Carbapenemase-producing Enterobacteriaceae, U.S. rivers.Our study was initiated by previous isolation of 30 imipenem-resistant, gram-negative rods from 7 of 16 U.S. rivers sampled from 1999 to 2001. Imipenem hydrolysis hydrolysis (hīdrŏl`ĭsĭs), chemical reaction of a compound with water, usually resulting in the formation of one or more new compounds. was detected in 22 of those isolates identified as Enterobacter asburiae. Random amplified polymorphism DNA analysis DNA analysis Any technique used to analyze genes and DNA. See Chromosome walking, DNA fingerprinting, Footprinting, In situ hybridization, Jeffries' probe, Jumping libraries, PCR, RFLP analysis, Southern blot hybridization. showed that these E. asburiae isolates were genetically indistinguishable. An identical clavulanic acid-inhibited [beta]-cactamase IMI-2 was identified from each isolate that shared 99% and 97% amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. identity with the chromosome-encoded [beta]-lactamases IMI-1 and NmcA, respectively, from E. cloacae clinical isolates. The blaIMI-2 gene was located on a self-transferable 66-kb plasmid. Sequence analysis of a cloned 5.5-kb DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. fragment obtained from 1 of the imipenem-resistant E. asburiae isolates identified an upstream LysR-type regulator gene regulator gene n. A gene that causes the production of a protein that represses the activity of another gene in an operon. that explained inducibility of IMI-2 expression. [beta]-Lactamase IMI-2 is the first inducible and plasmid-encoded carbapenemase. Identification of clonally related E. asburiae isolates from distant rivers indicates an environmental and enterobacterial reservoir for carbapenemase genes. ********** Carbapenems, such as imipenem and meropenem, are the most potent [beta]-lactam antimicrobial drugs for avoiding resistance in gram-negative rods. Resistance to carbapenems is rare in Enterobacteriaceae and may be mediated by 3 mechanisms: hyperproduction of an AmpC-type cephalosporinase combined with decreased drug permeability through the outer membrane, decreased affinity of penicillin-binding proteins that constitute target proteins for carbapenems, and carbapenem-hydrolyzing [beta]-lactamases (1-3). These rare carbapenemases may be either plasmid-mediated metallo-[beta]-lactamases (IMP- and VIM-type) or chromosomally encoded and clavulanate-inhibited enzymes (NmcA, IMI-1, Sme-1/Sme-2) (2,4 9). The latter group of enzymes shares consistent percentage of identity and belongs to the Ambler class A of [beta]-lactamases (2,10). Very recently, plasmid-mediated and clavulanate-inhibited carbapenernases have been reported as a source of nosocomial infections Nosocomial infections Infections that were not present before the patient came to a hospital, but were acquired by a patient while in the hospital. Mentioned in: Enterobacterial Infections, Staphylococcal Infections in U.S. hospitals (11-15). While the role of animals in the emergence of clinically important, antimicrobial-resistant strains has been extensively shown (e.g., in Salmonella spp.), the role of aquatic environment as a reservoir of antimicrobial-resistance genes is less established (16-21). A recent study described high levels of antimicrobial-resistant strains from U.S. rivers (22). We identified the imipenem-resistant, gram-negative strains recovered from that study and analyzed the molecular mechanism involved in carbapenem resistance of the imipenem-resistant enterobacterial strains. Clonally related Enterobacter asburiae strains were identified in midwestern U.S. rivers. E. asburiae naturally produces a cephalosporinase but no carbapenemase and may be responsible for nosocomial infections (23). Here, the strains expressed a novel plasmid-encoded and clavulanate-inhibited carbapenemase. Materials and Methods Bacterial Isolates A previous study identified 30 imipenem-resistant, gram-negative strains out of 1,861 ampicillin-resistant, gram-negative isolates from 7 out of 16 U.S. rivers that were sampled from 1999 to 2001 (22). Identification of these imipenem-resistant