Research letters The nucleotide sequences of the three types of tet(S)containing structure were deposited in GenBank with accession nos EF679789, EF682210 and EF682209.
Funding This work was supported by a grant NSC 94-2320-B-002-095 from the National Science Council of Taiwan.
Transparency declarations None to declare.
References
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkm455 Advance Access publication 20 November 2007
Intercontinental travels of patients and dissemination of plasmid-mediated carbapenemase KPC-3 associated with OXA-9 and TEM-1 Laurent Dortet1, Irina Radu1, Vale´rie Gautier2, Franc¸ois Blot3, Elisabeth Chachaty1 and Guillaume Arlet2,4* 1
Service de Microbiologie Me´dicale, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France; 2 Universite´ Pierre et Marie Curie-Paris 6, EA 2392, Laboratoire de Bacte´riologie, Paris, France; 3Service de Re´animation, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France; 4Assistance Publique—Hoˆpitaux de Paris, Hoˆpital Tenon, Service de Bacte´riologie, Paris, France Keywords: b-lactamases, carbapenems, Enterobacter cloacae *Correspondence address. Service de Bacte´riologie, Hoˆpital Tenon, 4 rue de la Chine, 75970 Paris Cedex 20, France. Tel: þ33-1-56-01-70-18; E-mail:
[email protected]
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1. Tsai JC, Hsueh PR, Chen HJ et al. The erm(T) gene is flanked by IS1216V in inducible erythromycin-resistant Streptococcus gallolyticus subsp. pasteurianus. Antimicrob Agents Chemother 2005; 49: 4347–50. 2. Brenciani A, Ojo KK, Monachetti A et al. Distribution and molecular analysis of mef(A)-containing elements in tetracycline-susceptible and -resistant Streptococcus pyogenes clinical isolates with effluxmediated erythromycin resistance. J Antimicrob Chemother 2004; 54: 991–8. 3. Ng LK, Martin I, Alfa M et al. Multiplex PCR for the detection of tetracycline resistant genes. Mol Cell Probes 2001; 15: 209–15. 4. Teng LJ, Hsueh PR, Wang YH et al. Determination of Enterococcus faecalis groESL full-length sequence and application for species identification. J Clin Microbiol 2001; 39: 3326– 31. 5. Perreten V, Schwarz F, Cresta L et al. Antibiotic resistance spread in food. Nature 1997; 389: 801– 2.
Sir, Among b-lactam antibiotics, carbapenems are those that have the highest stability towards the hydrolytic activity of most of the innate natural and acquired extended-spectrum b-lactamases prevalent within bacterial species involved in clinical practice. However, emergence of acquired carbapenem-hydrolysing b-lactamases, i.e. carbapenemases, which were first recognized in the early 1990s in some isolates of opportunistic Gram-negative bacilli, has been increasingly reported from various parts of the world during the last decade.1 Carbapenem-hydrolysing b-lactamases can be metallo-b-lactamases, expanded-spectrum oxacillinases or Ambler class A enzymes.1,2 KPC b-lactamases are class A carbapenemases, which were first detected in 2001 in a Klebsiella pneumoniae isolate from North Carolina.3 Soon afterwards, several reports documented the emergence of Enterobacteriaceae strains from various species producing KPC-2 and KPC-3 b-lactamase variants in the eastern USA and recently disseminated in other countries worldwide, such as France, Columbia, Israel and China.2 A 31-year-old man was admitted to the intensive care unit of the Institut