Journal of Antimicrobial Chemotherapy (2005) 56, 128–132 doi:10.1093/jac/dki175 Advance Access publication 25 May 2005

Carbapenemase-producing Klebsiella pneumoniae in Brooklyn, NY: molecular epidemiology and in vitro activity of polymyxin B and other agents Simona Bratu, Pooja Tolaney, Usha Karumudi, John Quale, Mohamad Mooty, Satyen Nichani and David Landman* 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

Objectives: To describe the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae in Brooklyn, NY and assess the in vitro activity of various antibiotic combinations. 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. Time–kill 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 time– kill studies involving 16 isolates, tigecycline demonstrated bacteriostatic activity and polymyxin B concentration-dependent bactericidal activity. The combination of polymyxin B at 0.5 · 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, b-lactamases, imipenem, tigecycline

Introduction Carbapenem antibiotics have been important agents for the management of Gram-negative infections, particularly when caused by difficult nosocomial pathogens. Carbapenems are considered to be the agents of choice for the treatment of infections due to Enterobacteriaceae possessing extended-spectrum b-lactamases (ESBLs). The prevalence of ESBL-producing Klebsiella pneumoniae has been rising in the United States,1 and is approaching 50% of isolates in some regions.2 When such high rates of ESBL-producing organisms are encountered, carbapenems become an increasingly important therapeutic option.

Over the past few years, a progressive increase in carbapenemresistant 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.4–6 Carbapenemhydrolysing b-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.7–11 These isolates are often resistant to multiple antibiotic classes, presenting clinicians with

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*Corresponding author. Tel: +1-718-270-3790; Fax: +1-718-270-2465; E-mail: [email protected] .............................................................................................................................................................................................................................................................................................................................................................................................................................

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Carbapenem-resistant Klebsiella pneumoniae 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.

Methods Between August 2003 and June 2004, isolates of K. pneumoniae were referred to the central research laboratory from the microbiology laboratories of 10 Brooklyn, NY hospitals. Isolates were referred because of their unusual resistance to carbapenem antibiotics. MIC testing was performed by the agar dilution or broth microdilution methods according to NCCLS guidelines12 for the following antibiotics: imipenem, meropenem, ertapenem, cefotetan, ceftazidime, cefepime, piperacillin plus tazobactam (4 mg/L), gentamicin, tobramycin, amikacin, ciprofloxacin, doxycycline, tigecycline, chloramphenicol, polymyxin B and rifampicin. In addition, imipenem was tested in combination with clavulanic acid (2 mg/L). Susceptibility breakpoints were as recommended by the NCCLS;12 a breakpoint of 2 mg/L has been recommended for polymyxin B13 and proposed for tigecycline. ATCC strains P. aeruginosa 27853, Staphylococcus aureus 29213 and Escherichia coli 35218 were included as controls. All isolates underwent ribotyping with the Riboprinter 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 b-lactamases was tested by PCR as previously described.15 The following primers were used: KPC forward: 50 -ATGTCACTGTATCGCCGTCT-30 and KPC reverse 50 -TTTTCAGAGCCTTACTGCCC-30 . 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: 50 -AGCTGAACTCCGCCATCC-30 . Sequences were identified by the BLAST program from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/BLAST/). Time–kill 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.

Results A total of 96 isolates were referred to the central research laboratory during the study period. The results of susceptibility testing are shown in Table 1. The isolates were highly resistant to the carbapenems with nearly all having an MIC > 32 mg/L. The addition of clavulanic acid to imipenem lowered the MIC twofold in approximately one-third of isolates. Whereas 45% and 66% were susceptible to amikacin and doxycycline, respectively, nearly all had MICs at the respective breakpoints of 16 and 4 mg/L. Over 90% remained susceptible to polymyxin B, and all were inhibited by tigecycline 2 mg/L (Table 1). PCR testing revealed the presence of a blaKPC gene in 95 of 96 isolates. Ribotyping of the 95 KPC-positive isolates revealed eight distinct ribotypes (Figure 1). However, 78/95 (82%) were a single type (ribotype 1), 12/95 (13%) were a second type (ribotype 2), and the remaining six isolates were unique types. Isolates belonging to ribotypes 1 and 2 were detected at 9/10 and 7/10 hospitals, respectively. PFGE generally correlated with the ribotyping results, but only identified five of eight strain types (Figure 1). Sixteen isolates were selected for further in vitro testing, including the six unique strains and 10 representative isolates of ribotypes 1 and 2. The ribotype results for the 16 isolates are shown