isolates was performed by conventional biochemical techniques (API-20E and API-NE systems [bioMerieux, Marcy-l'Etoile, France]), and confirmed by 16S rDNA sequencing (24). E. asburiae CIP (1) (Common Isochronous Packet) The packet format used in time-based (real time) FireWire transmission. See FireWire, IEC 61883 and mLAN. (2) (Common Industrial P 103358 and E. asburiae CIP 105006 were used as reference strains (Institut Pasteur strain collection, Paris, France). E. cloacae NOR-1 and E. cloacae 1413B were used as strains that produce the chromosome-encoded, clavulanate-inhibited carbapenemases NmcA and IMI-1, respectively (5,8). One of the E. asburiae isolates recovered from a river (strain MS7) was used for cloning experiments. Streptomycin-resistant Escherichia coli Escherichia coli (ĕsh'ərĭk`ēə kō`lī), common bacterium that normally inhabits the intestinal tracts of humans and animals, but can cause infection in other parts of the body, especially the urinary tract. DH10B strain was used in cloning and conjugation conjugation, in genetics conjugation, in genetics: see recombination. conjugation, in grammar conjugation: see inflection. experiments (kite Technologies, Eragny, France). Antimicrobial Agents and Resistance Study The antimicrobial agents and their sources were as follows: amoxicillin amoxicillin /amox·i·cil·lin/ (ah-mok?si-sil´in) a semisynthetic derivative of ampicillin effective against a broad spectrum of gram-positive and gram-negative bacteria. a·mox·i·cil·lin n. , ceftazidime, clavulanic acid clav·u·lan·ic acid n. A drug that inhibits the action of beta-lactamase produced by bacteria, thereby counteracting bacterial resistance to beta-lactam antibiotics. , and ticarcillin (GlaxoSmithKline, Nanterre, France); aztreonam (Bristol-Myers Squibb, Paris La Defense Paris La Defense - Une Ville En Concert was a concert held by musician Jean Michel Jarre on the district of La Defense Paris on Bastille Day, July 14, 1990. About 2,5 million people standing in front of the pyramidical stage all the way down to the Arc de Triomphe witnessed this , France); cephalothin cephalothin a first generation cephalosporin antibiotic. Sensitive organisms include many penicillin-resistant staphylococci. cephalothin Cefalotin® Infectious disease A parenteral semisynthetic derivative of cephalosporin C, and 3 (Eli Lilly, Saint-Cloud, France); piperacillin and tazobactam (Lederle, Les Oullins, France); cefotaxime (Aventis, Romainville, France); imipenem (without cilastatin) (Merck Sharp and Dohme, Paris, France); meropenem (AstraZeneca, Paris, France); ampicillin ampicillin (ăm'pĭsĭl`ĭn), a penicillin-type antibiotic that is effective against both gram-negative microorganisms and gram-positive microorganisms such as Escherichia coli. and streptomycin streptomycin (strĕp'tōmī`sĭn), antibiotic produced by soil bacteria of the genus Streptomyces and active against both gram-positive and gram-negative bacteria (see Gram's stain), including species resistant to other (Sigma, Paris, France). MICs were determined by an agar dilution technique on Mueller-Hinton (MH) agar (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) with an inoculum inoculum /in·oc·u·lum/ (-ok´u-lum) pl. inoc´ula material used in inoculation. in·oc·u·lum n. pl. of [10.sup.4] CFU CFU see colony-forming units. per spot (25). Carbapenemase activity was determined by UV spectrophotometry spectrophotometry Branch of spectroscopy dealing with measurement of radiant energy transmitted or reflected by a body as a function of wavelength. The measurement is usually compared to that transmitted or reflected by a system that serves as a standard. with culture extracts of each of the imipenem-resistant, gram-negative rods and imipenem (100 [micro]mol) as substrate, as reported previously (26). One unit of enzyme activity Enzyme activity A measure of the ability of an enzyme to catalyze a specific reaction. Mentioned in: Glucose-6-Phosphate Dehydrogenase Deficiency corresponded to the hydrolysis of 1 [micro]mol of substrate per min. Inducibility of the [beta]-lactamase expression was determined with imipenem and cefoxitin as [beta]-lactamase inducers, as described (27). Briefly overnight culture of each imipenem-resistant E. asburiae isolate was diluted (1:10) in a prewarmed trypticase soy broth, allowed to culture in an antimicrobial-free medium for 2 h, and further cultured for 6 h with cefoxitin (2-50 mg/L) or imipenem (10-50 mg/L). [beta]-Lactamase culture extracts were obtained after centrifugation Centrifugation A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal and sonication sonication /son·i·ca·tion/ (son?i-ka´shun) exposure to sound waves; disruption of bacteria by exposure to high-frequency sound waves. son·i·ca·tion n. , as detailed (26). Nucleic Acid nucleic acid, any of a group of organic substances found in the chromosomes of living cells and viruses that play a central role in the storage and replication of hereditary information and in the expression of this information through protein synthesis. Techniques and Conjugation Genotype comparison of the imipenem-resistant E. asburiae strains was performed by using the random amplified polylnorphism detection (RAPD RAPD Randomly Amplified Polymorphic DNA RAPD relative afferent pupillary defect (ophthalmology; aka Marcus-Gunn Pupil) ) technique as described with primer 6MW (CCGACTCGAG NNNNNNATGTGG) and primers UBC UBC Uniform Building Code UBC University of British Columbia UBC Union of the Baltic Cities UBC United Brotherhood of Carpenters UBC Universal Battery Charger UBC Union of Baltic Cities UBC Universal Bibliographic Control UBC Used Beverage Cans 245 and UBC 282 (26,28,29). Transfer of the imipenem resistance marker from each imipenem-resistant E. asburiae isolate to E. coli E. coli: see Escherichia coli. E. coli in full Escherichia coli Species of bacterium that inhabits the stomach and intestines. E. coli can be transmitted by water, milk, food, or flies and other insects. DH10B was attempted by using the immobilization Immobilization Definition Immobilization refers to the process of holding a joint or bone in place with a splint, cast, or brace. This is done to prevent an injured area from moving while it heals. filter mating out technique, as described (26). Briefly, equal volume (0.1 mL) of overnight cultures of each E. asburiae isolate and E. coli DH 10B were put onto a paper filter that was placed on an MH agar plate. Twenty-four hours later, the filter was removed, washed with water (0.2 mL), and the bacterial suspension was spread onto MH agar plates containing ampicillin (100 mg/L) and streptomycin (50 nag/L) for selecting transconjugants after 24 h (26). Plasmid extraction was performed for each E. asburiae strain and their transconjugants and compared to reference plasmid sizes of E. coli NCTC NCTC National Conservation Training Center NCTC National Counterterrorism Center (9/11 Commission Report) NCTC National Cable Television Cooperative NCTC National Collection of Type Cultures (UK laboratory) 50192 by using the Kieser technique designed to extract large size plasmids (30,31). Whole-cell DNA of Enterobacter spp. reference strains and of an E. asburiae strain MS7 was extracted as described (26). Southern hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun) 1. crossbreeding; the act or process of producing hybrids. 2. molecular hybridization 3. of plasmid DNA (26) of the transconjugants was performed as described by the manufacturer with the ECL (Emitter-Coupled Logic) A digital circuit composed of bipolar transistors in which the emitter ends are wired together. ECL gates switch faster than TTL gates, but consume more power. See TTL, I2L and bipolar. 1. nonradioactive kit (Amersham, Les Ulis, France). An 818-bp internal probe for [bla.sub.IMI-1] was obtained by using primers IMI-A (5'-ATAGCCATCCTTGTTTAGCTC-3') and IMI-B (5'-TCTGCGATTACTTTATCCTC-3') and standard polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction Polymerase chain reaction (PCR) ) amplification procedures (5,26). Primers designed to hybridize hy·brid·ize intr. & tr.v. hy·brid·ized, hy·brid·iz·ing, hy·brid·iz·es 1. To produce or cause to produce hybrids; crossbreed. 