Enterobacter cloacae outbreak and emergence of quinolone resistance gene in Dutch hospital.An outbreak of Enterobacter cloacae Enterobacter cloacae is a clinically significant Gram-negative, facultatively-anaerobic, rod-shaped bacterium. infections with variable susceptibility to fluoroquinolones occurred in the University Medical Center Utrecht The Universitary Medical Center Utrecht (Dutch: Universitair Medisch Centrum Utrecht) or UMCU is the main hospital of the city of Utrecht. It is affiliated with the Universiteit Utrecht. in the Netherlands in 2002. Our investigation showed that a qnrA1 gene was present in 78 (94%) of 83 outbreak isolates and that a qnrA1-encoding plasmid transferred to other strains of the same species and other species. The earliest isolate carrying this same plasmid was isolated in 1999. qnrA1 was located in a complex integron consisting of the intl1, aadB, qacE[DELTA]1, sul1, orf orf (orf) a contagious pustular viral dermatitis of sheep, communicable to humans.
see contagious ecthyma.
ORF Oral rehydration fluid
orf 513, qnrA1, ampR, qacE[DELTA]1, and sul1 genes that were not described previously. On the same plasmid, 2 other class 1 integrons were present. One was a new integron associated with the [bla.sub.CTX-M-9] extended-spectrum [beta]-lactamase.
Multidrug-resistance among Enterobacteriaceae, including resistance to quinolones, is increasing. Although quinolone resistance is predominantly caused by chromosomal mutations, it may also result from a plasmid-encoded qnr-gene (1). The QnrA determinant, a 218-amino acid protein, protects DNA gyrase DNA gyrase (ji´ras) a type II DNA topoisomerase. and topoisomerase IV Topoisomerase IV is one of two type-II topoisomerases in bacteria, the other being DNA gyrase. Like gyrase, topoisomerase IV is able to pass one double-strand of DNA through another double-strand of DNA, thereby changing the linking number of DNA by two in each enzymatic step. from the inhibitory activity of quinolones (2). However, expression of qnrA alone is frequently insufficient to reach Clinical and Laboratory Standards Institute breakpoints for ciprofloxacin ciprofloxacin /cip·ro·flox·a·cin/ (sip?ro-flok´sah-sin) a synthetic antibacterial effective against many gram-positive and gram-negative bacteria; used as the hydrochloride salt.
n. resistance. Since first identified in 1994 in the United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. , qnrA-like genes have been sporadically identified in Enterobacteriaceae worldwide (3-9).
At the end of 2002, an outbreak of aminoglycoside-resistant Enterobacter cloacae infections with variable susceptibility for ciprofloxacin was detected in the University Medical Center Utrecht (UMCU UMCU Unmanned Mobile Combat Unit ), the Netherlands, involving >80 patients (10). The first aim of this study was to test the hypothesis that the variable susceptibility to ciprofloxacin of the outbreak strain was associated with plasmid-mediated qnrA and if so, to characterize the gene's molecular background and determine its ability to transfer in vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment.
In an artificial environment outside a living organism. as well as in vivo in vivo /in vi·vo/ (ve´vo) [L.] within the living body.
Within a living organism.
in vivo adv. . Maximum circumstantial evidence circumstantial evidence
In law, evidence that is drawn not from direct observation of a fact at issue but from events or circumstances that surround it. If a witness arrives at a crime scene seconds after hearing a gunshot to find someone standing over a corpse and holding a for horizontal transfer in vivo with the outbreak strain as donor would be obtained if the following observations were made: 1) different species or strains collected from the same patient harbored the same qnrA-encoding plasmid; 2) this same qnrA-encoding plasmid was not found in patients without an epidemiologic link to the outbreak. The second aim of this study was to determine to what extent the qnrA gene is an emerging resistance problem in our hospital.
