Detection of aac(6 0 )-Ib-cr in KPC-producing Klebsiella pneumoniae isolates from Tel Aviv, Israel

Journal of Antimicrobial Chemotherapy (2009) 64, 718 722 doi:10.1093/jac/dkp272 Advance Access publication 4 August 2009 Detection of aac(6 0 )-Ib-cr in KPC-producing Klebsiella pneumoniae isolates from Tel Aviv, Israel Inna Chmelnitsky 1 *, Orit Hermesh 1, Shiri Navon-Venezia 1, Jacob Strahilevitz 2 and Yehuda Carmeli 1 1 Division of Epidemiology, Tel Aviv Medical Center, Tel Aviv, Israel; 2 Department of Clinical Microbiology and Infectious Diseases, Hadassah-Hebrew University, Jerusalem, Israel Received 2 March 2009; returned 15 April 2009; revised 28 June 2009; accepted 5 July 2009 Objectives: We aimed to evaluate the occurrence and characteristics of plasmid-mediated quinolone resistance (PMQR) genes in KPC-producing (KPC-P) Klebsiella pneumoniae (Kpn) isolates in Tel Aviv Medical Center, Israel. Methods: Forty-seven KPC-P Kpn isolates were studied. Antibiotic susceptibilities were determined by Vitek 2, Etest or agar dilution. b-lactamases and PMQR determinants were detected by PCR. For plasmid characterization, transformation, transconjugation, restriction mapping and Southern blot analysis were performed. Results: Six out of 47 (13%) KPC-P isolates carried aac(6 0 )-Ib-cr. Acquisition of aac(6 0 )-Ib-cr-encoding plasmids increased the MIC of ciprofloxacin by 2-fold. In five of six KPC-P isolates, aac(6 0 )-Ib-cr and bla KPC-2 were encoded on the same plasmid. Conclusions: The most prevalent PMQR gene in the studied KPC-P K. pneumoniae isolates is aac(6 0 )- Ib-cr. The co-existence of PMQR genes with KPC on the same plasmid poses a serious epidemiological, clinical and public-health threat. Keywords: b-lactamases, carbapenem resistance, quinolone resistance, plasmids Introduction Multiple plasmid-mediated quinolone resistance (PMQR) determinants, including the five qnr genes qnra, qnrb, qnrs, qnrc and qnrd, 1 5 the aminoglycoside acetyltransferase-encoding enzyme variant aac(6 0 )-Ib-cr 6 and the efflux-pump-encoding qepa, 7 are all involved in resistance to fluoroquinolones. These genes confer low-level quinolone resistance that can facilitate the selection of chromosomal mutations and high-level resistance. 8 With the increasing prevalence of plasmid-encoded Klebsiella pneumoniae carbapenemase (KPC) in various parts of the world, PMQR genes were sought on the same plasmids. The co-existence of qnra and qnrb with bla KPC has been reported in K. pneumoniae from China and the USA. 9 We recently reported the co-existence of qnrb2 and bla KPC-2 on a single plasmid in Enterobacter cloacae from Israel. One of these isolates also carried aac(6 0 )-Ib-cr on a separate plasmid. 10 Approximately 6% of the K. pneumoniae isolates in the Tel Aviv Medical Center carry KPC (S. Navon-Venezia, M. Schwaber and Y. Carmeli, unpublished results). We aimed to evaluate systematically the occurrence of the PMQR determinants qnr and aac(6 0 )-Ib-cr among KPC-producing K. pneumoniae clinical isolates in the Tel Aviv Medical Center. Materials and methods Bacterial strains, susceptibility and genetic relatedness Forty-seven KPC-producing (KPC-P) K. pneumoniae single patient isolates collected in Tel Aviv Medical Center, a 1200 bed tertiary care teaching hospital, between the years 2004 and 2006 were studied. Isolates were molecularly characterized and antibiotic susceptibilities were determined by Vitek 2 (BioMerieux Inc., Marcy, France). MICs of carbapenems, and ciprofloxacin and levofloxacin were determined via Etest (AB Biodisk, Solna, Sweden), and of nalidixic acid by agar dilution (CLSI guidelines 11 ). Genetic relatedness was determined using PFGE after SpeI restriction. PFGE DNA macrorestriction patterns were compared using GelComparII software (Applied Maths, Belgium).... *Corresponding author. Tel: þ972-3-6974892; Fax: þ972-3-6974332; E-mail: innach@tasmc.health.gov.il... 718 # The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