2. to the ends of the [bla.sub.NmcA], [bla.sub.IMI-1], and [bla.sub.Sme-1/Sme-2] genes were used for standard PCR amplification experiments (5,7,8) with plasmid DNA of each imipenem-resistant E. asburiae isolate and of their transconjugants as templates. Cloning experiments were then performed with BamHI restricted whole-cell DNA of E. asburiae MS7 followed by ligation ligation /li·ga·tion/ (li-ga´shun) the application of a ligature. tubal ligation sterilization of the female by constricting, severing, or crushing the uterine tubes. of DNA fragments into the BamHI-site of cloning vector cloning vector n. An autonomously replicating plasmid having regions into which foreign DNA can be inserted. pGB2 (32). Recombinant plasmids were transformed by electroporation electroporation (i·lekˈ·trō·p  ·rāˑ·sh into E. coli DH 10B electrocompetent
cells (26). E. coli DH10B harboring recombinant plasmids was selected on
MH agar plates containing ampicillin (100 mg/L) and streptomycin (100
mg/L).DNA sequencing of both strands of PCR fragments amplified with the primers for [bla.sub.IMI-1] and plasmid DNA of E. asburiae isolates as templates and of the cloned fragment of a recombinant plasmid was determined with an Applied Biosystems sequencer See MIDI sequencer. (music) sequencer - Any system for recording and/or playback of music via a programmable memory which stores music not as audio data, but as some representation of notes. (AB1377). The nucleotide sequences and the deduced protein sequences were analyzed with software available on the Internet from the National Center for Biotechnology Information The National Center for Biotechnology Information (NCBI) is part of the United States National Library of Medicine (NLM), a branch of the National Institutes of Health. The NCBI is located in Bethesda, Maryland and was founded in 1988. Web site (http://www.ncbi.nlm.nih.gov/BLAST). Results Bacterial Identification Twenty-nine of the 30 imipenem-resistant isolates substantially hydrolyzed imipenem, i.e., 10.5 [+ or -] 1.6 U/mg of protein of culture extracts. These isolates were a single Aeromonas hydrophila isolate, 6 Stenotrophomonas maltophilia isolates known to naturally produce carbapenemases, and 22 Enterobacter spp. isolates identified as E. asburiae that were further analyzed. As reported in Table 1, E. asburiae strains were isolated at different times from several rivers in the midwest. Other tested rivers had ampicillin-resistant isolates that were not imipenem-resistant (Figure). These rivers were Arkansas (Little Rock), Canadian (Oklahoma City), Hudson (New York New York, state, United States New York, Middle Atlantic state of the United States. It is bordered by Vermont, Massachusetts, Connecticut, and the Atlantic Ocean (E), New Jersey and Pennsylvania (S), Lakes Erie and Ontario and the Canadian province of ), Chicago (Chicago), Colorado (Glenwood Springs), Missouri (Parkville), Cuyahoga (Cleveland), Mississippi (New Orleans, St. Louis), Ohio (Cincinnati, Louisville, Pittsburgh, Wheeling), Platte (Grand Island), Scioto (Columbus), Wabash (Terre Haute), and White (Indianapolis). RAPD analysis was then performed to compare all imipenem-resistant E. asburiae isolates. Using a series of different primers, this genotyping technique identified clonally indistinguishable E. asburiae isolates, although they were from various geographic origins (data not shown). [beta]-Lactam Resistance Marker The imipenem resistance marker was transferred from each imipenem-resistant E. asburiae isolate to E. coli DH10B by conjugation. Plasmid analysis identified a 66-kb plasmid (pNat) from cultures of each imipenem-resistant E. asburiae isolate, whereas this plasmid was not isolated from E. cloacae and E. asburiae reference strains (data not shown). PCR experiments with primers for the [bla.sub.IMI-1] gene were positive with plasmid DNA of each E. asburiae isolate and transconjugants as templates, whereas primers designed to amplify [bla.sub.NmcA] and [bla.sub.Sme-1/Sme-2] failed to give PCR product. The Southern blot analysis South·ern blot analysis n. An electrophoretic procedure used to separate and identify DNA sequences. confirmed that the [bla.sub.IMI