Materials and Methods
A total of 1,167 isolates were tested for a qnrA gene. Group I consisted of 178 E. cloacae pulsed-field gel electrophoresis gel electrophoresis
Electrophoresis performed in a gel composed of agarose, polyacrylamide, or starch. (PFGE PFGE Pulsed-Field Gel Electrophoresis ) typed isolates obtained from January 2001 to August 2003 from 159 patients (10). Of these, 83 tobramycin-resistant isolates obtained from 83 patients belonged to 1 clonal lineage (cluster I, outbreak strain). Five of these patients also carried a tobramycin-susceptible variant of the clonal lineage ([I.sup.A]). The remaining 95 E. cloacae isolates contained 5 small clusters of 2 isolates each (III-VII), 1 cluster with 6 isolates (VIII), 1 cluster with 3 isolates (II), and 70 unique strains.
Groups II and III consisted of aminoglycoside-resistant, gram-negative bacteria identified in the hospital database that were other than the outbreak strain; these bacteria were isolated from patients with an outbreak strain (group II) as well as from patients not involved in the outbreak but admitted in the same period (January 2001-August 2003) (group III In the periodic table Group III covered what are now called
Identification and Susceptibility Testing
Identification and susceptibility testing of isolates obtained through 2000 were performed by using the VITEK1 System with AMS AMS - Andrew Message System R09.1 software (bioMerieux, Marcy-L'etoile, France); isolates obtained after 2000 were tested by using the Phoenix 100 Automated Microbiology System version V3.22 software (Becton Dickinson BD (NYSE: BDX), is a medical technology company that manufactures and sells medical devices, instrument systems and reagents. Founded in 1897 and headquartered in Franklin Lakes, New Jersey, BD employs 27,000 people in nearly 50 countries. Biosciences, Sparks, MD, USA). For susceptibility testing, Clinical and Laboratory Standards Institute guidelines were used (12). In the conjugation conjugation, in genetics
conjugation, in genetics: see recombination.
conjugation, in grammar
conjugation: see inflection. experiments, MICs were determined by using Etest (AB Biodisk, Solna, Sweden).
Genotyping and Characterization of [beta]-Lactamases
E. cloacae isolates were typed by PFGE. Citrobacter freundii Citrobacter freundii Microbiology A Citrobacter opportunistic pathogen Management Cephalothin, aminoglycosides , 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. , and Klebsiella pneumoniae Klebsiella pneu·mo·ni·ae
Friedlander's bacillus. were typed by PFGE and random amplified polymorphic polymorphic - polymorphism DNA DNA: see nucleic acid.
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. (13). To determine the kind of [beta]-1actamases the outbreak strain expressed, isoelectric focusing isoelectric focusing,
n the ordering and concentration of substances according to their isoelectric points. (IEF (Information Engineering Facility) A fully integrated set of CASE tools from Sterling Software that runs on PCs and MVS mainframes. It generates COBOL code for PCs, MVS mainframes, VMS, Tandem, AIX, HP-UX and other Unix platforms. ) was performed with Phastgels (pH gradient 3-9) with the PhastSystem (Pharmacia AB, Uppsala, Sweden) (14). [beta]-lactamases of isoelectric isoelectric /iso·elec·tric/ (i?so-e-lek´trik) showing no variation in electric potential.
showing no variation in electric potential. pH (pI) 5.6 (TEM-1), pI 7.6 (SHV-2A), and pI 8.2 ([bla.sub.CTX-M-9]) and a broad range pI calibration set (Amersham Biosciences, Little Chalfont Little Chalfont is a village in south east Buckinghamshire, United Kingdom. It is situated in a small group of villages called The Chalfonts which also consists of Chalfont St Giles and Chalfont St Peter. The villages are sandwiched between High Wycombe and Rickmansworth. , UK) were used. [beta]-lactamases were detected with nitrocefin (Oxoid, Basingstoke, UK).