aac(6 0 )-Ib-cr in KPC-producing K. pneumoniae PMQR and b-lactamase screening PCR using primers specific for bla KPC (F: ATGTCACTGTATCGCC GTCT; R: TTTTCAGAGCCTTACTGCCC) followed by sequencing was performed. Screening for qnra, qnrb and qnrs was carried out using multiplex PCR with primers described by Robicsek et al. 12 Screening with primers specific for qnrc (F: GGGTTGTAC ATTTATTGAATCG; R: CACCTACCCATTTATTTTCA), qnrd 5 and qepa (F: AACTGCTTGAGCCCGTAGAT; R: GTCTACGCC ATGGACCTCAC) was performed. The presence of aac(6 0 )-Ib-cr was determined by PCR followed by BtsCI (New England BioLabs, Beverly, MA, USA) digestion and sequencing. Gene fragments surrounding the quinolone resistance-determining region in gyra and parc were amplified using primers gyra6 and gyra613r (F: CGA CCTTGCGAGAGAAAT; R: GTTCCATCAGCCCTTCAA), and parc91 and parc583r (F: TACGTCATCATGGACAGG; R: GCCA CTTCACGCAGGTTG), respectively, and sequenced. Screening for the presence of extended-spectrum b-lactamase (ESBL) genes in PMQR-positive isolates was carried out. Amplification using primers recognizing the bla SHV -group (F: TTTATCGGCCYTCACT CAAGG; R: GCTGCGGGCCGGATAACG) and the bla TEM -group (F: KACAATAACCCTGRTAAATGC; R: AGTATATATGAGT AAACTTGG) genes was performed. Obtained amplicons were sequenced. PCR with primers specific for bla CTX-M-2 (F: ATGA TGACTCAGAGCATTCG; R: TTATTGCATCAGAAACCGTG), bla CTX-M-25 (F: CACACGAATTGAATGTTCAG; R: TCACTCCACA TGGTGAGT) and bla CTX-M-9 (F: GTGACAAAGAGAGTGCAA CGG; R: ATGATTCTCGCCGCTGAAGCC) groups was performed. The bla CTX-M-3 -group genes were amplified using the primers CTX-M-3FF (GTTGTTGTTATTTCGTATCTTCC) and CTX-M-3FR (CGATAAACAAAAACGGAATG), and sequenced. Plasmid analysis and transfer of PMQR genes Plasmids were isolated using the NucleoBond PC 100 midi kit (Macherey-Nagel, Düren, Germany). Transformation was performed by electroporation (Electroporator 2510, Eppendorf, Hamburg, Germany) into Escherichia coli strain DH10B (Invitrogen, Carlsbad, CA, USA). Transformants were selected on LB agar plates containing 100 mg/l ampicillin. Transformed colonies were screened for the presence of aac(6 0 )-Ib-cr via PCR followed by BtsCI digestion. The presence of bla CTX-M-15, bla KPC and bla SHV in the transformed colonies was verified by PCR. Size estimation of transformed plasmids was carried out by treatment with S1 nuclease (Promega, Madison, WI, USA), followed by PFGE as described previously. 13 The Lambda Ladder PFG marker (New England Biolabs, Ipswich, MA, USA) was used as a molecular size marker. Transconjugation experiments were performed with isolate 475, using the filter-mating method. The rifampicin-resistant K. pneumoniae strain 29RR was used as a recipient strain. Transconjugants were selected on Mueller Hinton plates containing 400 mg/l rifampicin and 8 mg/l ceftazidime. Obtained colonies were screened for the presence of aac(6 0 )-Ib-cr via PCR followed by BtsCI digestion and subjected to PFGE genotyping to confirm the occurrence of transconjugation. For Southern blot analysis, S1-digested DNA from donors and transformants followed by electrophoresis was transferred to a Hybond N þ membrane (Amersham