Adler, H.

Fenner, L.

Frei, R.

Hohler, D.

Oezcan, S.

Schultheiss, E.

Walter, P.

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5. Naas T, Nordmann P, Vedel G et al. Plasmid-mediatedcarbapenem-hydrolyzing b-lactamase KPC in a Klebsiella pneumoniaeisolate from France. Antimicrob Agents Chemother 2005; 49: 4423–4.6. Woodford N, Tierno PM Jr, Young K et al. Outbreak of Klebsiellapneumoniae producing a new carbapenem-hydrolyzing class Ab-lactamase, KPC-3, in a New York medical center. Antimicrob AgentsChemother 2004; 48: 4793–9.Journal of Antimicrobial Chemotherapydoi:10.1093/jac/dkm472Advance Access publication 7 December 2007Plasmid-mediated AmpC b-lactamases inEnterobacteriaceae lacking inducible chromosomalampC genes: prevalence at a Swiss universityhospital and occurrence of the different moleculartypes in SwitzerlandH. Adler*, L. Fenner, P. Walter, D. Hohler, E. Schultheiss,S. Oezcan and R. FreiMicrobiology Laboratory, University Hospital Basel,Petersgraben 4, 4031 Basel, SwitzerlandKeywords: resistance genes, antibiotic resistance, plasmids*Corresponding author. Tel: þ41-61-2654248; Fax: þ41-61-2655355; E-mail: hadler@uhbs.chSir,Since 1989, plasmid-mediated AmpC b-lactamases have beenknown to exist in various species lacking inducible chromoso-mal ampC genes such as Klebsiella spp., Escherichia coli,Proteus mirabilis and Salmonella. They descend from chromo-somal ampC genes and fall into six phylogenetic groups.Origins are the ampC genes of Hafnia alvei, Morganella morga-nii, Citrobacter freundii, Enterobacter cloacae and twounknown organisms.1 Organisms producing plasmid-mediatedAmpC b-lactamases raise special concerns because of the highrate of clinical failure among infected patients.2 We report hereon the prevalence of plasmid-mediated AmpC b-lactamases inisolates of Enterobacteriaceae lacking an inducible chromosomalampC gene at University Hospital Basel and the occurrenceof different molecular types of plasmid-mediated AmpC b-lactamases in Switzerland.Between 27 January 2006 and 27 January 2007, a total of 3217consecutive clinical isolates of various species ofEnterobacteriaceae lacking inducible chromosomal ampC genes(i.e. 2434 E. coli, 174 Klebsiella oxytoca, 459 Klebsiella pneumo-niae, 8 Klebsiella spp., 134 P. mirabilis, 7 Salmonella entericassp. enterica and 1 Shigella flexneri) were screened for resistanceto cefoxitin with the disc diffusion test according to CLSI guide-lines.3 Isolates with an inhibition zone diameter of ,18 mm wereconsidered putative AmpC producers and were stored at 2708Cfor further investigation. Additionally, 45 clinical isolates sus-pected to harbour a plasmid-mediated AmpC b-lactamase werecollected from 5 laboratories situated in Switzerland. ampC geneswere identified by a ampC multiplex PCR with primers specificfor the genes of six different phylogenetic groups.1 For sequen-cing, the ampC genes were amplified by PCR using primer pairsas described previously for blaCMY and blaDHA, respectively.4,5 Inaddition, blaCMY-31 was amplified using a second pair of primers.6PCR products were purified using Montage PCR Units (Millipore,Zug, Switzerland) and sequencing reactions were carried out usinga BigDye Terminator v1.1 Cycle Sequencing Kit (AppliedBiosystems, Rotkreuz, Switzerland) as described by the manufac-turer. Sequencing products were purified with Dye Ex 2.0 Spin Kit(Qiagen, Hombrechtikon, Switzerland) and electrophoresis wasperformed with the 3130 Genetic Analyzer (Applied Biosystems).The isoelectric point (pI) of the new b-lactamase was determinedby isoelectric focusing, applying the supernatants of crude soniccell 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 aspI markers. A filter paper containing 2.5% flucloxacillin(GlaxoSmithKline, Munchenbuchsee, Switzerland) as an inhibitorof the AmpC b-lactamase was applied for 2 min to one of the gelsbefore staining with nitrocefin (Oxoid, Basel, Switzerland).Of 3217 consecutive