NosocomialTransmission of CTX-M-15 and OXA-30 β-lactamase-producing Escherichia coli in a Neurosurgical Intensive Care Unit

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1 Available online at NosocomialTransmission of CTX-M-15 and OXA-30 β-lactamase-producing Escherichia coli in a Neurosurgical Intensive Care Unit Yang-Ree Kim, 1 Sang-Il Kim, 1 Ji-Young Lee, 2 Yeon-Joon Park, 3 Kyo-Young Lee, 3 and Moon-Won Kang 1 Departments of Internal Medicine, 1 Infection Control, 2 and Clinical Pathology, 3 College of Medicine, The Catholic University of Korea, Seoul, Korea Abstract. Extended-spectrum β-lactamase-producing E. coli were recovered from 16 patients in a neurosurgical intensive care unit; surveillance cultures revealed that the organism could be transmitted through the contaminated environment and the hands of health care personnel. All isolates from the patients, environment, and health care personnel showed the same pulsed-field gel electrophoresis pattern. Isoelectric focusing, PCR analysis,and sequencing demonstrated that this E. coli harbored CTX-M-15 and OXA-30 β-lactamases that were transferred by conjugation. This is the first report of nosocomial transmission caused by E. coli that harbor the combination of CTX-M-15 and OXA-30. This can be a threat because the organisms can hydrolyze cefotaxime, ceftazidime, and cefepime. (received 2 March 2005; accepted 9 March 2005) Keywords: CTX-M-15, OXA-30, β-lactamases, Escherichia coli, nosocomial transmission Introduction The increasing incidence of extended-spectrum β- lactamases (ESBLs) in members of the family Enterobacteriaceae has become a serious clinical problem on a worldwide scale [1]. Although the most common ESBLs in members of Enterobacteriaceae are mutants of the classical TEM and SHV enzymes [2], other types of ESBLs are emerging. These non-tem/shv ESBLs include the Ambler class A CTX-M family. The CTX-M type β- lactamases are increasingly found in enterobacterial species throughout the world; more than half have been reported within the last 4 yr [3]. These are generally more active against cefotaxime and show little activity against ceftazidime. Phylogenetically, they are grouped into 5 clusters based on their amino Address correspondence to Yeon-Joon Park, M.D., Department of Clinical Pathology, College of Medicine, The Catholic University of Korea, Kangnam St. Mary s Hospital, 505 Banpo-dong, Seocho-ku, Seoul, , Korea; tel ; fax ; yjpk@catholic.ac.kr. acid identities: CTX-M-1 cluster (CTX-M-1, -3, -10, -11, -12, and -15); CTX-M-2 cluster (CTX- M-2, -4, -5, -6, -7, -20, and TOHO-1); CTX-M-8 cluster (CTX-M-8); CTX-M-9 cluster (CTX-M-9, -13, -14, -16, -17, -19, and TOHO-2); and CTX- M-25 cluster (CTX-M-25 and -26). CTX-M-15 differs from CTX-M-3 by a single amino acid change, Asp240Gly, and displays catalytic activity toward ceftazidime [4]. The class D oxacillinase has spread in P. aeruginosa, but is uncommon in Enterobacteriaceae [5,6]. They are composed of 5 groups (I-V) [7]. Group I includes OXA-5, -7, -10, -13 and OXA- 10-derived ESBLs (OXA-11, -14, -16, -17, -19), and group II includes OXA-2, -3, -15, and -20. Group III includes OXA-1, -4, -30, and -31. Groups IV and V consist of a single enzyme OXA-9 and LCR- 1, respectively. Although several OXA-derived ESBLs usually hydrolyze more ceftazidime than cefepime, bla OXA-30 differs from bla OXA-1 by having a single mutation at codon 131, AGA GGA (arginine to glycine), that confers resistance to cefepime rather than to ceftazidime [8] /05/ $ by the Association of Clinical Scientists, Inc. 11 Kim