Biosciences, Buckinghamshire, UK) and UV cross-linked. A 481 bp amplicon of aac(6 0 )-Ib-cr (nucleotides 97 578) and a complete nucleotide sequence of bla KPC-2 (882 bp) were used to generate probes for aac(6 0 )-Ib-cr and bla KPC-2, respectively. Probes were labelled using random primer DNA labelling mixture (Biological Industries, Beit Haemek, Israel). Results Prevalence of PMQR determinants and antibiotic susceptibilities Forty-seven KPC-P K. pneumoniae isolates were characterized, of which 43 (91%) were resistant to quinolones. Two isolates were resistant to levofloxacin and had an intermediate ciprofloxacin MIC (2 mg/l). The qnr groups and the qepa gene were not detected via PCR screening. PCR followed by digestion with BtsCI showed the restriction pattern of an aac(6 0 )-Ib-cr variant in six of the isolates (12.8%). Sequencing confirmed the presence of aac(6 0 )-Ib-cr by revealing two known mutations (W102R and D179Y) in all six isolates. These aac(6 0 )-Ib-cr-carrying isolates belonged to three different genetic clones. Two isolates (belonging to clone P) were susceptible to quinolones and four isolates (belonging to clones B and S) were resistant. Susceptibility data for the other antimicrobial agents are presented in Table 1. All KPC-producing aac(6 0 )-Ib-cr-carrying isolates carried bla KPC-2. Two strains, belonging to two different clones (B and P), also carried bla CTX-M-15. All isolates belonging to clones B and S carried bla SHV-1 ; isolates 469 and 475 (clone P) carried bla SHV-27. Genes belonging to the bla TEM family and to bla CTX-M groups other than bla CTX-M-15 were not detected via PCR screening. All four quinolone-resistant isolates (clones B and S) had, in addition to PMQR genes, two mutations in gyra and one mutation in parc (Table 1). Plasmid analysis Transconjugation of the aac(6 0 )-Ib-cr-carrying plasmid derived from K. pneumoniae isolate 475 was successful. The MIC of ciprofloxacin for the transconjugant increased 2.6-fold (from 0.38 to 1 mg/l), relative to the recipient clone 29RR. The transconjugant carried a plasmid with aac(6 0 )-Ib-cr and bla KPC-2. The bla SHV-27 gene was not transferred by transconjugation. Transformation experiments succeeded for all KPC-producing isolates, except for K. pneumoniae isolate 475. MICs of ciprofloxacin in the transformants increased by 1.5- to 2-fold as compared with the E. coli DH10B recipient strain, whereas MICs of levofloxacin did not change upon plasmid acquisition, as expected. MICs of carbapenems increased in most of the transformants, although they did not reach the same MICs as those of the donors (Table 1). PCR analysis of transformants revealed that in isolates belonging to clone S, aac(6 0 )-Ib-cr co-existed with bla KPC-2 on the same plasmid. In isolate 469 (clone P), aac(6 0 )-Ib-cr co-existed with bla KPC-2 and bla CTX-M-15 on the same plasmid. In clone B, isolate aac(6 0 )-Ib-cr co-existed with bla CTX-M-15 on the same plasmid, whereas bla KPC-2 was carried on a different plasmid. The bla SHV genes were not transferable (Table 1). The three isolates derived from clone S carried aac(6 0 )-Ibcr-encoding plasmids similar in size (migrated between lambda marker bands 48.5 97 kb) (Figure 1a and b, lanes 525 D,T, 526 D,T and 531 D,T), but different in size from aac(6 0 )-Ib-cr-encoding plasmids of clone B (48.5 kb) (Figure 1a and b, lanes 365 D,T1) and of clone P (97 kb) (Figure 1a and b, lanes 469 D,T). Southern blot analysis of donors and transformants hybridized with aac(6 0 )-Ib-cr or bla KPC-2 probes confirmed that 719