Gustave-Roussy in November 2005 with sepsis related to intra-abdominal suppuration. The patient had previously undergone a total gastrectomy for intractable gastric bleeding episodes during a trip to New York and had stayed for 3 weeks in the intensive care unit of a local hospital. Blood cultures taken at admission in our hospital yielded a K. pneumoniae isolate fully susceptible to antimicrobial agents usually active against this species. Specimens taken during subsequent surgical debridement of abdominal abscesses yielded an Enterobacter cloacae isolate resistant to all b-lactam antibiotics including imipenem (Table 1), aminoglycosides, sulphonamides and fluoroquinolones and an Enterococcus faecium isolate resistant to ampicillin and vancomycin, and which carried the vanA gene (detected by PCR). A double disc diffusion assay with imipenem and amoxicillin/ clavulanate discs showed a small increase in imipenem activity against the E. cloacae isolate, whereas no synergistic effect was observed with EDTA (data not shown), thus excluding the presence of a metallo-b-lactamase. Using ceftazidime (2 mg/L)/ sodium azide (400 mg/L) as selective agent and azide-resistant Escherichia coli C600 as recipient strain, a conjugative experiment was performed. It allowed the transfer en bloc of resistance to b-lactam antibiotics including imipenem (Table 1), aminoglycosides and sulphonamides. Plasmid analysis showed that both the clinical and the transconjugant strains carried a unique plasmid, more than 126 kb in size. The gene encoding for KPC b-lactamase was detected by PCR in both the donor and recipient strains. Sequencing of the amplified fragment showed it to display 100% identity with blaKPC-3 (GenBank accession number AF395881). PCR amplification with specific primers and sequencing analysis showed that both the clinical isolate and the transconjugant carried blaTEM-1. The genetic environment of these b-lactamase-encoding genes was further investigated using a cloning approach. Total DNA of the transconjugant strain was partially digested with the endonuclease Sau3A, and fragments were ligated into a pACYC184 vector (Fermentas, St Re´my Les Chevreuse, France) before transformation of the recipient strain, E. coli DH10B. By selection on medium supplemented with ceftazidime (2 mg/L),
Research letters Table 1. MICs of b-lactams for a clinical strain of E. cloacae, a transconjugant strain (Tc E. coli C600), E. coli C600, recombinant E. coli DH10B producing KPC-3 enzyme (KPC-3 clone), recombinant E. coli DH10B producing TEM-1 enzyme (TEM-1 clone), recombinant E. coli DH10B producing OXA-9 enzyme (OXA-9 clone) and E. coli DH10B MIC (mg/L) for b-Lactamsa
a
Tc E. coli C600
E. coli C600
E. coli DH10B (blaKPC-3)
E. coli DH10B (blaTEM-1)
E. coli DH10B (blaOXA-9)
E. coli DH10B
.128 .128 .128 .128 .256 .256 .256 .64 .32 .64 .32 32 32 .64 .32 .16 2
.128 .128 .128 .128 .256 64 16 64 0.25 64 3 8 0.25 64 2 .16 0.19
4 2 8 ND 0.5 0.5 ND 0.06 ND 0.12 ND 0.012 ND 0.12 0.06 0.12 ND
.128 .128 .128 .128 .256 128 .256 32 32 32 32 64 8 .256 .32 8 2
.128 16 .128 32 128 4 1.5 0.064 0.064 0.06 0.06 0.012 0.012 0.06 0.032 0.19 0.19
16 2 64 8 48 0.5 1.5 0.064 0.064 0.06 0.06 0.012 0.012 1.5 0.064 0.19 0.19
2 2 1 1 1 0.5 ND ,0.06 ND ,0.06 ND 0.06 ND 0.06 ND 0.06 ND
CLA, clavulanic acid at a fixed concentration of 2 mg/L; TZB, tazobactam at a fixed concentration of 4 mg/L.