2 298 In this paper, we report nosocomial transmission caused by E. coli harboring a combination of two ESBLs (CTX-M-15 and OXA-30) in a neurosurgical intensive care unit (NSICU) at our medical center. Materials and Methods Bacterial strains. In October 2003, 2 multi-resistant E. coli were isolated from the respiratory secretions of 2 patients in the NSICU. In the following 3 months, a total of 16 nonduplicate, multi-resistant E. coli clinical strains were isolated from 16 patients in the NSICU. The isolates were recovered from sputum (14 isolates) and urine (2 isolates). Biochemical identification of the isolates was performed by an NEG Combo 32 panel and Walk-Away 96 SI System (Dade Behring, Sacramento, CA) according to the manufacturer s instructions. The bacterial strains were stored in skim milk at -76 C. MIC determination and phenotypic ESBL confirmatory test. The minimum inhibitory concentrations (MIC) of amikacin, gentamicin, cefuroxime, ceftazidime, cefotaxime, aztreonam, cefepime, meropenem, and imipenem were determined by an agar dilution method according to NCCLS guidelines. Antimicrobial disks (BBL, Cockeysville, MD) for cefotaxime (30 µg), ceftazidime (30 µg), aztreonam (30 µg), cefepime (30 µg), and amoxicillin/clavulanic acid (20 µg /10 µg) were used for phenotypic confirmation of ESBL [9]. Isoelectric focusing. Crude β-lactamase preparations, which were derived from sonicated bacterial cultures of the E. coli isolates, were assessed for the β-lactamase pis and the general inhibitor profile by isoelectric focusing (IEF). IEF was performed at room temperature using a Bio-Rad mini-isoelectric focusing III apparatus (Bio-Rad, Richmond, CA). The enzymes were visualized by staining the gel with 0.5 mm nitrocefin solution (BBL, Cockeysville, MD). An inhibition assay was performed to define the characteristics of the β-lactamases by overlaying the gel with filter paper containing 0.3 mm cloxacillin (Sigma- Aldrich, St. Louis, MO) or 0.3 mm clavulanic acid (GlaxoSmithKline, Philadelphia, PA) and 1 mm nitrocefin in 0.1 M phosphate buffer (ph 0.7). The isoelectric points of enzymes from E. coli isolates were estimated by comparison with TEM-1, TEM-10, SHV-1, SHV-5, and CMY-1. Conjugal transfer. Conjugal experiments were performed with a representative strain and a sodium azide-resistant E. coli J53 as a recipient. The transconjugants were selected on a nutrient agar containing sodium azide (150 mg/l) and either ceftazidime (2 mg/l) or cefotaxime (2 mg/l). From the transconjugants, plasmid DNA was purified from bacterial cells by an alkaline lysis procedure using the QIAGEN Plasmid Midi Kit (Quiagen, Hilden, Germany). PCR analysis and sequencing. A search for bla TEM-1, bla SHV- 1, bla CTX-M-common, bla PER-1, bla GES-1, bla OXA-group I, bla OXA-group II, bla OXA-group III genes and class I integron Table 1. Primers used for PCR analysis. Target Primer bla TEM-1 5'-AAGCCATACCAAACGACGAG-3' 5'-ATTGTTGCCGGG AAGCTAGA-3' bla SHV-1 5'-TATCCCTGTTAGCCACCCTG-3' 5'-CCACTGCAGCA GCTGC(A/C)TT-3' bla CTX-M-common 5'-CGCTTTATGCGCAGACGA-3' 5'-GATTCTCGCCG CTGAAGC-3' bla PER-1 5'-ATGAATGTCATTATAAAAGC-3' 5'-AATTTGGGCTT AGGGCAGAG-3' bla GES-1 5'-ATGCGCTTCATTCACGCAC-3 5'- CTATTTGTCCG TGCTCAGG-3' bla OXA group I 5 -TCTTTCGAGTACGGCATTAGC-3 5 -CCAATGATGCCCTCACTTTCC-3 bla OXA group II 5 -GCCAAAGGCACGATAGTTGT-3 5 -GCGTCCGAGTTGACTGCCGG-3 bla OXA group III 5 -AGCCGTTAAAATTAAGCCC-3 5 -CTTGATTGAAGGGTTGGGCG-3 Class I integron 5 -CS 5 -GGCATCCAAGCAGCAAGC-3 3 -CS 5 -AAGCAGACTTGACCTGAT-3 in the clinical isolates was performed by PCR amplification using the primer sets listed in Table 1. A combination of 5 -CS or 3 -CS primers and OXA-1F or OXA-1B primers, respectively, with both primers reading out from bla OXA-1, was used to determine the genetic content of the class I integron. Freshly isolated colonies were suspended in distilled water and adjusted to 0.5 McFarland, which was then boiled for 10 