Detecting and Characterizing Resistance Genes
Target DNA for 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.
polymerase chain reaction
Polymerase chain reaction (PCR) ) assays was extracted by heating bacterial suspensions for 10 min at 95[degrees]C. qnrA, [bla.sub.CTX-M], and aadB were detected by PeR with primers and annealing annealing (ənēl`ĭng), process in which glass, metals, and other materials are treated to render them less brittle and more workable. temperatures described in the Table. The outbreak strain carried an integron containing an aadB gene encoding aminoglycoside resistance (17). Primers were developed to detect aadB gene and the downstream 3'-conserved segment (CS) of the integron in the same PCR (aadB-3'CS). PCR assays were performed for 30 or 35 cycles. The AmpC PCR tests were performed as described earlier, except that a single PCR format was used (18).
The [bla.sub.CTX-M] gene from E. cloacae 02-477 was sequenced by using CTX-M-9 group sequence primers (Table). The flanking regions flanking regions
noncoding sequences on either side of the coding region of a gene that contain various regulatory sequences (motifs). of the qnrA gene and the [bla.sub.CTX-M-9] gene were determined by using a PeR and DNA sequencing DNA sequencing
The determination of the sequence of nucleotides in a sample of DNA. strategy based on the sequences from In7, In36, In37, In60, and an integron from E. coli E. coli: see Escherichia 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. O159 (5,9,19-22). To confirm that the gene cassettes were part of a complex integron with qnrA or [bla.sub.CTX-M-9], we used the Expand Long Template PCR system (Roche, Woerden, the Netherlands) that employed primers to amplify sequences between the qnrA or [bla.sub.CTX-M-9] and the possible gene cassettes. All amplified products were (partly) sequenced for confirmation. Sequencing was performed with Qiagen Quick (Qiagen, Westburg b.v., Leusden, the Netherlands) purified PCR products by using the BigDye Terminator v1.1 Cycle Sequencing Ready Reaction Kit and a 3100 capillary DNA sequencer A DNA sequencer is an instrument used to automate the DNA sequencing process.
DNA sequencers have become more important due to large genomics projects and the need to increase productivity. (Applied Biosystems, Nieuwerkerk a/d Yssel, the Netherlands).
For conjugation experiments, an E. coli K12 and a tobramycin-susceptible clinical E. cloacae (03-702) isolate of PFGE cluster [I.sup.A] were used as recipients. An E. cloacae (02-477) belonging to PFGE cluster I was used as donor. Conjugation was performed as described (23). MacConkey agar plates containing tobramycin tobramycin /to·bra·my·cin/ (to?brah-mi´sin) an aminoglycoside antibiotic derived from a complex produced by Streptomyces tenebrarius, (8 [micro]g/mL) were used for counter selection, and transconjugants were selected on colony form. Conjugation was confirmed by a qnrA-specific PCR. Secondly, transconjugant E. coli C02-477A was used as a donor for qnrA-negative E. cloacae 03-702 belonging to cluster [I.sup.A]. Transconjugants were selected by using 15 [micro]g/mL ampicillin-clavulanic acid and 5 [micro]g/mL tobramycin. Transconjugants were characterized as described above.
Detecting Resistance Genes on Plasmid by Southern Hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.
2. molecular hybridization
Plasmids were isolated with the Qiagen Plasmid Maxi Kit (Qiagen). Plasmid DNA was separated on 1% PFGE agarose agarose
more highly purified form of agar with similar uses to agar and widely used in the separation of nucleic acid fragments. (Bio-Rad Laboratories, Richmond, CA, USA) in 0.5x Tris-borate-EDTA, 0.05 mmol/L thiourea thiourea
a goitrogenic agent used in industry as a photographic fixative. Mode of action is as for thiouracil. buffer at 14[degrees]C in CHEF DR-II apparatus (Bio-Rad). Run time was 22 h with a voltage of 6 V/cm and a linearly ramped pulse time of 30 to 70 s. The DNA was blotted and hybridized. The probes were PCR amplification products obtained with primers used to detect aadB-3'-CS, [bla.sub.CTX-M-9], and qnrA genes (Table). Products were labeled with the AlkPhosDirect Reaction Kit (Amersham Biosciences) and detected with CPD-Star (Amersham Biosciences).
qnrA1 in Outbreak Strain
For 78 (94%) of the 83 E. cloacae isolates in cluster I (outbreak strain), the qnrA-specific PCR was positive. To confirm results from the PCR, 2 fragments were sequenced. The obtained sequences were identical to the published sequence of qnrA1 (GenBank accession no. AY070235).