we obtained a clone resistant to imipenem (Table 1) that produced the KPC-3 enzyme. Analysis of the blaKPC-3 cloning fragment (GenBank accession number AM774409) showed that blaKPC-3 displayed the same genetic environment as blaKPC-2 in Salmonella Cubana 4707 (GenBank accession number AF481906). By selection on ticarcillin (50 mg/L), we obtained two different phenotypes of resistance to b-lactams. Sequencing of the inserts showed the presence of blaTEM-1 or blaOXA-9 (Table 1). Accurate intragenic region amplifications of the OXA-9 fragment suggested blaTEM-1 and blaOXA-9 to be part of Tn1331, as has been previously described for the multiresistance plasmid pJHCMW1 of K. pneumoniae (GenBank accession number AF479774).4 This is the first KPC-3 b-lactamase-producing E. cloacae strain recovered from a patient in France. Interestingly, a KPC-2-producing K. pneumoniae strain has been recently recovered from another patient admitted to a hospital in Paris.5 A common feature of these two patients was that they stayed in intensive care units of New York City hospitals before their admission to a French hospital. More recently, K. pneumoniae and E. coli isolates harbouring blaKPC-2 were reported in Columbia, Israel and China, with no discernible linkage to the USA.2 Furthermore, as recently described, our KPC-producing strain was found to accumulate other b-lactam resistance enzymes (OXA-9 and TEM-1).2 Emergence of KPC b-lactamase-producing strains of Enterobacteriaceae and, recently, of Pseudomonaceae is worrisome, because they are resistant to all b-lactam antibiotics and often also to most other classes of the available antibacterial agents.2 Moreover, some reports have focused on the ability of
some KPC b-lactamase-producing K. pneumoniae strains to cause outbreaks in medical centres in New York City.6 Evidence of intercontinental transfer of KPC b-lactamaseproducing strains through patient travel stresses the risk for the rapid worldwide dissemination of these recently recognized carbapenemases.
Funding This work was partially funded by grants from the Faculte´ de Me´decine Pierre et Marie Curie, Universite´ Paris 6 and from the European Community (6th PCRD Contract: LSHM-CT 2003-503335).
Transparency declarations None to declare.
References 1. Nordmann P, Poirel L. Emerging carbapenemases in Gram-negative aerobes. Clin Microbiol Infect 2002; 8: 321–31. 2. Walther-Rasmussen J, Hoiby N. Class A carbapenemases. J Antimicrob Chemother 2007; 60: 470–82. 3. 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. 4. Sarno R, McGillivary G, Sherratt DJ et al. Complete nucleotide sequence of Klebsiella pneumoniae multiresistance plasmid pJHCMW1. Antimicrob Agents Chemother 2002; 46: 3422–7.
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Amoxicillin AmoxicillinþCLA Ticarcillin TircarcillinþCLA Piperacillin PiperacillinþTZB PiperacillinþCLA Cefotaxime CefotaximeþCLA Ceftazidime CeftazidimeþCLA Cefepime CefepimeþCLA Aztreonam AztreonamþCLA Imipenem ImipenemþCLA
E. cloacae
Research letters 5. Naas T, Nordmann P, Vedel G et al. Plasmid-mediated carbapenem-hydrolyzing b-lactamase KPC in a Klebsiella pneumoniae isolate from France. Antimicrob Agents Chemother 2005; 49: 4423–4. 6. Woodford N, Tierno PM Jr, 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.
Journal of Antimicrobial Chemotherapy doi:10.1093/jac/dkm472 Advance Access publication 7 December 2007
Plasmid-mediated AmpC b-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes: prevalence at a Swiss university hospital and occurrence of the different molecular types in Switzerland H. Adler*, L. Fenner, P. Walter, D. Hohler, E. Schultheiss, S. Oezcan and R. Frei
Keywords: resistance genes, antibiotic resistance, plasmids *Corresponding author. Tel: þ41-61-2654248; Fax: þ41-612655355; E-mail:
[email protected] Sir, Since 1989, plasmid-mediated AmpC b-lactamases have been known to exist in various species lacking inducible chromosomal ampC genes such as Klebsiella spp., Escherichia coli, Proteus mirabilis and Salmonella. They descend from chromosomal ampC genes and fall into six phylogenetic groups. Origins are the ampC genes of Hafnia alvei, Morganella morganii, Citrobacter freundii, Enterobacter cloacae and two unknown organisms.1 Organisms producing plasmid-mediated AmpC b-lactamases raise special concerns because of the high rate of clinical failure among infected patients.2 We report here on the prevalence of plasmid-mediated AmpC b-lactamases in isolates of Enterobacteriaceae lacking an inducible chromosomal ampC gene at University Hospital Basel and the occurrence of different molecular types of plasmid-mediated AmpC blactamases in Switzerland. Between 27 January 2006 and 27 January 2007, a total of 3217 consecutive clinical isolates of various species of Enterobacteriaceae lacking inducible chromosomal ampC genes (i.e. 2434 E. coli, 174 Klebsiella oxytoca, 459 Klebsiella pneumoniae, 8 Klebsiella spp., 134 P. mirabilis, 7 Salmonella enterica ssp. enterica and 1 Shigella flexneri) were screened for resistance to cefoxitin with the disc diffusion test according to CLSI guidelines.3 Isolates with an inhibition zone diameter of ,18 mm were considered putative AmpC producers and were stored at 2708C for further investigation. Additionally, 45 clinical isolates suspected to harbour a plasmid-mediated AmpC b-lactamase were collected from 5 laboratories situated in Switzerland. ampC genes were identified by a ampC multiplex PCR with primers specific
Table 1. Occurrence of various molecular types of AmpC Molecular type of AmpC CMY-2 CMY-2 CMY-2 DHA-1 CMY-31
457
Number of isolates
Species
12 1 1 2 1
E. coli K. pneumoniae P. mirabilis E. coli K. pneumoniae
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Microbiology Laboratory, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland
for the genes of six different phylogenetic groups.1 For sequencing, the ampC genes were amplified by PCR using primer pairs as described previously for blaCMY and blaDHA, respectively.4,5 In addition, blaCMY-31 was amplified using a second pair of primers.6 PCR products were purified using Montage PCR Units (Millipore, Zug, Switzerland) and sequencing reactions were carried out using a BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Rotkreuz, Switzerland) as described by the manufacturer. Sequencing products were purified with Dye Ex 2.0 Spin Kit (Qiagen, Hombrechtikon, Switzerland) and electrophoresis was performed with the 3130 Genetic Analyzer (Applied Biosystems). The isoelectric point (pI) of the new b-lactamase was determined by isoelectric focusing, applying the supernatants of crude sonic cell extracts to Phast gels (GE HealthCare, Fairfield, CT, USA) with a pH gradient of 3–9 in a Phast system (GE HealthCare). Extended-spectrum b-lactamases (ESBLs) with known pI values (TEM-1, TEM-12, SHV-12 and CTX-M15) were included as pI markers. A filter paper containing 2.5% flucloxacillin (GlaxoSmithKline, Munchenbuchsee, Switzerland) as an inhibitor of the AmpC b-lactamase was applied for 2 min to one of the gels before staining with nitrocefin (Oxoid, Basel, Switzerland). Of 3217 consecutive clinical isolates that were obtained at our hospital, 124 (3.8%; 103 E. coli, 3 K. oxytoca and 18 K. pneumoniae) were resistant to cefoxitin and were thus considered putative AmpC producers. Among these, five isolates (all of them E. coli) carrying an ampC gene known to be plasmid-encoded were found by ampC multiplex PCR. Thus, the prevalence of plasmid-mediated AmpC b-lactamases at University Hospital Basel was 0.16% for Enterobacteriaceae lacking an inducible chromosomal ampC gene (0.2% for E. coli). Overall, plasmid-mediated AmpC b-lactamases were found in 17 isolates obtained from five Swiss laboratories. Details are given in Table 1. Fourteen of the isolates were E. coli, two were K. pneumoniae and one was P. mirabilis. Fifteen of the plasmid-mediated AmpC b-lactamases were CIT enzymes, a phylogenetic group that has its origin in the chromosomal ampC gene of C. freundii. Fourteen of them were CMY-2. For one isolate of K. pneumoniae, sequencing of the full gene revealed a unique sequence that has been designated CMY-31 (GenBank accession no. EF622224). The derived amino acid sequence differed from CMY-2 by one amino acid (Q235R). Isoelectric focusing revealed a b-lactamase with a pI between 8.8 and 9.0. Enzyme activity of CMY-31 was inhibited by flucloxacillin which is indicative of an AmpC b-lactamase. Enzyme activity of the ESBLs that served as pI markers was retained. This is the first report of the isolation and characterization of CMY-31, a new b-lactamase which is closely related to CMY-2. DHA-1, a plasmid-mediated AmpC b-lactamase that has its origin in the chromosomal ampC gene
