MICROBIAL DRUG RESISTANCE Volume 20, Number 2, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/mdr.2013.0102
Epidemiology and Molecular Characterization of Extended-Spectrum Beta-Lactamase-Producing Enterobacter spp., Pantoea agglomerans, and Serratia marcescens Isolates from a Bulgarian Hospital Rumyana Donkova Markovska,1 Temenuga Jekova Stoeva,2 Kalina Dineva Bojkova,2 and Ivan Gergov Mitov1
Forty-two extended-spectrum beta-lactamase (ESBL)-producing isolates of Enterobacter aerogenes, Enterobacter cloacae, Pantoea agglomerans, and Serratia marcescens, collected consecutively during the period January–November 2011 from the University Hospital in Varna, Bulgaria, were studied to characterize their ESBLs by isoelectric focusing, group-specific PCR, and sequencing. The epidemiological relationship was evaluated by random amplified polymorphic DNA analysis (RAPD). Transferability of ESBL genes was determined by conjugation experiments. Plasmid analysis was done by replicon typing and PstI fingerprinting. The overall rate of ESBL production was 20%. The most widespread enzyme was CTX-M-3, found in 64%. It was dominant in E. aerogenes (100%) and S. marcescens (83%). SHV-12, CTX-M-3, and CTX-M-15 were found among E. cloacae isolates in 50%, 35%, and 45%, respectively. Three main CTX-M-3-producing epidemic clones of E. aerogenes and S. marcescens have been detected. Among E. cloacae isolates, six different RAPD profiles were discerned. The plasmids harboring blaCTX-M-3 belonged to IncL/M type and demonstrated similar PstI fingerprinting profiles. IncFII plasmids were detected in two CTX-M-15-producing E. cloacae isolates. Our results demonstrate wide intrahospital dissemination of clonal E. aerogenes and S. marcescens isolates, carrying IncL/M conjugative plasmids.
Introduction
P
lasmid-mediated extended-spectrum beta-lactamases (ESBLs) first appeared in the mid-1980s in Europe, mainly in Klebsiella pneumoniae associated with nosocomial outbreaks. Later, ESBLs have been found predominantly not only in Klebsiella spp. and Escherichia coli, but also in other species such as Enterobacter spp., Serratia spp., Citrobacter spp., Proteus spp., and Salmonella spp.4,5 Enterobacter spp. and Serratia spp. are recognized as important causes of nosocomial infections.3,28 These organisms are associated with outbreaks in hospital settings. They produce inducible AmpC beta-lactamases that in derepressed mutants have constitutive expression.4 The most common ESBLs in Enterobacter spp. and Serratia spp. belong to TEM and SHV groups, although there is a trend of increased incidence of CTX-M-producing isolates.1,3,14,18,22–25 CTX-M betalactamases are characterized by preferential hydrolysis of cefotaxime rather than ceftazidime, though some CTX-M types, such as CTX-M-15, may actually hydrolyze ceftazidime.4
Dissemination of specific clones or clonal groups in nosocomial settings is one of the main reasons for the increase of most of the widespread ESBLs belonging to TEM (TEM-4, TEM-24, TEM-52), SHV (SHV-5, SHV-12), and CTX-M (CTX-M-3, CTX-M-9, CTX-M-14, and CTX-M-15) families.3,12,14,16,17,22–25 Different plasmid families are detected in Enterobacteriaceae and also play a major role in the dissemination of ESBL genes.7,8 Several groups such as IncFI, IncFII, IncA/C, IncL/M, IncN, and IncI1 carrying ESBL genes or acquired AmpC genes are reported to be ‘‘epidemic resistance plasmids,’’ being worldwide detected in Enterobacteriaceae of different origin and sources.7,8,15 The detection of successful clones and plasmids is an essential first step for preventing their spread. The European Antimicrobial Resistance Surveillance Network reports for 2011 high rates of invasive E. coli and K. pneumoniae isolates resistant to oxyimino-cephalosporins in Bulgaria—23% and 81%, respectively (www.ecdc.europa .eu/en/publications/Publications/antimicrobial-resistancesurveillance-europe-2011.pdf). In Bulgaria, a wide survey on
1
Department of Medical Microbiology, Faculty of Medicine, Medical University of Sofia, Sofia, Bulgaria. Department of Microbiology, Medical University, Varna, Bulgaria.