Table 1. Susceptibility results for 96 isolates of K. pneumoniae from 10 Brooklyn, NY hospitals Antibiotic Imipenem Meropenem Ertapenem Cefotetan Ceftazidime Cefepime Piperacillin/tazobactam Gentamicin Tobramycin Amikacin Ciprofloxacin Doxycycline Tigecycline Rifampicin Chloramphenicol Polymyxin B

Susceptible

Intermediate

Resistant

MIC50

MIC90

Range

0% 1% 0% 59% 2% 40% 0% 61% 3% 45% 2% 66% 100% 4% 7% 91%

1% 0% 0% 18% 0% 30% 1% 6% 3% 52% 0% 10% 0% 7% 0%

99% 99% 100% 23% 98% 30% 99% 33% 94% 3% 98% 24% 0% 89% 93% 9%

>32 >32 >32 16 >32 16 >128 2 >16 32 >8 4 0.5 16 >32 2

>32 >32 >32 >64 >32 >32 >128 >16 >16 32 >8 >32 1 >16 >32 2

8–>32 4–>32 8–>32 1–>64 2–>32 2–>32 64–>128 £0.25–>16 0.25–>16 0.5–>64 £0.125–>8 1–>32 0.25–2 £0.25–>16 £1–>32 0.5–16

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Ribotype # 1 PFGE type mw A

1 A

1 A

1 A

3 A

4 A

5 C

1 A

1 2 A mw B

2 B

2 B

6 B

2 B

7 D

8 E mw

kb 485 388 291 242 194 145 97 45

Figure 1. Ribotyping (top) and PFGE (bottom) results for 16 isolates of carbapenem-resistant K. pneumoniae possessing KPC-2.

in Figure 1. Representative isolates from ribotypes 1 and 2 were selected to include some that were susceptible and resistant to gentamicin and doxycycline. Two of the isolates were resistant to polymyxin B (MICs 8 and 16 mg/L) and the remainder had MICs of 0.5–2 mg/L. Sequencing of the blaKPC gene revealed that all 16 possessed KPC-2. The results of time–kill 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 concentrationdependent 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 · 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 · 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.

Discussion Over the past decade, carbapenem resistance has become a serious problem that primarily affected non-lactose-fermenting bacteria. Carbapenem-resistant Enterobacteriaceae have remained uncommon. In a previous report involving
600 isolates of K. pneumoniae collected during a citywide survey in Brooklyn in 2003, nine KPC-producing isolates were recovered.2 The current report provides evidence that these multiresistant pathogens are an increasing problem. Isolates from nearly 100 unique patients were referred during an 11 month period. The isolates were selected at the discretion of the individual microbiology laboratories and did not necessarily include all carbapenem-resistant isolates. In addition, the presence of carbapenem resistance in KPC-producing K. pneumoniae may be missed by automated microdilution susceptibility testing.2 These factors suggest that an even greater number of patients may have been affected during the study period. 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

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Carbapenem-resistant Klebsiella pneumoniae Table 2. Results of time–kill studies against 16 isolates of carbapenem-resistant K. pneumoniae

Antibiotic (concentration, mg/L) None Imipenem (4) Imipenem (4) + clavulanate (2) Rifampicin (1) Imipenem (4) + rifampicin (1) Cefotetan (8) Doxycycline (4) Tigecycline (2) Gentamicin (2)—gent.-resistant Gentamicin (2)—gent. susceptible Polymyxin B (0.5) Polymyxin B (1) Polymyxin B (2) Polymyxin B (4) Polymyxin B (1) + rifampicin (1) Polymyxin B (1) + imipenem (4) Polymyxin B (0.5 · MIC) Polymyxin (0.5 · MIC) + rifampicin Polymyxin (0.5 · MIC) + imipenem Polymyxin (0.5 · MIC) + rifampicin + imipenem

Change (mean – SD) log cfu/mL at 4 h +2.3 –0.04 –0.1 +2.3 +0.4 +0.7 +1.1 –0.4 +1.8 –3.6 –1.6 –3.3 –4.1 –4.3 –4.0 –3.4 –2.5 –3.6 –2.6 –3.3

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 b-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 time–kill 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.