Table 1. PFGE, resistance genes and antibiotic susceptibilities of the six PMQR-carrying KPC-producing K. pneumoniae (Kpn) clinical isolates and their transformants 720 MIC (mg/l) Bacteria PFGE PMQR gyra parc bla genes NAL CIP LVX AMK GEN TOB ATM IPM MEM CRO FEP CAZ Kpn 365 B aac(6 0 )-Ib-cr S83F D87N S80I KPC-2, SHV-1, CTX-M-15.256.32.32 16 16 16 64 24 4 64 64 64 365-T a 1 aac(6 0 )-Ib-cr CTX-M-15 2 0.004 0.006 8 16 16 64 0.25 0.032 64 8 64 365-T2 KPC-2 2 0.002 0.006 2 1 1 64 8 1 8 2 16 Kpn 525 S aac(6 0 )-Ib-cr S83F D87N S80I KPC-2, SHV-1.256.32.32 16 1 16 64 24 16 64 16 64 525-T aac(6 0 )-Ib-cr KPC-2 2 0.004 0.006 16 1 8 64 8 1.5 8 2 16 Kpn 526 S aac(6 0 )-Ib-cr S83F D87N S80I KPC-2, SHV-1.256.32.32 4 1 8 64 24.32 64 8 32 526-T aac(6 0 )-Ib-cr KPC-2 2 0.004 0.004 16 0.025 8 64 6 1.5 16 2 16 Kpn 531 S aac(6 0 )-Ib-cr S83F D87N S80I KPC-2, SHV-1.256.32.32 16 1 16 64.32.32 64 32 64 531-T aac(6 0 )-Ib-cr KPC-2 2 0.003 0.006 16 1 16 64 4 1.5 16 2 16 Kpn 469 P aac(6 0 )-Ib-cr no mutations KPC-2, SHV-27, 4 0.064 0.064 4 16 16 64 1.5 2 64 64 64 detected CTX-M-15 469-T aac(6 0 )-Ib-cr KPC-2, CTX-M-15 2 0.004 0.006 8 16 16 64 4 1 64 16 64 Recipient DH10B 2 0.002 0.006 2 1 1 1 1 0.25 1 1 1 Kpn 475 P aac(6 0 )-Ib-cr no mutations KPC-2, SHV-27 32 0.38 0.38 2 16 16 64 8 24 64 8 64 detected 475-Tc b aac(6 0 )-Ib-cr KPC-2 256 1 0.38 16 16 16 64 8 12 32 2 16 29RR 256 0.38 0.25 2 1 1 1 0.25 0.25 1 1 1 Chmelnitsky et al. NAL, nalidixic acid; CIP, ciprofloxacin; LVX, levofloxacin; AMK, amikacin; GEN, gentamicin; TOB, tobramycin; ATM, aztreonam; IPM, imipenem; MEM, meropenem; CRO, ceftriaxone; FEP, cefepime; CAZ, ceftazidime. a Transformant. b Transconjugant.

aac(6 0 )-Ib-cr in KPC-producing K. pneumoniae kb 291.0 242.5 194.0 145.5 97.0 48.5 (a) (b) 365 469 525 526 531 M D T1 T2 D T D T D T D T aac(6 ')-Ib-cr varies between species; it has been reported to be more prevalent in E. coli than in K. pneumoniae or Enterobacter spp. The presence of PMQR determinants, aac(6 0 )-Ib-cr and qnr, together with the KPC gene among nosocomial pathogens, is of great importance as it further limits the usefulness of quinolones in the treatment of these extremely drug-resistant strains. The co-existence of aac(6 0 )-Ib-cr with bla CTX-M-15, with or without bla TEM-1 and bla OXA-1, has been previously described in E. coli and K. pneumoniae strains. 9 Here, we extended this finding to an even more difficult-to-treat pathogen, and found the co-existence of aac(6 0 )-Ib-cr and bla KPC-2 on the same plasmid in two distinct clones of K. pneumoniae clinical isolates. The co-carriage provides an evolutionary benefit to these strains in an antibiotic-rich environment, and leads to their selection under both b-lactam and quinolone pressure. In this respect, aac(6 0 )-Ib-cr provides an even greater advantage by also conferring resistance to certain aminoglycosides. The aac(6 0 )Ib-cr-carrying plasmid was transconjugable. This provides an advantage in the dissemination of the aac(6 0 )-Ib-cr gene between clones, and even between species, which may contribute to its spread. For all these reasons, increasing awareness of the presence of PMQR determinants in clinical strains, and especially in extremely drug-resistant strains such as KPC-P Klebsiella strains, is necessary. (c) bla KPC-2 Acknowledgements This work was presented in part at the Forty-eighth Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 2008 (Abstract C1-128). We would like to express our gratitude to Ms Moran Goren for providing us with the sequence for the bla CTX-M-3 -group primers and to Ms Azita Leavitt for