IMI International Masonry Institute (Washington, DC) IMI Israel Military Industries IMI Institute of the Motor Industry IMI International Market Insight IMI Imposto Municipal Sobre Imóveis (Portugal) ]-like gene was located on the natural plasmid pNat (data not shown). Sequencing PCR products with primers hybridizing at the ends of the [bla.sub.IMI-1] gene and plasmid DNA of each imipenem-resistant E. asburiae isolate identified the same [beta]-lactamase IMI-2 in all cases. This novel enzyme had 2 amino acid substitutions (tyrosine to histidine histidine (hĭs`tĭdēn), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein. at position Ambler 105 and asparagine asparagine (əspâr`əjēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of mammalian proteins. to aspartic acid aspartic acid (əspär`tĭk), organic compound, one of the 20 amino acids commonly found in animal proteins. Only the l-stereoisomer participates in the biosynthesis of proteins. at position Ambler 35) compared to the chromosomally encoded carbapenemase IMI-1 (5). [beta]-Lactamase IMI-1 had been isolated from an E. cloacae isolate from Minnesota close to locations where IMI-2-producing isolates have been found (5). However, the [bla.sub.IMI-2] gene was not just a point-mutant derivative of the [bla.sub.IMI-1] gene, since these genes differ by 11 nucleotide substitutions. [beta]-Lactamase IMI-2 was also related to NmcA (97% amino acid identity) (8). Cloning BamHI-restricted DNA of whole-cell DNA of E. asburiae MS7 gave recombinant plasmid pIMI-2 that had a 5.5-kb insert that allowed identification of the surrounding sequence of the [bla.sub.IMI-2] gene. A gene encoding a LysR-type regulator named IMIR-2 was found just upstream of [bla.sub.IMI-2]. It shared 95% amino acid identity with IMIR-1, which is located upstream of the [bla.sub.IMI-1] gene (5). The surrounding sequences of [bla.sub.IMI-2] shared significant nucleotide identity with transposable transposable /trans·pos·a·ble/ (trans-poz´ah-b'l) capable of being interchanged or put in a different place or order. elements. Part of an open reading frame that shared 97% nucleotide identity with that of the transposase gene tnpA of the transposon transposon /trans·po·son/ (trans-po´zon) a small mobile genetic (DNA) element that moves around the genome or to other genomes within the same cell, usually by copying itself to a second site but sometimes by splicing itself out of its Tn2501 (Tn3 family) was identified downstream of [bla.sub.IMI-2] (33). Upstream of imiR-2, a 142-bp sequence shared 76% nucleotide identity with part of the insertion sequence insertion sequence n. Any of several discrete DNA sequences that repeat at various sites on a bacterial chromosome, on certain plasmids, and on bacteriophages and that can move from one site to another on the chromosome, to another plasmid in the same IS2. Susceptibility Testing and Expression of Resistance MICs of several [beta]-lactams, including carbapenems for the IMI-2--positive E. asburiae MS7 and for E. coli DH10B expressing the [bla.sub.IMI-2] gene were high (Table 2). The MICs of [beta]-lactams for all imipenem-resistant clinical isolates were identical (data not shown). Much higher level of resistance to aztreonam than to expanded-spectrum cephalosporins Cephalosporins Definition Cephalosporins are medicines that kill bacteria or prevent their growth. Purpose Cephalosporins are used to treat infections in different parts of the body—the ears, nose, throat, lungs, sinuses, and was found for the IMI-2-positive strains, as reported for the other producers of class A carbapenemases (2). The activity of [beta]-lactamase IMI-2 was partially inhibited by clavulanate and tazobactam. Induction studies showed increase of [beta]-lactamase expression from 17- to 30-fold (170 to 300 U/mg of protein) (for each E. asburiae isolate when imipenem (50 mg/L) and cefoxitin (50 mg/L) were used as inducers, respectively. These induction results were consistent with location and functionality of a LysR-type regulator gene upstream of the [bla.sub.IMI-2] gene in the imipenem-resistant E. asburiae isolates. No other