clinical isolates that were obtained at ourhospital, 124 (3.8%; 103 E. coli, 3 K. oxytoca and 18K. pneumoniae) were resistant to cefoxitin and were thus con-sidered putative AmpC producers. Among these, five isolates (allof them E. coli) carrying an ampC gene known to beplasmid-encoded were found by ampC multiplex PCR. Thus,the prevalence of plasmid-mediated AmpC b-lactamases atUniversity Hospital Basel was 0.16% for Enterobacteriaceaelacking an inducible chromosomal ampC gene (0.2% for E. coli).Overall, plasmid-mediated AmpC b-lactamases were foundin 17 isolates obtained from five Swiss laboratories. Details aregiven in Table 1. Fourteen of the isolates were E. coli, twowere K. pneumoniae and one was P. mirabilis. Fifteen of theplasmid-mediated AmpC b-lactamases were CIT enzymes, aphylogenetic group that has its origin in the chromosomalampC gene of C. freundii. Fourteen of them were CMY-2. Forone isolate of K. pneumoniae, sequencing of the full generevealed a unique sequence that has been designated CMY-31(GenBank accession no. EF622224). The derived amino acidsequence differed from CMY-2 by one amino acid (Q235R).Isoelectric focusing revealed a b-lactamase with a pI between8.8 and 9.0. Enzyme activity of CMY-31 was inhibited by flu-cloxacillin which is indicative of an AmpC b-lactamase.Enzyme activity of the ESBLs that served as pI markers wasretained. This is the first report of the isolation and character-ization of CMY-31, a new b-lactamase which is closelyrelated to CMY-2. DHA-1, a plasmid-mediated AmpCb-lactamase that has its origin in the chromosomal ampC geneTable 1. Occurrence of various molecular types of AmpCMolecular type of AmpC Number of isolates SpeciesCMY-2 12 E. coliCMY-2 1 K. pneumoniaeCMY-2 1 P. mirabilisDHA-1 2 E. coliCMY-31 1 K. pneumoniaeResearch letters457of M. morganii, was detected in two isolates of E. coli.DHA-1 is an inducible plasmid-mediated AmpC b-lactamasewhose emergence raises concerns because the mortality ofpatients infected with organisms that produce DHA-1 has beenshown to be higher than that of patients infected with organ-isms that produce CMY-1.2In conclusion, we have demonstrated that plasmid-mediatedAmpC b-lactamases have emerged in Switzerland. The preva-lence of 0.16% is still low. However, the occurrence of DHA-1,an inducible type of enzyme, raises clinical concerns.Additionally, a novel plasmid-mediated AmpC b-lactamase,which was designated CMY-31, was found.FundingThis study was funded by the Department of LaboratoryMedicine, University Hospital Basel.Transparency declarationsNone to declare.References1. Perez-Perez FJ, Hanson ND. Detection of plasmid-mediatedAmpC b-lactamase genes in clinical isolates by using multiplex PCR.J Clin Microbiol 2002; 40: 2153–62.2. Pai H, Kang CI, Byeon JH et al. Epidemiology and clinical fea-tures of bloodstream infections caused by AmpC-type-b-lactamase-producing Klebsiella pneumoniae. Antimicrob Agents Chemother 2004;48: 3720–8.3. Clinical and Laboratory Standards Institute. PerformanceStandards for Antimicrobial Susceptibility Testing: SixteenthInformational Supplement M100-S16. CLSI, Wayne, PA, USA, 2006.4. Winokur PL, Brueggemann A, DeSalvo DL et al. Animal andhuman multidrug-resistant, cephalosporin-resistant salmonella isolatesexpressing a plasmid-mediated CMY-2 AmpC b-lactamase. AntimicrobAgents Chemother 2000; 44: 2777–83.5. Muratani T, Kobayashi T, Matsumoto T. Emergence and preva-lence of b-lactamase-producing Klebsiella pneumoniae resistant tocephems in Japan. Int J Antimicrob Agents 2006; 27: 491–9.6. Liebana E, Gibbs M, Clouting C et al. Characterization ofb-lactamases responsible for resistance to extended-spectrum cepha-losporins in Escherichia coli and Salmonella enterica strains from food-producing animals in the United Kingdom. Microb Drug Resist 2004;10: 1–9.Journal of Antimicrobial Chemotherapydoi:10.1093/jac/dkm483Advance Access publication 21 December 2007Prevalence and