Susceptibility testing showed that 87% of the 83 outbreak isolates were resistant or intermediate resistant to ciprofloxacin (43% resistant, 43% intermediate resistant), 100% were resistant to tobramycin, 63% to gentamicin gentamicin /gen·ta·mi·cin/ (jen?tah-mi´sin) an aminoglycoside antibiotic complex isolated from bacteria of the genus Micromonospora, , 2% to amikacin, 100% to ceftriaxone ceftriaxone /cef·tri·ax·one/ (cef?tri-ak´son) a semisynthetic, ß–resistant, third-generation cephalosporin effective against a wide range of gram-positive and gram-negative bacteria, used as the sodium salt. , 12% to trimethoprim-sulfamethoxazole, and 0% to carbapenems. A total of 81 (98%) of the 83 isolates harbored an aadB containing integron.
IEF showed the presence of a [beta]-lactamase with a pI of [approximately equal to] 8.2, which suggested the presence of either an AmpC [beta]-lactamase or a CTX-M type extended-spectrum [beta]-lactamase. No AmpC-specific amplification products were obtained. Eighty-two (99%) of the 83 isolates harbored a [bla.sub.CTX-M] gene. DNA sequencing showed the presence of [bla.sub.CTX-M-9].
The plasmid (pQC) of conjugant con·ju·gant
Either of a pair of organisms, cells, or gametes undergoing conjugation. E. coli C02-477A was isolated, and its size was estimated at 180 kb by agarose gel electrophoresis Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, RNA, or protein molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). . Southern blotting that used specific probes confirmed that pQC contained the qnrA1 gene, the [bla.sub.CTX-M-9] gene, and the integron with an aadB gene cassette (data not shown). Sequences flanking the qnrA1 and [bla.sub.CTX-M-9] genes were comparable with 3 previously described class 1 integrons (Figure 1). The first integron (In-UMCU-1 accession no. AY987395), containing the qnrA1 gene, had the same additional structures as In36, orf513, qnrA1, ampR, plus a second copy of the 3'-conserved segment. The In36 integron contained the gene cassettes drf16 and aadA2, while In-UMCU-1 contained only the aadB gene cassette. In addition, the DNA sequences between the second sul1 gene and orf5 (bp 9606-9624 of In36) differed from the sequence of In-UMCU-1 (5). The second integron (In-UMCU-2, accession no. DQ108615), which contained [bla.sub.CTX-M-9], was comparable to In60, but In60 contained the drf16 and aadA2 gene cassettes, while In-UMCU-2 contained the aadB gene cassette (21). The third integron (In-UMCU-3, accession no. DQ019420), which contained the gene cassettes sat, psp, and aadA2, was described previously in an enterotoxigenic en·ter·o·tox·i·gen·ic
Of or being an organism containing or producing an enterotoxin.
Enterotoxigenic E. coli O159 isolated in Japan (22). PCR amplification of the aadA2 gene of the donor, recipient, and transconjugants indicated that this third integron was also located on pQC.
Evidence for Transfer of qnrA in vitro
In vitro conjugation experiments showed that pQC could be transferred both from and to the outbreak strain (Figure 2). pQC was successfully transferred from E. cloacae 02-477 to recipient E. coli K12. The resulting transconjugant E. coli was subsequently used as donor to transfer pQC to a type [I.sup.A] E. cloacae (03-702), which resulted in a successful transfer of pQC. pQC conferred increased ciprofloxacin MICs (from 6- to 10-fold) and resistance to tobramycin, tetracycline tetracycline (tĕ'trəsī`klēn), any of a group of antibiotics produced by bacteria of the genus Streptomyces. They are effective against a wide range of Gram positive and Gram negative bacteria, interfering with protein , and ceftriaxone to the transconjugants (Figure 2). Acquisition and loss of pQC were associated with 2 changes in the PFGE pattern.