2
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132 ESBL-producing Enterobacteriaceae detected the production of SHV-12, CTX-M-3, and CTX-M-15 ESBLs in Enterobacter spp. and Serratia spp. during the period 1999–2003.19 In 2003, an outbreak of hospital infections in Sofia with CTX-M-3 Serratia marcescens was also reported.16 Results from local surveillance programs in Varna University Hospital indicated an increase in the rate of ESBL-producing Enterobacter spp.: from 28% in 2007 to 36% and 39% in 2010 and 2011, respectively (data not published). These worrying findings prompted more detailed investigations. The aims of this study were to evaluate the epidemiological relationship between ESBL-producing clinical isolates of Enterobacter spp., Pantoea agglomerans, and S. marcescens isolated in the University Hospital in Varna city during the period January 2011–November 2011 and to characterize the ESBLs, to perform conjugation experiments, and to analyze the plasmids harboring the ESBL genes. Materials and Methods Bacterial isolates Varna University Hospital is a 960-bed hospital that is located in Eastern Bulgaria. Between January 2011 and November 2011, a total of 205 consecutive and clinically significant isolates of Enterobacter cloacae (n = 124), Enterobacter aerogenes (n = 46), P. agglomerans (n = 14), and S. marcescens (n = 21) were obtained from hospitalized patients. Of these, 15 E. aerogenes, 20 E. cloacae, 1 P. agglomerans, and 6 S. marcescens isolates were detected to be ESBL producers by double-disk synergy test (DDST) with disks ceftazidime, cefotaxime, cefepime, and amoxicillin/clavulanic acid in a distance of 20 mm.17 The isolates were recovered from various clinical specimens, predominantly from patients in pediatric wards. Patient data, including hospital unit, date and site of isolation, are shown in Table 1. Species identification was performed by API 20 E (bioMe´rieux). Antimicrobial susceptibility testing Antimicrobial susceptibility was determined by disk diffusion method according to CLSI guidelines.9 Isoelectric focusing and bioassay Beta-lactamase production was analyzed by isoelectric focusing (IEF) as previously described.21 The hydrolytic activity of individual beta-lactamase bands was assessed by bioassay.2 Detection of ESBL encoding genes and nucleotide sequencing PCR was performed to detect the presence of blaSHV, blaTEM, and blaCTX-M genes as previously described.19 P. agglomerans isolates and 20 representative Enterobacter spp., S. marcescens isolates, selected according to their antimicrobial susceptibility profile, IEF, and random amplified polymorphic DNA analysis (RAPD) type, were chosen for sequencing of their bla genes. PCR amplification products obtained with oligonucleotides binding to the flanking region of the betalactamase genes19 were subjected to sequencing at Eurofins MWG Operon. The nucleotide and deduced amino-acid sequences were analyzed, and multiple alignments were per-
MARKOVSKA ET AL. formed using Chromas Lite 2.01 (Technelysium Pty Ltd.) and DNAMAN 4.11 Software (Lynnon BioSoft). Molecular typing Whole-cell DNA was prepared using the Illustra Bacteria Genomic DNA Prep Kit (GE Healthcare) and used in RAPD with ERIC 1 and ERIC 2A primers and RAPD-4 for S. marcescens isolates.29 Conjugation experiments, plasmid analysis, and replicon typing Conjugative plasmid transfer was performed on Mueller— Hinton agar using E. coli K12:W3110 lac - resistant to rifampicin and E. coli K12:W3110 lac + resistant to nalidixic acid as recipient strains. Transconjugants were selected on MacConkey agar containing 2 mg/L ceftazidime or cefotaxime and 50 mg/ L rifampicin or nalidixic acid. Plasmid DNA of transconjugant isolates was extracted with the Plasmid MidiPrep Kit (Quantum Prep BioRad Laboratories) and subjected to plasmid fingerprint analysis by restriction with PstI (Amersham Biosciences). The size of transferred