min. The supernatant, which was obtained after centrifugation at 12,000 rpm for 10 min, was used as the template DNA. The PCRs were performed in 50 µl volumes containing 50 ng of DNA, 25 pm each of primer, 100 µm dntps, 2.5 U of Takara Ex Taq (Takara, Shiga, Japan), and PCR buffer under the following conditions: 94 C for 10 min, 35 cycles of 94 C for 1 min, 55 C for 1 min, 72 C for 3 min, and a final extension at 72 C for 10 min. The PCR products were purified with a PCR purification column (Qiagen, Hilden, Germany). The sequencing reactions were performed with an ABI PRISM 3700 automated sequencer (Applied Biosystems, Foster City, CA). The nucleotide and deduced protein sequences were analyzed using software from National Center of Biotechnology Information Web site ( Surveillance cultures. Cultures of the environment and the hands of the health care personnel were performed. The environmental surveillance cultures were carried out from the ventilator circuits, side rails and side tables of patients, humidifiers, and keeping places of the suction connect line. Genomic typing. Pulsed-field gel electrophoresis (PFGE) was carried out according to the manufacturer s protocol (Bio-Rad Laboratories, Richmond, CA). Briefly, the whole-cell DNA of the E. coli isolate was digested with the XbaI restriction enzyme for 4 hr at 50 C. Electrophoresis was performed with a CHEF DRIII apparatus (Bio-Rad) through a 1% agarose gel in 0.5x Tris-borate-EDTA buffer using a pulse time ranging from 5 to 11 Kim

CTX-M-15 and OXA-30 β-lactamase-producing E. coli 299 60 sec at a voltage of 200 V for 20 hr at 14 C. A bacteriophage λ-dna ladder (Bio-Rad) was used as the DNA molecular weight marker.

3 CTX-M-15 and OXA-30 β-lactamase-producing E. coli sec at a voltage of 200 V for 20 hr at 14 C. A bacteriophage λ-dna ladder (Bio-Rad) was used as the DNA molecular weight marker. Results and Discussion This study began with the observation that multiresistant E. coli isolates were recovered from the sputum specimens from patients in the NSICU, which is an unusual finding. During a 3-mo period, 16 clinical isolates showing identical antibiograms according to the MicroScan NEG Combo 32 panel (Dade Behring, Sacramento, CA) were isolated. The MICs of the various antimicrobial agents are listed in Table 2. A double disk synergy test confirmed that they produced ESBL. Isoelectric focusing showed two β-lactamase bands of pi 8.6 and 7.3. The former was inhibited by clavulanic acid and the latter was weakly inhibited by clavulanic acid. The isolates did not cause significant diseases such as pneumonia or urinary tract infections. As an infection control program, the health care personnel were informed of the need for the appropriate use of gloves, and the need for hand-washing was reinforced. In addition, the procedure for storing Table 2. MICs of the various antibiotics for E. coli strain. Antibiotics MIC (µg/ml) MIC (µg/ml) of transconjugant Amikacin 16 2 Gentamicin Cefuroxime Ceftazidime cefotaxime Aztreonam Cefepime Meropenem Imipenem suction connect lines was changed. The E. coli isolates have not been isolated since these infection control measures were applied. The PCR search revealed that the E. coli isolates all contained the amplification products for the bla CTX-M -like genes and the bla OXA-1 -like gene, whereas no amplicons were produced for the other primers. Sequence analysis of these bla CTX-M -like and bla OXA-1 -like gene PCR products showed 100% homology of its DNA with that of bla CTX-M-15 and bla OXA-30 (GenBank Accession No. AY and No. AF255921, respectively), using a BLAST search. Fig. 1. PFGE pattern of the 11 clinical E. coli isolates (lanes 1-11), 2 isolates from keeping places for suction connect lines (lanes 12, 13), and 1 isolate from the hand of a health care worker (lane 14). 11 Kim