characterization ofmacrolide-lincomycin-streptogramin B-resistantStaphylococcus aureus in Korean hospitalsYoung-Hee Jung1, Kwang Wook Kim1, Kyeong Min Lee1,Jae Il Yoo1, Gyung Tae Chung1, Bong Soo Kim2,Hyo-Sun Kwak3 and Yeong Seon Lee1*1Division of Antimicrobial Resistance, Center for InfectiousDisease Research, National Institute of Health, 194, Tongil-Lo, Eunpyung-Gu, Seoul 122-701, Republic of Korea;2Division of Biodefence Research, Center for InfectiousDisease Research, National Institute of Health, 194, Tongil-Lo, Eunpyung-Gu, Seoul 122-701, Republic of Korea; 3FoodMicrobiology Team, Center for Food Safety Evaluation,Korea Food and Drug Administration, 194, Tongil-Lo,Eunpyung-Gu, Seoul 122-704, Republic of KoreaKeywords: macrolide resistance genes, double disc diffusion,non-tertiary hospitals*Corresponding author. Tel: þ82-2-380-1478; Fax: þ82-2-380-1550; E-mail: yslee07@nih.go.krSir,Resistance to macrolide, lincosamide and streptogramin B(MLSB) antibiotics in Staphylococcus spp. is mediated by amethylase encoded by erythromycin ribosome methylation (erm)genes or ATP transporter efflux pumps encoded by the msr ormef genes. The methylation of the 50S ribosomal subunit byerm gene products causes constitutive or inducible resistance toMLSB antibiotics. Constitutively resistant MLSB (MLSBc)strains and inducibly resistant MLSB (MLSBi) strains expresserm genes, erm(A) or/and erm(C). In this study, we investigatedand characterized the constitutive and inducible resistance, andthe susceptibility to clindamycin of Staphylococcus aureus iso-lated from non-tertiary hospitals.We collected 508 S. aureus strains from 157 hospitals nation-wide from January to June 2005 in Korea. Antimicrobial suscep-tibility to oxacillin, erythromycin, clindamycin and quinupristin/dalfopristin and the MICs were evaluated according to theguidelines of the CLSI. Of the 508 S. aureus isolates, 46.1%(234 isolates) were resistant to oxacillin, 55.1% (280 isolates) wereresistant to erythromycin, which comprised 98.3% (230/234)methicillin-resistant S. aureus (MRSA) strains and 18.2%(50/274) methicillin-susceptible S. aureus (MSSA) strains, and37.4% (190 isolates) were resistant to clindamycin. No strainswere susceptible to erythromycin and resistant to clindamycin.One hundred and eighty-six MRSA (80.9%) and four MSSA(8.0%) were MLSBc (resistant to erythromycin and clindamycin;Table 1). D-test according to Steward’s method was used for iso-lates with erythromycin resistance and clindamycin susceptibility.1Forty-three MRSA (18.7%) and 40 MSSA (80.0%) were MLSBi(showing the D form on the D-test), and 1 MRSA (0.4%) and 6MSSA (12.0%) were MLSBs (susceptible to clindamycin).The MLSB resistance genes, erm(A), erm(B), erm(C),msr(A), msr(B) and mef, were detected using a PCR method.2erm(A) was detected in 250 strains, erm(C) in 14 strains, erm(B)in 1 strain, msr(A) in 3 strains, erm(A) and erm(C) in 2 strains,erm(A) and msr(A) in 3 strains and no genes in 7 strains.Most MLSBc and MLSBi strains carried the erm(A) orerm(C) gene (Table 1). The erm(A) gene (in 250 isolates) wasdetected far more frequently than erm(C) (in 14 strains).However, the erm(C) gene was distributed more frequently inMLSBi strains (12 isolates) than in MLSBc strains (2 isolates).Using OLIGO primers designed by Arthur et al.,3 the PCR pro-ducts were detected in three MLSBc or MLSBi strains lackingerm(A), erm(B) or erm(C). It was possible that the three strainscarry an erm gene besides erm(A), erm(B) or erm(C). msr(A)Research letters458

Plasmid-mediated AmpC β-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes: prevalence at a Swiss university hospital and occurrence of the different molecular types in Switzerland

Plasmid-mediated AmpC β-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes: prevalence at a Swiss university hospital and occurrence of the different molecular types in Switzerland

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