plasmid types was estimated by a comparison with reference plasmids (154, 66, 38, and 7 kb), isolated from E. coli NCTC 50192. Plasmid incompatibility groups were determined using PCR-based replicon typing scheme described by Carattoli et al.6 Statistical analysis Differences in the resistance rates were assessed with the chi-square test or Fisher’s exact test. Results Detection and rate of ESBLs The screening DDST, performed with all 205 isolates, detected 42 ESBL producers: 20 E. cloacae, 15 E. aerogenes, 1 P. agglomerans, and 6 S. marcescens isolates. The rate of ESBL production in the studied group of isolates was 16% in E. cloacae, 33% in E. aerogenes, 7% in P. agglomerans, and 29% in S. marcescens. Overall rate of ESBL production was 20%. Interestingly, only two E. cloacae isolates with constitutive production of AmpC enzymes were detected, with no synergy effect demonstrated. IEF and bioassay Beta-lactamases with different isoelectric points (pIs) (5.4, 7.4, 8.2, 8.4, and 8.8) were found (Table 1). Thirty-seven isolates produced a single ESBL. According to their ceftazidime - or cefotaxime-hydrolyzing activity, the enzymes were assigned to different groups of beta-lactamases. Beta-lactamases that did not hydrolyze ceftazidime or cefotaxime were suspected to be TEM-1 (pI 5.4). The enzymes with pI 8.2, which were able to hydrolyze ceftazidime, probably belonged to SHV group, while the cefotaxime-hydrolyzing beta-lactamases with pIs of 8.4, 8.8 were suspected to be CTX-M enzymes. Detection of ESBL-encoding genes and nucleotide sequencing ESBL-group-specific PCR detected the presence of blaSHV and blaCTX-M genes in 9 and 37 isolates, respectively, and
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Date of isolation
Serratia marcescens 516 15.01.2011 517 16.01.2011 519 21.01.2011 520 25.01.2011 523 27.01.2011 528 10.03.2011 Enterobacter Aerogenes 526 14.02.2011 540 14.02.2011 541 26.05.2011 542 13.06.2011 543 14.06.2011 544 17.06.2011 549 13.08.2011 513 11.01.2011 515 10.01.2011 531 28.03.2011 532 30.03.2011 535 12.04.2011 551 21.09.2011 552 21.09.2011 555 27.09.2011
Isolate
Conjunctiva swab Pus Urine Urine Nasal swab Urine Blood Urine Urine Urine Throat swab Urine Blood Blood Blood
PICU Pediatric PICU Pediatric Pediatric Pediatric PICU Pediatric Pediatric Pediatric Pediatric Pediatric Pediatric Pediatric Pediatric ward ward ward ward ward ward ward ward
2 3 2 1 1 1 2 1
ward 3 ward 1 ward 3
ward 3
Blood Blood Blood Blood Blood Sputum
Site of isolation
PICU Pediatric ward 3 Pediatric ward 3 Pediatric ward 3 PICU Pulmology
Ward
8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4 8.4
5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 8.4 8.4 8.4 8.4 8.4 7.4, 8.8
IEFa
CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3
CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-15
ESBL type
A A A A A A A A A A A A B B B
W W W W W V
RAPD
A
A A A
A A A A A A
A
A
A
A
Plasmid fingerprinting
L/M L/M L/M L/M L/M L/M L/M L/M L/M L/M L/M L/M L/M L/M
L/M L/M L/M L/M L/M
Replicon typing
Table 1. Characteristics of Serratia marcescens, Enterobacter cloacae, Enterobacter aerogenes, and Pantoea agglomerans Isolates Analyzed in This Study
G, G, G, G, G, G,
AM, AM, AM, AM, AM, T
T, T, T, T, T,
T/S T/S T/S T/S T/S
(continued)
Susceptible/no transfer TB, G, AM TB, G, AM TB, G, AM TB, G, AM TB, G, AM TB, G, AM TB, G, AM, T/S TB, G, AM, T/S TB, G, AM, T/S TB, G, AM, T/S TB, G, AM, T/S TB, G, AM, T/S TB, G, AM, T/S TB, G, AM, T/S
TB, TB, TB, TB, TB, TB,
Phenotype of the donor isolateb
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Date of isolation Urine Throat swab Urine Pus Urine Urine Urine Urine Urine Urine CSF Throat swab Sputum Throat swab Urine sputum Throat swab Urine Pus cvc cvc
Cardiac surgery
Site of isolation
PICU PICU Ambulatory Cardiac surgery Endocrinology Endocrinology Urology Cardiac surgery Pediatric ward 3 Pediatric ward 3 Neurology Hematology Hematology Neonatology Neurology Pulmology Hematology Neurology Cardiac surgery Cardiac surgery