[FIGURE 2 OMITTED]
Evidence for Transfer of qnrA1 in vivo
Different species or strains collected from the same patient harbored the same pQC. From 22 of the 53 patients with an outbreak strain, 35 other tobramycin-resistant, gram-negative clinical isolates were available. Eleven different strains obtained from 11 patients were positive for qnrA1, [bla.sub.CTX-M-9], and aadB-3'-CS. These comprised 4 different species: C. freundii (n = 1), Enterobacter aerogenes (n = 1), E. coli (n = 7), and K. pneumoniae (n = 2). Plasmid isolation from 6 E. coli and 1 K. pneumoniae yielded a plasmid of the same size as the pQC in the outbreak strain. Because of its large size and possibly a very low copy number, only small amounts of plasmid DNA could be isolated. These amounts were insufficient to perform further comparative analyses by restriction fragment analysis or Southern blotting.
Some E. cloacae strains with a strong epidemiologic link to the outbreak strain were also pQC positive. All isolates belonging to clusters III, VII, and VIII contained pQC as well as 5 E. cloacae isolates with a unique genotype. Plasmid isolation of 3 strains again showed a plasmid of the same size as the outbreak pQC. Three of the 5 unique isolates were obtained from patients who also harbored the outbreak strain.
The qnrA gene, the aadB-containing integron, and the [bla.sub.CTX-M-9] could not be detected in PFGE cluster [I.sup.A], which is closely related to the outbreak strain (Figure 3). The loss of these genes was associated with increased susceptibility to ciprofloxacin, tobramycin, ceftriaxone, and tetracycline. In addition, an identical change in the PFGE pattern was observed, as in the in vitro experiments. These results suggest that the host may lose pQC in vivo.
[FIGURE 3 OMITTED]
qnrA1 Recent Emergence as Clinical Problem
pQC was not found in isolates obtained from patients without an epidemiologic link to the outbreak. No qnrA1 gene was detected in any of 83 aminoglycoside-resistant gram-negative organisms (44 E. coli, 19 K. pneumoniae, 4 Proteus mirabilis Proteus mirabilis Microbiology A gram-negative pathogen linked to UTIs, wound infections Habitat P mirabilis may be found in water,
soil, feces , 2 Klebsiella oxytoca, 2 E. cloacae, 1 Enterobacter sp., 7 C. freundii, 4 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, ↓ ) obtained from 74 patients admitted to wards not involved in the outbreak during the outbreak period. Neither was qnrA1 detected in any of the 269 UMCU isolates or the 84 community isolates.
Only 1 qnrA1-positive isolate was found in the 514 European isolates. This qnrA1-positive isolate was an E. cloacae organism isolated in 1999 at a surgical ward at UMCU that belonged to cluster III. The other 2 cluster III isolates were isolated at the same surgical ward during the outbreak period.
We report a nosocomial nosocomial /noso·co·mi·al/ (nos?o-ko´me-il) pertaining to or originating in a hospital.
1. Of or relating to a hospital.
2. outbreak with an Rplasmid-encoded qnrA1 gene. This plasmid (pQC) was first detected in an E. cloacae isolated in 1999 and subsequently in another E. cloacae strain that caused a large outbreak in our hospital, starting in 2001. Strong evidence is provided that this outbreak strain was the source from which pQC disseminated to other strains of the same species and other species by horizontal gene transfer “HGT” redirects here. For other uses, see HGT (disambiguation).