4 300 A PCR amplification of class I integron gave a negative result and it coincides with a case of Salmonella enterica producing CTX-M-15 and OXA-30, in which the bla OXA-30 was not located in class I integron [10]. The resistance was transferred by conjugation and the transconjugant exhibited a similar resistance pattern to the donor except that the MICs of amikacin, ceftazidime, aztreonam, and cefepime were decreased 2- to 3-fold. However, the MICs of cerfotaxime, ceftazidime, aztreonam, and cefepime were still in the resistant range (Table 2). PCR amplification of the transconjugant, using the primers for bla CTX-M and bla OXA-1, gave the expected PCR products (data not shown). These findings indicate that the CTX-M-15 and OXA-30 were located in conjugative plasmids and confer resistance to cefotaxime, ceftazidime, aztreonam, and cefepime. From the surveillance cultures, 4 E. coli isolates were identified (3 from the keeping places of the suction tips and 1 from the hands of a health care worker). All 16 clinical isolates and the 4 isolates from the surveillance cultures had the same biotype ( ) by the MicroScan GN Combo. The PFGE profiles of the XbaI-restricted DNA of the 16 clinical E. coli isolates and the 4 isolates from the surveillance cultures were identical, which suggested clonal spread of the strain (Fig. 1). The first CTX-M β-lactamase was isolated in Germany from E. coli in 1989 [11], and has rapidly spread into many parts of the world. In Korea, the prevalence of the CTX-M enzymes in E. coli and K. pneumoniae is 1.9% and 1.5%, respectively; CTX- M-3, -14, and -15 were reported for E. coli and CTX-M-3 and -14 were reported for K. pneumoniae [12]. They confer a high-level resistance to cefotaxime, ceftriaxone, aztreonam, and have only marginal effects on the MIC of ceftazidime, which is the opposite pattern to that of most TEM- and SHV-type ESBLs [13]. CTX-M-15 was first discovered in India in 1999; it is an Asp240Gly variant of CTX-M-3 with higher resistance to ceftazidime than other CTX-M-type enzymes [14]. The class D oxacillinase has spread in P. aeruginosa and A. baumannii, but is uncommon in Enterobacteriaceae [5, 6]. The bla OXA-30 differs from bla OXA-1 by having a mutation at codon 131, AGA GGA (arginine to glycine), which confers resistance to cefepime rather than to ceftazidime [8]. The cefepime MIC was 64 µg/ml in this study, which is higher than in other reports (MICs of 1-4 µg/ml) [8,15], but coincides with that reported by Weil et al [10], where an S. enterica isolate harbored CTX-M-15 and OXA-30 concurrently and the cefepime MIC was >32 µg/ml. The production of OXA-30 β-lactamase has been rarely encountered in members of the family Enterobacteriaceae (Shigella flexneri, E. coli, and Salmonella typhimurium) [8,10,15,16] and this is the first report of an E. coli harboring an OXA-type enzyme in Korea. However, this rare occurrence may be partly due to lack of effort to detect the enzyme. The β-lactamase may have spread to members of the family Enterobacteriaceae considering the facts that OXA-30 is frequently co-produced with CTX- M-15 and that CTX-M is a rapidly growing family of ESBLs that are associated with ISEcp1-like insertion sequences [10,17]. Although ESBL is widespread in Enterobacteriaceae, strains with 2 or more ESBLs are still uncommon, and most of the combinations reported are TEM- or SHV-derived enzymes. We report here the nosocomial transmission of E. coli harboring a new combination of ESBLs (CTX-M-15 and OXA- 30) in the neurosurgical intensive care unit at our medical center. This combination may be a grave threat because it was transferred by conjugation and it exhibits high levels of resistance to extendedspectrum cephalosporins including cefepime. There appears to be a need for surveillance for this enzyme in enterobacterial species. Acknowledgements. The authors gratefully acknowledge technical assistance by Jung Jun Park and Kanggyun Park. We thank Professor Hyunjoo Pai for generous gifts of the pi control strains. References 1. Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14: Kim