Ward
8.4 8.8 8.8 8.8 8.8 8.2 8.2 8.2 8.2 8.2 8.2, 8.2, 8.2, 7.4, 8.2,
8.4 8.4
8.4 8.8 8.8 8.2, 8.8 8.8
5.4, 7.4, 8.8
5.4, 5.4, 8.4 8.4 8.4 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4,
IEFa
CTX-M-15
CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-3 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 SHV-12 SHV-12 SHV-12 SHV-12 SHV-12 SHV-12, CTX-M-3 SHV-12, CTX-M-15 SHV-12, CTX-M-15 SHV-12, CTX-M-15 SHV-12, CTX-M-15
ESBL type
—
f f e e e e c c e e d d d d d d d i g g
RAPD
NT
M
NT
L/M
L
M
FIIAs FIIAs
L/M L/M L/M L/M L/M
Replicon typing
S S
A A
A A
Plasmid fingerprinting
TB, G, CP, T, T/S
TB, G, AM, T/S TB, G, AM, T/S TB, G, AM, T/S TB, G, AM, T/S TB, G, AM T/S TB, G, AM, CP, T, T/S, TB, G, T TB, G, CP, T, T/S TB, G, CP, T, T/S TB, G, AM, CP, T, T/S, TB, G, AM, CP, T, T/S, TB, G, AM, CP, T, T/S, TB, G, AM, CP, T, T/S, TB, G, AM, CP, T, T/S, TB, G, AM, CP, T, T/S, TB, G, AM, CP, T, T/S, TB, G, AM, CP, T, T/S TB, G, CP, T, T/S TB, G, AM, CP, T, T/S
Phenotype of the donor isolateb
CL CL CL CL CL CL CL
CL
b
The bold pIs indicate ceftazidime or cefotaxime hydrolysis in bioassay; underlined pIs indicate successful conjugation transfer. Resistance of the donor isolate to nonbeta-lactam antibiotics, transferrable resistance is underlined. AM, amikacin; CP, ciprofloxacin; G, gentamicin; TB, tobramycin; T/S, trimethoprim/sulfamethoxazole; CL, chloramphenicol; T, tetracycline; NT, nontypeable; CSF, cerebrospinal fluid; PICU, pediatric intensive care unit; cvc, central venous catheter; IEF, isoelectric focusing; ESBL, extended-spectrum beta-lactamase; RAPD, random amplified polymorphic DNA analysis; pIs, isoelectric points.
a
Enterobacter Cloacae 521 10.02.2011 546 22.05.2011 524 14.02.2011 525 14.02.2011 522 08.02.2011 529 21.03.2011 553 15.07.2011 554 20.07.2011 538 18.04.2011 539 18.04.2011 514 03.01.2011 518 21.01.2011 530 28.03.2011 533 29.03.2011 547 01.06.2011 527 21.2.2011 534 08.04.2011 550 22.08.2011 537 18.04.2011 548 09.08.2011 Pantoea Agglomerans 545 30.06.2011
Isolate
Table 1. (Continued)
ESBL-PRODUCING ENTEROBACTER SPP. AND S. MARCESCENS IN BULGARIA confirmed the IEF data. The sequence analysis proved the presence of blaSHV-12, blaCTX-M-3, and blaCTX-M-15. The overall rate of CTX-M enzymes was 88%. The most widespread enzyme was CTX-M-3, found in 64% (n = 27). It was dominant in E. aerogenes (100%, n = 15) and S. marcescens (83%, n = 5). SHV-12, CTX-M-15, and CTX-M-3 were found among E. cloacae isolates in 50% (n = 10), 45% (n = 9), and 35% (n = 7), respectively. Co-production of SHV-12 and CTX-M-15 (n = 4), as well as CTX-M-3 and SHV-12 (n = 1) was detected in the same group. P. agglomerans and one S. marcescens isolate produced CTX-M-15 ESBL. Molecular typing Among E. aerogenes isolates, two different RAPD profiles were detected (A, B), of which profile A was predominantly found in 12 isolates. All E. aerogenes isolates with RAPD A profile were recovered from pediatric wards between January 2011 and August 2011 (Table 1). Among S. marcescens (n = 6), five isolates showed an identical RAPD profile (W) and were isolated in January 2011 (Table 1). Among E. cloacae isolates (n = 20), six different RAPD profiles (c, d, e, f, g, i) were discerned, each with one to seven members (Table 1). All SHV-12-producing E. cloacae isolates exhibited the same RAPD profile. Conjugation experiments, plasmid analysis, and replicon typing Conjugation experiments were successful with 29 isolates, belonging to all four species (Table 1). The conjugation rate for CTX-M-3 producers was high—92%. The genetic transfer experiments with blaSHV-12 were unsuccessful. Attempts to transfer blaCTX-M-15 from 9 blaCTX-M-15-positive isolates were successful in 44% (n = 4) (Table 1). The most prevalent antibiotic resistance phenotype of the transconjugants was resistant to tobramycin, gentamicin, and