCGCCGTCT-3' and KPC reverse 5'-TTTTCAGAGCCTTACTGCCC-3'. Bi-directional sequencing was performed on selected blaKPC-positive isolates using the automated fluorescent dye-terminator sequencing system (Applied Biosystems). The following additional internal primer was included for sequencing of blaKPC: 5'-AGCTGAACTCCGCCATCC-3'. Sequences were identified by the BLAST program from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/BLAST/).
Timekill studies were performed as previously described.16 The following antibiotics were tested: imipenem 4 mg/L, cefotetan 8 mg/L, gentamicin 2 and 4 mg/L, rifampicin 1 mg/L, doxycycline 4 mg/L, tigecycline 2 mg/L, and imipenem plus clavulanic acid 2 mg/L. In addition, gentamicin 2 mg/L was combined with imipenem, doxycycline, or tigecycline. Finally, polymyxin 0.25, 0.5, 1, 2 and 4 mg/L was tested alone and combined with rifampicin, imipenem, and both agents. The concentrations tested were chosen to reflect clinically relevant levels. Antibiotic carryover was eliminated by diluting the final cultures 200-fold in pour plates. Separate experiments demonstrated no antibiotic carryover effect at this dilution. Bactericidal activity was defined as a decrease of 3 log cfu/mL in 24 h. Synergy was defined as a
100-fold increase in killing at 24 h by a combination compared with the most active single agent, assuming no significant inhibition by at least one agent. Antagonism was defined as a
100-fold decrease in killing at 24 h by a combination compared with the most active single agent.
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Results |
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The results of timekill studies for various antibiotics and antibiotic combinations are summarized in Table 2. At 24 h, imipenem, rifampicin, doxycycline, cefotetan, and the combination of imipenem plus clavulanate produced no significant inhibition. Tigecycline was bactericidal against two isolates, and generally produced a bacteriostatic effect. Gentamicin was rapidly bactericidal against gentamicin-susceptible isolates. The combinations of gentamicin with imipenem, doxycycline, or tigecycline produced no interaction. Polymyxin B demonstrated concentration-dependent killing, and was bactericidal against most strains at 2 or 4 mg/L. The combination of polymyxin B plus rifampicin was bactericidal against 15/16 isolates at 1 mg/L polymyxin, was synergic for 15/16 isolates at 0.5 x MIC of polymyxin, and was consistently the most active regimen. For the isolate with a polymyxin B MIC of 16 mg/L, a decrease of 2 log cfu/mL was demonstrated with the combination of rifampicin with subinhibitory concentrations of polymyxin B. The combination of polymyxin B (0.5 x MIC) with imipenem was synergic for 10/16 isolates but was antagonistic for three isolates. The addition of imipenem to the combination of polymyxin B plus rifampicin had no effect.
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Discussion |
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The carbapenem-hydrolysing enzymes KPC-1, KPC-2 and KPC-3 have been demonstrated to reside on plasmids,7,10,11 facilitating the transmission of carbapenem resistance among strains. Indeed, ribotyping of this collection revealed that eight unrelated strains possessed KPC-2. KPC-2 has also been found in two strains of Enterobacter sp. and one strain of K. oxytoca in New York City.2,15 Nevertheless, the fingerprinting data demonstrate that the problem is primarily clonal in nature. The presence of this highly resistant clone in most regional hospitals suggests that joint efforts at patient identification and infection control will be important to contain the spread of this problem.
The isolates collected in this study were not only broadly resistant to ß-lactams, but also to fluoroquinolones and variably to aminoglycosides. In many cases, none of the commonly used antibiotics are likely to be useful and effective therapeutic regimens have not been defined. Although many isolates were susceptible to doxycycline, the MICs of these isolates were at the NCCLS breakpoint of 4 mg/L. In timekill studies, doxycycline was ineffective at a clinically relevant concentration. Therefore, the use of doxycycline against these strains cannot be recommended. Gentamicin demonstrated bactericidal activity for gentamicin-susceptible isolates, and may be considered for infections likely to respond to aminoglycoside therapy. Tigecycline, an experimental glycylcycline, was generally bacteriostatic at 2 mg/L and may prove to be useful. Clinical data on the use of tigecycline in this setting are lacking.