5 CTX-M-15 and OXA-30 β-lactamase-producing E. coli Nordmann P, Guibert M. Extended-spectrum betalactamases in Pseudomonas aeruginosa. J Antimicrob Chemother 1998;42: Bonnet R. Growing group of extended-spectrum betalactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004;48: Poirel L, Gniadkowski M, Nordmann P. Biochemical analysis of the ceftazidime-hydrolyzing extendedspectrum β-lactamase CTX-M-15 and of its structurally related β-lactamase CTX-M-3. J Antimicrob Chemother 2002;50: Aubert D, Poirel L, Chevalier J, Leotard S, Pages JM, Nordmann P. Oxacillinase-mediated resistance to cefepime and susceptibility to ceftazidime in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2001;45: Marumo K, Takeda A, Nakamura Y, Nakaya K. Detection of OXA-4 β-lactamase in Pseudomonas aeruginosa isolated by genetic methods. J Antimicrob Chemother 1999;43: Bert F, Branger C, Lambert-Zechovsky N. Identification of PSE and OXA β-lactamases in Pseudomonas aeruginosa using PCR-restriction fragment length polymorphism. J Antimicrob Chemother 2002;50: Dubois V, Arpin C, Quentin C, Texier-Maugein J, Poirel L, Nordmann P. Decreased susceptibility to cefepime in a clinical strain of Escherichia coli related to plasmid- and integron-encoded OXA-30 β-lactamase. Antimicrob Agents Chemother 2003;47: National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial disc susceptibility testing; Fourteenth Informational Supplement M100- S14, NCCLS. Villanova, PA, Weil FX, Perrier-Gros-Claude JD, Demartin M, Coignard S, Grimont PAD. Characterization of extended-spectrum β-lactamase (CTX-M-15)-producing strains of Salmonella enterica isolated in France and Senegal. FEMS Microbiol Lett 2004;238: Bauernfeind A, Grimm H, Schweighart S. A new plasmidic cefotaximase in a clinical isolate of Escherichia coli. Infection 1990;18: Bae IK, Woo GJ, Jeong SH, Park KO, Cho BK, Kim DM, Kwon SB, Kim HJ, Kang HK. Prevalence of CTX- M-type extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates in Korea. Kor J Clin Microbiol 2004;7: Tzouvelekis LS, Tzelepi E, Tassios PT, Legakis NJ. CTX- M-type β-lactamases: an emerging group of extendedspectrum enzymes. Int J Antimicrob Agents 2000;14: Karim A, Poirel L, Nagarajan S, Nordmann P. Plasmidmediated extended-spectrum β-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol Lett 2001;201: Antunes P, Machado J, Carlos Sousa J, Peixe L. Dissemination amongst humans and food products of animal origin of a Salmonella typhimurium clone expressing an integron-borne OXA-30. J Antimicrob Chemother 2004;54: Siu LK, Lo JYC, Yuen KY, Chau PY, Ng MH, Ho PL. β- lactamases in Shigella flexneri isolates from Hong Kong and Shanghai and a novel OXA-1-like β-lactamase, OXA- 30. Antimicrob Agents Chemother 2000;44: Gangoue-Pieboji J, Miriagou M, Vourli S, Tzelepi E, Ngassam P, Tzouvelekis LS. Emergence of CTX-M-15 producing Enterobacteria in Cameroon and characterization of a bla CTX-M-15 -carrying element. Antimicrob Agents Chemother 2005;49: Kim

SUMMARY. Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M,

SUMMARY. Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M, SUMMARY Key words: antibioticresistance, Enterobacteriaceae, ESBL, CTX-M, The doctoral thesis entitled Prevalence of Enterobacteriaceae producing extendedspectrum beta-lactamases (ESBL) isolated from broilers