amikacin – trimethoprim/ sulfamethoxazole and was associated with blaCTX-M-3. Plasmid replicon typing revealed two incompatibility groups—IncL/M and IncFII. (Table 1). The plasmids harboring the blaCTX-M-3 gene belonged to IncL/M group. Plasmid fingerprinting showed that all tested plasmids (except one) from IncL/M group were with similar PstI fingerprinting profiles (Table 1). Only four transconjugants expressed CTX-M-15, and their plasmids belonged to repFIIAs type or were nontypeable. They showed two PstI profiles (Table 1). All donor strains with SHV-12 production showed HI2 replicon type. The size of plasmids from IncL/M and IncFII types was *90 kbp and—66 kbp, respectively. Antimicrobial susceptibility testing The isolates were resistant to tobramycin (95%), gentamicin (95%), and, to a lesser extent, to trimethoprime/sulfamethoxazole (83%). The overall resistance rate to amikacin (79%) was high, mainly due to the higher resistance rate among CTX-M-3 producers (92%). The isolates that produced CTX-M-15 or/and SHV-12 demonstrated significantly higher resistance rate to tetracycline and ciprofloxacin than CTX-M-3 producers (100% vs. 19% and 88% vs. 15%) ( p < 0.001). Only imipenem retained activity against all isolates (100% susceptibility), followed by pipercillin/tazobactam (90% susceptibility) and cloramphenicol (81%).
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Discussion This study provides epidemiological data on ESBLproducing Enterobacter spp., P. agglomerans, and S. marcescens, isolated in 2011 in the University Hospital in one of the most populated Bulgarian cities—Varna. The rate of ESBL production in the studied group of isolates was 16% for E. cloacae, 33% for E. aerogenes, and 7% and 29% for P. agglomerans and S. marcescens, respectively. The overall ESBL rate was 20%. These results are higher than those found in Germany (7%) for Enterobacter spp.24 and in Algeria for Enterobacter spp. and Serratia spp. (7%, 6.8%).26 In contrast, authors from Poland report much higher ESBL rates for Serratia spp. (70.8%), but similar to our results for E. cloacae (18.9%).12 However, the clinical nature of the samples gives only a partial view of the real situation, and further investigation is needed to estimate the ESBLs prevalence in systematic fecal samples. The overall rate of CTX-M enzymes in this study was 88%. Previous investigations in Varna city also detected a high rate of CTX-M production in K. pneumoniae and E. coli— 97%.20 The high ESBL rate in this study, especially in E. aerogenes and S. marcescens, was due to CTX-M-3 production, which is in concordance with other investigators.12,22,25,26 The predominance of CTX-M-3 enzyme in our hospital can be explained with the detection of two major epidemic clones (A and W) among E. aerogenes and S. marcescens. These clones were found predominantly in pediatric wards and in the pediatric intensive care unit between January and August 2011. An outbreak in these wards can be suggested, although the investigation of the hospital environment did not find a source of contamination. CTX-M-3 producers were susceptible to ciprofloxacin and tetracycline, which are not appropriate therapeutic choices for children. Piperacillin/ tazobactam and carbapenems were the only therapeutic options. In our study, meropenem was successfully used for therapy in the case of S. marcescens outbreak in the pediatric wards in January 2011. Our finding is similar to other investigations that found S. marcescens and Enterobacter spp. clones carrying CTX-M-3 enzymes.12,22,25,26 In this study, blaCTX-M-3, detected in E. aerogenes, S. marcescens, and E. cloacae isolates was transferable by conjugative IncL/M plasmids with *90 kbp size (type A, Table 1) and similar PstI fingerprinting profiles. This plasmid was presented in both clonal and nonclonal strains of E. cloacae, suggesting a horizontal gene transfer, which