Horizontal gene transfer (HGT), also Lateral gene transfer (LGT), is any process in which an organism transfers genetic material to another cell that is not its offspring. . The qnrA1 gene was not detected in any of the hospital isolates (1994-2003) tested without an epidemiologic link to the outbreak strain, indicating that qnrA1 is a new emerging resistance trait in our hospital.
pQC contained 3 different class 1 integrons. One integron was identical to an integron detected in an E. coli O159 from Japan (22). The 2 other integrons were complex integrons, In-UMCU-1 and In-UMCU-2, which were not described previously. Complex integrons are composed of a 5'-CS, gene cassettes, 3'-CS, qac[DELTA]E, sulI, additional genes, qac[DELTA]E, and sulI. These additional genes differ from gene cassettes by lacking a 59-bp element and having their own promoter (24). The qnrA1 gene in In-UMCU-1 was also present as an additional gene, as was the case for the 3 previous characterized qnrA1 genes in In36, In37, and the complex integron of pQR1 (5,9). The sequences of these genes were identical for In-UMCU-1, In 36, and In37, and slightly different for the integron on pQR1. The gene cassette content of the 4 integrons, however, was different, although all 4 possessed a gene encoding aminoglycoside resistance. All qnrA1-positive isolates reported in the literature also show resistance to cephalosporins Cephalosporins Definition
Cephalosporins are medicines that kill bacteria or prevent their growth.
Cephalosporins are used to treat infections in different parts of the body—the ears, nose, throat, lungs, sinuses, and (1,4-9,25-27). Therefore, qnrA1 seems to be closely associated with resistance to cephalosporins and aminoglycosides. How these comparable but different complex integrons arose is unclear. Either the same additional genes became associated with different integrons or the gene cassettes in an original complex integron were exchanged.
Our study confirmed previous findings that the presence of qnrA1 does not necessarily lead to MICs above Clinical and Laboratory Standards Institute breakpoints for resistance to eiprofloxacin (1,3,7,25). Therefore, the presence of qnrA1 had no therapeutic consequences for the patients from whom these isolates were obtained. However, the increased MIC may provide the host bacterium a selective advantage in an environment of low concentrations of quinolones, increasing the bacterial numbers and therefore the absolute chance of a chromosomal mutation encoding resistance (7,25). The presence of a qnrA-carrying plasmid might even enhance the mutation rate encoding quinolone resistance (1). Furthermore, acquisition of qnrA by a host bacterium that already contains quinolone resistance mechanisms may raise MICs above the LCSI LCSI Logo Computer Systems Inc. (Canada) breakpoints (25,28,29). As shown in this study, the same plasmid may cause fluctuation in susceptibility in MICs in different recipients because of variation in porin Porin can be:
In conclusion, in a hospital setting the qnrA gene is advantageous for the host bacterium. Because of this gene's location on promiscuous R-plasmids, it is likely to emerge worldwide.
We thank George A. Jacoby and Fred C. Tenover for providing control strains for detecting extended AmpCs and extended-spectrum [beta]-lactamases.
Mr Paauw is a doctoral candidate at the Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, UMCU, the Netherlands. His research is focused on detecting and characterizing genetic features that can enhance virulence, resistance, and epidemic behavior of Enterobacteriaceae.