supplying us with the rifampicin-resistant recipient strain K. pneumoniae 29RR. Figure 1. PFGE of KPC-P isolates and their transformants digested with S1 nuclease. The positions of the molecular size marker (Lambda Ladder PFG marker) are indicated to the left of the gel (a). Hybridization with aac(6 0 )-Ib-cr (b) and KPC-2 (c). D, donor; T, transconjugant. aac(6 0 )-Ib-cr and bla KPC-2 were encoded on different plasmids in clone B, and on the same plasmid in clones S and P (Figure 1b and c). In clone S, two additional plasmids hybridized with the aac(6 0 )-Ib-cr probe with lower intensity. Discussion The most prevalent PMQR gene among carbapenem-resistant K. pneumoniae isolates included in this study is aac(6 0 )-Ib-cr and was found in 6/47 (13%) of all isolates tested. In most isolates (5/6) it is co-carried with the bla KPC-2 gene on the same plasmid. Among the PMQR-carrying isolates, the bla CTX-M-15 gene is the most prevalent ESBL gene. The aac(6 0 )-Ib-cr gene is involved in resistance to fluoroquinolones as well as to aminoglycosides. 6 The gene has been detected in various locations, including the USA, Canada, Argentina, Portugal, France, China and Spain. 9 Its prevalence Funding This work was supported by the Research Fund of Chief Scientist Office, Ministry of Health (Jerusalem, Israel), and by the Morasha 1833/07 grant from the Israel Science Foundation awarded to Dr Jacob Strahilevitz. Transparency declarations None to declare. References 1. Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet 1998; 351: 797 9. 2. Jacoby GA, Walsh KE, Mills DM et al. qnrb, another plasmidmediated gene for quinolone resistance. Antimicrob Agents Chemother 2006; 50: 1178 82. 3. Hata M, Suzuki M, Matsumoto M et al. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b. Antimicrob Agents Chemother 2005; 49: 801 3. 721

Chmelnitsky et al. 4. Wang M, Guo Q, Xu X et al. New plasmid-mediated quinolone resistance gene, qnrc, found in a clinical isolate of Proteus mirabilis. Antimicrob Agents Chemother 2009 53: 1892 7. 5. Cavaco LM, Hasman H, Xia S et al. qnrd, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin. Antimicrob Agents Chemother 2009; 53: 603 8. 6. Robicsek A, Strahilevitz J, Jacoby GA et al. Fluoroquinolonemodifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006; 12: 83 8. 7. Perichon B, Courvalin P, Galimand M. Transferable resistance to aminoglycosides by methylation of G1405 in 16S rrna and to hydrophilic fluoroquinolones by QepA-mediated efflux in Escherichia coli. Antimicrob Agents Chemother 2007; 51: 2464 9. 8. Martinez-Martinez L, Pascual A, Garcia I et al. Interaction of plasmid and host quinolone resistance. J Antimicrob Chemother 2003; 51: 1037 9. 9. Martinez-Martinez L, Eliecer Cano M, Rodriguez-Martinez JM et al. Plasmid-mediated quinolone resistance. Expert Rev Anti Infect Ther 2008; 6: 685 711. 10. Chmelnitsky I, Navon-Venezia S, Strahilevitz J et al. Plasmid-mediated qnrb2 and carbapenemase gene bla KPC-2 carried on the same plasmid in carbapenem-resistant ciprofloxacin-susceptible Enterobacter cloacae isolates. Antimicrob Agents Chemother 2008; 52: 2962 5. 11. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Nineteenth Informational Supplement M100-S19. CLSI, Wayne, PA, USA, 2009. 12. Robicsek A, Strahilevitz J, Sahm DF et al. qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. Antimicrob Agents Chemother 2006; 50: 2872 4. 13. Li L, Lim CK. A novel large plasmid carrying multiple b-lactam resistance genes isolated from a Klebsiella pneumoniae strain. J Appl Microbiol 2000; 88: 1038 48. 722