– – – – – – – – – – – – – – – – – – – –

0.3 1.4 1.6 0.4 1.4 2.2 1.2 1.6 1.8 1.2 1.8 1.4 1.4 1.1 1.0 1.7 1.0 1.2 1.6 1.0

Change (mean – SD) log cfu/mL at 24 h

No. with decrease ‡3 log cfu/mL at 24 h

– – – – – – – – – – – – – – – – – – – –

0/16 0/16 0/16 0/16 0/16 0/16 0/16 2/16 0/9 7/7 2/16 7/16 12/16 13/16 15/16 11/16 0/16 14/16 10/16 13/16

+3.0 +2.7 +2.7 +2.7 +2.7 +2.6 +2.7 –0.06 +2.6 –4.9 +1.7 –0.9 –3.4 –3.9 –4.8 –3.2 +2.1 –4.4 –2.0 –4.2

0.4 0.7 0.9 0.5 0.6 0.3 0.7 1.9 0.5 0.5 2.2 3.6 2.7 2.5 1.0 3.2 0.7 1.9 3.7 2.1

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.

Acknowledgements This work was supported by grants from Merck & Co., Inc, WyethAyerst Pharmaceuticals, Elan Pharmaceuticals, and Pfizer, Inc.

References 1. National Nosocomial Infections Surveillance. System Report, data summary from January 1992 through June 2003, issued August 2003. Am J Infect Control 2003; 31: 481–98. 2. Bratu S, Landman D, Haag R et al. Rapid spread of carbapenemresistant 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: 1515–20. 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: 214–20. 5. Hyunjoo P, Kim JW, Kim J et al. Carbapenem resistance mechanisms in Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother 2001; 45: 480–4.

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Bratu et al. 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 b-lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother 1997; 41: 563–9. 7. Yigit H, Queenan AM, Anderson GJ et al. Novel carbapenemhydrolyzing b-lactamase, KPC-1, from a carbapenem-resistant strain of Klebsiella pneumoniae. Antimicrob Agents Chemother 2001; 45: 1151–61. 8. Bradford PA, Bratu S, Urban C et al. Emergence of carbapenemresistant Klebsiella spp. possessing the class A carbapenem-hydrolyzing KPC-2 and inhibitor-resistant TEM-30 b-lactamases in New York City. Clin Infect Dis 2004; 39: 55–60. 9. Moland ES, Black JA, Ourada J et al. Occurrence of newer b-lactamases in Klebsiella pneumoniae isolates from 24 U.S. hospitals. Antimicrob Agents Chemother 2002; 46: 3837–42. 10. Moland ES, Hanson ND, Herrera VL et al. Plasmid-mediated, carbapenem-hydrolysing b-lactamase, KPC-2, in Klebsiella pneumoniae isolates. J Antimicrob Chemother 2003; 51: 711–4. 11. Woodford N, Tierno Jr, PM, Young K et al. Outbreak of Klebsiella pneumoniae producing a new carbapenem-hydrolyzing class A b-lactamase, KPC-3, in a New York Medical Center. Antimicrob Agents Chemother 2004; 48: 4793–9. 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: 183–90. 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: 2233–9. 15. Bratu S, Landman D, Alam M et al. Recovery of KPC carbapenemhydrolyzing enzymes in Enterobacter spp. from Brooklyn, NY. Antimicrob Agents Chemother 2005; 49: 776–8. 16. Landman D, Mobarakai NK, Quale JM. Novel antibiotic regimens against Enterococcus faecium resistant to ampicillin, vancomycin, and gentamicin. Antimicrob Agents Chemother 1993; 37: 1904–8. 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: 1008–11. 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: e154–60. 19. Karabinis A, Paramythiotou E, Mylona-Petropoulou D et al. Colistin for Klebsiella pneumoniae-associated sepsis. Clin Infect Dis 2004; 38: e7–9.

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