antimicrobial resistance marker was carried by natural plasmid pNat. Discussion This report indicates that several U.S. rivers may be a reservoir for broad-spectrum carbapenemases. Here, we report a novel clavulanic-acid inhibited Ambler class A [beta]-lactamase IMI-2 that has an usual spectrum of hydrolysis for this type of [beta]-lactamase, including penicillins, carbapenems, and aztreonam (2). [beta]-Lactamase IMI-2 is closely related to several Ambler class A carbapenemases whose genes are chromosomally located, including [bla.sub.IMI-1] and [bla.sub.NmcA], and found in several clinical isolates (5,8). While this work was in progress, a clinical case of an NmcA-producing E. cloacae isolate was reported from Seattle (34). An extended epidemiologic survey epidemiologic survey, n See research, epidemiologic survey. identified Sme-1 type-producing Serratia marcescens Serratia marcescens Microbiology The type-species of the gram-negative Serratia, widely present in the environment, and occasional cause of hospital-acquired infections Asssociations Contaminated fluids, equipment, cleaning solutions, hands, ↓ isolates from the West Coast to the East Coast, which indicates that these isolates may also represent a reservoir for carbapenemases in Enterobacteriaceae (9). Thus, identification of carbapenemase genes in enterobacterial strains from rivers may have clinical importance. In the present study, the [beta]-lactamase gene was plasmid-encoded and was adjacent to mobile sequences that may play an additional role in gene transfer. The E. asburiae isolates were clonally related and may correspond to a single clone, although they were obtained from distantly related midwestern rivers. The reason for the presence of these antimicrobial-resistant strains in this region is unknown. Taking into account the small number of specimens withdrawn from the rivers and the selection technique for imipenem-resistant isolates (ampicillin- and not imipenem-containing plates), the prevalence of carbapenemase-producing enterobacterial strains may be high in the environment, at least in the Midwest. Cloning experiments led to identification of a regulatory gene from an E. asburiae strain (found in the other E. asburiae strains as well [data not shown]) that explained inducibility of carbapenemase expression. Whatever the level of imipenem resistance is, failure of an imipenem-containing regimen may occur when treating infections caused by similar carbapenemase-producing strains, as deduced from results obtained with an animal model of pneumonia (35). Finally, this study raises the question of the importance of this reservoir in Enterobacteriaceae as well as the origin of this plasmid-located carbapenemase gene that may be transferred among other enterobacterial pathogens.
Table 1. Origin and date of isolation of imipenem-resistant
Enterobacter asburiae environmental isolates
River (city) Isolate Date
Arkansas River (Wichita, KS) E. asburiae AK1 September 1999
Kansas River (Topeka, KS) E. asburiae K1-K5 September 2000
Des Moines River E. asburiae DM1-DM8 August 2001
(Des Moines, IA)
Mississippi River E. asburiae MS1-MS8 August 2001
(Minneapolis, MN)
Table 2. MICs (mg/L) of [beta]-lactams for several carbapenemase
producers and reference strain Escherichia coli DH10B
Enterobacter E. cloacae Escherichia coli
asburiae MS7 1413B DH10B (pNat)
[beta]-Lactam(s) * ([dagger]) ([dagger]) ([double dagger])
Amoxicillin >512 >512 >512
Amoxicillin + CLA >512 >512 >512
Ticarcillin 128 >256 128
Ticarcillin + CLA 16 >256 16
Piperacillin 16 >256 8
Piperacillin + TZB 4 >256 2
Cephalothin 512 >256 64
Cefotaxime 0.06 1 0.06
Ceftazidime 0.12 2 0.06
Aztreonam 4 8 4
Imipenem >64 >64 16
Meropenem 32 4 2
E. coli DH10B
(pIMI-2) E. coli
[beta]-Lactam(s) * ([double dagger]) DH10B
Amoxicillin >512 4
Amoxicillin + CLA >512 4
Ticarcillin 256 4
Ticarcillin + CLA 32 4
Piperacillin 128 2
Piperacillin + TZB 16 2
Cephalothin 512 4
Cefotaxime 1 0.06
Ceftazidime 0.5 0.25
Aztreonam 64 0.12
Imipenem >64 0.06
Meropenem 32 0.06
* CLA, clavulanic acid at a fixed concentration of 2 mg/L; TZB,
tazobactam at a fixed concentration of 4 mg/L.