Increasingly, clinicians are using polymyxins as agents of last resort for multiresistant Gram-negative pathogens. Preliminary data on the use of colistin for carbapenem-resistant A. baumannii and P. aeruginosa suggest that this agent is useful.17,18 A single case has also been reported of the successful use of colistin for sepsis due to carbapenem-resistant K. pneumoniae.19 This study provides evidence of concentration-dependent bactericidal activity of polymyxin B against a collection of multiresistant K. pneumoniae isolates. The combination of polymyxin B plus rifampicin was synergic against nearly all isolates, including two isolates that were resistant to polymyxin B. Polymyxin B plus rifampicin may remain a treatment option for such strains. Whether this combination proves to be clinically effective, and whether use of the combination can prevent the emergence of polymyxin resistance, remains to be determined.
Carbapenem-resistant K. pneumoniae possessing KPC enzymes appear to be spreading through hospitals in New York City. The outbreak is characterized by the presence of multiple clones, with one dominant strain affecting most hospitals. In vitro data suggest that polymyxin B ± rifampicin, or possibly tigecycline alone, may prove to be useful therapeutic options. Coordinated infection control efforts will be important to help contain the spread of these pathogens.
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Acknowledgements |
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References |
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2. Bratu S, Landman D, Haag R et al. Rapid spread of carbapenem-resistant Klebsiella pneumoniae in New York City: A new threat to our antibiotic armamentarium. Arch Intern Med 2005; 49: in press.
3.
Landman D, Quale JM, Mayorga D et al. Citywide clonal outbreak of multiresistant Acinetobacter baumannii and Pseudomonas aeruginosa in Brooklyn, NY: the preantibiotic era has returned. Arch Intern Med 2002; 162: 151520.
4. Quale J, Bratu S, Landman D et al. Molecular epidemiology and mechanisms of carbapenem resistance in Acinetobacter baumannii endemic in New York City. Clin Infect Dis 2003; 37: 21420.[CrossRef][ISI][Medline]
5.
Hyunjoo P, Kim JW, Kim J et al. Carbapenem resistance mechanisms in Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 2001; 45: 4804.
6. Bradford PA, Urban C, Mariano N et al. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC ß-lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother 1997; 41: 5639.[Abstract]
7.
Yigit H, Queenan AM, Anderson GJ et al. Novel carbapenem-hydrolyzing ß-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother 2001; 45: 115161.
8. Bradford PA, Bratu S, Urban C et al. Emergence of carbapenem-resistant Klebsiella spp. possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 ß-lactamases in New York City. Clin Infect Dis 2004; 39: 5560.[CrossRef][ISI][Medline]
9.
Moland ES, Black JA, Ourada J et al. Occurrence of newer ß-lactamases in Klebsiella pneumoniae isolates from 24 U.S. hospitals. Antimicrob Agents Chemother 2002; 46: 383742.
10.
Moland ES, Hanson ND, Herrera VL et al. Plasmid-mediated, carbapenem-hydrolysing ß-lactamase, KPC-2, in Klebsiella pneumoniae isolates. J Antimicrob Chemother 2003; 51: 7114.
11.
Woodford N, Tierno Jr, PM, Young K et al. Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A ß-lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother 2004; 48: 47939.
12. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: 14th Informational Supplement. NCCLS document M100-S14. NCCLS, Wayne, PA, USA, 2004.
13.
Gales AC, Reis AO, Jones RN. Contemporary assessment of antimicrobial susceptibility testing methods for polymyxin B and colistin: review of available interpretative criteria and quality control guidelines. J Clin Microbiol 2001; 39: 18390.
14.
Tenover FC, Arbeit RD, Goering RV et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995; 33: 22339.
15.
Bratu S, Landman D, Alam M et al. Recovery of KPC carbapenem-hydrolyzing enzymes in Enterobacter spp. from Brooklyn, NY. Antimicrob Agents Chemother 2005; 49: 7768.
16. Landman D, Mobarakai NK, Quale JM. Novel antibiotic regimens against Enterococcus faecium resistant to ampicillin, vancomycin, and gentamicin. Antimicrob Agents Chemother 1993; 37: 19048.[Abstract]
17. Levin AS, Barone AA, Penco J et al. Intravenous colistin as therapy for nosocomial infections caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. Clin Infect Dis 1999; 28: 100811.[ISI][Medline]
18. Linden PK, Kusne S, Coley K et al. Use of parenteral colistin for the treatment of serious infection due to antimicrobial-resistant Pseudomonas aeruginosa. Clin Infect Dis 2003; 37: e15460.[CrossRef][ISI][Medline]
19. Karabinis A, Paramythiotou E, Mylona-Petropoulou D et al. Colistin for Klebsiella pneumoniae-associated sepsis. Clin Infect Dis 2004; 38: e79.[CrossRef][ISI][Medline]