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FEMS Federation of European Materials Societies
FEMS Fabrication Engineering Management System
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A gene that causes the production of a protein that represses the activity of another gene in an operon. ampR, originated from Morganella morganii Morganella morganii
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Armand Paauw, * Ad C. Fluit, * Jan Verhoef, * and Maurine A. Leverstein-van Hall *
* University Medical Center, Utrecht, the Netherlands
Address for correspondence: Armand Paauw, Eijkman-Winkler Institute for Microbiology, Infectious Diseases and Inflammation, UMCU, Room G04.614 PO Box 85000, 3508 GA, Utrecht, the Netherlands; email: A.Paauw@umcutrecht.nl
Table. Oligonucleotides used for polymerase chain reaction amplification and sequencing 5'-3' Target Primer sequences QnrA gnrAR AGG AAG CGC CGC TGA GAT TG gnrAF CTA TGC CGA TCT GCG CGA TG aadB-3'CS aadB TGG AGG AGT TGG ACT AT 3'CS AAG CAG ACT TGA CCT GA [bla.sub. ctx-m-uni-F CGA TGT GCA GTA CTX-M-]: CCA GTA A most ctx-m-uni-R ATA TCG TTG GTG GTG CC [bla.sub. ctx-m-2F ATG ATG ACT CAG CTX-M-]-: AGC ATT CG 2,4,5,6,7, ctx-m-2R TTA TTG CAT CAG 20,Toho-1 AAA CCG TG [bla.sub. ctx-m-10-1F ATG GTT AAA AAA CTX-M-]: TCA CTG CG 3,10,11,12, ctx-m-10-4R AAA CCG TTG GTG 152,225 ACG AT [bla.sub. ctx-m-9F AGA CGA GTG CTX-M-]: CGG TGC AGC AA 9,13,14,15, ctx-m-9R GAT TCT CGC CGC 16,17,18, TGA AGC CA 19,24, Toho-2 and -3 Sequence ctx-m-9-1 F TGG TGA CAA AGA [bla.sub. GAG TGC AAC G CTX-M-9] ctx-m-9-MF GGA GGC GTG group ACG GCT TTT ctx-m-9-MR AAA AGC CGT CAC GCC TCC ctx-m-9-4R TCA CAG CCC TTC GGC GAT GenBank 5'-3' accession Target sequences no. QnrA AGG AAG CGC AY070235 CGC TGA GAT TG CTA TGC CGA TCT AY070235 GCG CGA TG aadB-3'CS TGG AGG AGT AY173047 TGG ACT AT AAG CAG ACT TGA M73819 CCT GA [bla.sub. CGA TGT GCA GTA U95364 CTX-M-]: CCA GTA A most ATA TCG TTG GTG U95364 GTG CC [bla.sub. ATG ATG ACT CAG X92507 CTX-M-]-: AGC ATT CG 2,4,5,6,7, TTA TTG CAT CAG X92507 20,Toho-1 AAA CCG TG [bla.sub. ATG GTT AAA AAA X92506 CTX-M-]: TCA CTG CG 3,10,11,12, AAA CCG TTG GTG X92506 152,225 ACG AT [bla.sub. AGA CGA GTG AJ416345 CTX-M-]: CGG TGC AGC AA 9,13,14,15, GAT TCT CGC CGC AJ416345 16,17,18, TGA AGC CA 19,24, Toho-2 and -3 Sequence TGG TGA CAA AGA AJ416345 [bla.sub. GAG TGC AAC G CTX-M-9] GGA GGC GTG AJ416345 group ACG GCT TTT AAA AGC CGT CAC AJ416345 GCC TCC TCA CAG CCC TTC AJ416345 GGC GAT Annealing Nucleotide temperature Target positions ([degrees]C) QnrA 762-743 56 482-501 aadB-3'CS 251-267 55 1342- 1326 [bla.sub. 214-232 50 CTX-M-]: 751-735 most [bla.sub. 6-25 58 CTX-M-]-: 889-870 2,4,5,6,7, 20,Toho-1 [bla.sub. 63-82 60 CTX-M-]: 934-918 3,10,11,12, 152,225 [bla.sub. 217-236 67 CTX-M-]: 989-970 9,13,14,15, 16,17,18, 19,24, Toho-2 and -3 Sequence 133-154 [bla.sub. 576-593 CTX-M-9] 593-576 group 1007-990 Amplicon Target size (bp) Source QnrA 281 This study This study aadB-3'CS 432 This study (15) [bla.sub. 538 This study CTX-M-]: This study most [bla.sub. 884 (16) CTX-M-]-: (16) 2,4,5,6,7, 20,Toho-1 [bla.sub. 872 This study CTX-M-]: This study 3,10,11,12, 152,225 [bla.sub. 773 This study CTX-M-]: This study 9,13,14,15, 16,17,18, 19,24, Toho-2 and -3 Sequence This study [bla.sub. This study CTX-M-9] This study group This study