([dagger]) Enterobacter asburiae MS7 produces acquired
([beta]-lactamase IMI-2, whereas E. cloacae 1413B produces
acquired [beta]-lactamases TEM-1 and IMI-1 (5).
([double dagger]) Natural plasmid pNat harbors the [bla.sub.IMI-2]
gene, whereas pIMI-2 is a recombinant plasmid that has the same
[beta]-lactamase gene.
Acknowledgement We thank K. Bush for providing E. cloacae 1413B that produced the chromosome-encoded, clavulanate-inhibited carbapenemase IMI-1. This work was tended by a grant from the Ministere de l'Education Nationale et de la Recherche, (UPRES EA 3539) Universite Paris XI, Paris, France, and by the European Community (6th PCRD PCRD Postgraduate Centre for Refugee Doctors (UK) , LSHM-CT-2003-503-335). References (1.) Lee EH, Nicolas MH, Kitzis MD, Pialoux G, Collatz E, Gutmann L. Association of two resistance mechanisms in a clinical isolate of Enterobacter cloacae with high level resistance to imipenem. Antimicrob Agents Chemother. 1991;35:1093-8. (2.) Nordmann P, Poirel L. Emerging carbapenemases in gram-negative aerobes. Clin Microbiol Infect. 2002;8:321-31. (3.) De Champs C, Henquell C, Guelon D, Sirot D, Gazuy N, Sirot J. Clinical and bacteriological bac·te·ri·ol·o·gy n. 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A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene. 1984;31:165-71. (33.) Michiels T, Cornelis G, Ellis K, Grinsted J. Tn2501, a component of the lactose transposon Tn951, is an example of a new category of class II transposable elements. J Bacteriol. 1987; 169:624-31. (34.) Pottumarthy S, Smith-Moland E, Juretschko S, Swanzy SR, Thomson KS, Fritsche TR. NmcA carbapenem-hydrolyzing enzyme in Enterobacter cloacae in North America. Emerg Infect Dis. 2003;9:999-1002 (35.) Mimoz O, Leotard S, Jacolot A, Padoin C, Louchahi K, Petitjean O, et al. Efficacies of imipenem, meropenem, cefepime, and ceftazidime in rats with experimental pneumonia due to a carbapenem-hydrolyzing [beta]-lactamase-producing strain of Enterobacter cloacae. Antimicrob Agents Chemother. 2000;44:885-90. Address for correspondence: Patrice Nordmann, Service de Bacteriologie-Virologie, Hopital de Bicetre, 78 Rue du General Leclerc, 94275 Le Kremlin-Bicetre, France; fax: 33-1-45-21-63-40; email: nordmann. patrice@bct.ap-hop-paris.fr Cecile Aubron, * Laurent Poirel, * Ronald J. Ash, ([dagger}) and Patrice Nordmann * * University Paris XI, Paris, France; and ([dagger]) Washburn University, Topeka, Kansas, USA Dr. Aubron is studying antimicrobial resistance mechanisms at the Hospital Bicetre, South-Paris Medical School, University Paris XI, France. She is a resident specializing in infections diseases. |
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