mediates the dissemination of CTX-M-3 enzymes. IncL/M plasmids carrying the blaCTX-M-3 gene have been often reported in Enterobacteriacae, isolated in different countries (Poland, France, Belgium, Korea, and Russia).7,8,10,13 In this study, E. cloacae isolates are characterized with a relatively lower ESBL rate (16%). This result is in concordance with the lack of wide dissemination of particular clones or plasmids among E. cloacae with the exception of SHV-12 producers. The ESBLs identified in E. cloacae were predominantly SHV-12 and CTX-M-15. CTX-M-15 was coproduced with SHV-12 in four isolates. CTX-M-15 was also detected in single P. agglomerans and S. marcescens isolates. The conjugation experiments were successful only with CTXM-15-producing E. cloacae isolates but at a low rate. The transferability of blaCTX-M-15 was associated with identical IncFII with a size of 66 kbp and non-typeable plasmids. Recent studies have reported a dramatic increase in CTX-M-15-
136 producing isolates predominantly in E. coli, K. pneumoniae, but also in Enterobacter spp.1,14,15,23,27 BlaCTX-M-15 gene in E. coli and K. pneumoniae has been identified mostly on IncFII plasmids.7,8 Some authors report an association between blaCTX-M-15 and conjugative plasmids belonging to IncF, IncL/M, and IncA/C groups in Enterobacter spp.13 Very recently, Dolejska et al.11 detected IncFIIK plasmids carrying blaCTX-M-15 in Enterobacteriaceae, isolated from rectal swabs from children in oncology wards. IncF plasmids have also been associated with the spread of blaKPC, blaAmpC, and determinants coding resistance to quinolones and aminoglycosides.8 Interestingly, we found different plasmids (A and S) to be carried by strains from the same E. cloacae clone in three different wards. This finding underlines the great complexity of the ESBL dissemination in the hospital and different spread profiles according to the environment. The conjugation experiments with blaSHV-12-positive E. cloacae were unsuccessful, but HI2 replicons were detected in the donor isolates. The unsuccessful conjugation experiments suggest the probable chromosomal location of the replicon or location on a nonconjugative plasmid. In conclusion, the identification of blaCTX-M-3, blaCTX-M-15, and blaSHV-12 in our study confirms the wide geographical distribution of CTX-M-3, CTX-M-15, and SHV-12 ESBLs among Enterobacter and Serratia species. Our results demonstrate the high prevalence of CTX-M-3 enzyme due to hospital dissemination of clonal E. aerogenes and S. marcescens isolates, carrying IncL/M conjugative plasmids. Funding This work was partially supported by a grant from the Medical University, Sofia, Bulgaria (N 1/2011) (I.M., R.M.). Disclosure Statement The authors declare that there are no conflicts of interest. References 1. Anastay, M., E. Lagier, V. Blanc, and H. Chardon. 2013. Epidemiology of extended spectrum beta-lactamases (ESBL) Enterobacteriaceae in a General Hospital, South of France, 1999–2007. Pathol. Biol. (Paris). 61:38–43. 2. Bauernfeind, A., I. Schneider, R. Jungwirth, H. Sahly, and U. Ullmann. 1999. A novel type of AmpC Beta-Lactamase, ACC-1, produced by a Klebsiella pneumoniae causing nosocomial pneumonia. Antimicrob. Agents Chemother. 43: 1924–1931. 3. Biendo, M., B. Canarelli, D. Thomas, F. Rousseau, F. Hamdad, C. Adjide, G. Laurans, and F. Eb. 2008. Successive emergence of extended-spectrum beta-lactamaseproducing and carbapenemase-producing Enterobacter aerogenes isolates in a university hospital. J. Clin. Microbiol. 46:1037–1044. 4. Bradford, P.A. 2001. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin. Microbiol. Rev. 14:933–951. 5. Canto´n, R., A. Novais, A. Valverde, E. Machado, L. Peixe, F. Baquero, and T.M. Coque. 2008. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe. Clin. Microbiol. Infect. 14:144–153.
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Address correspondence to: Rumyana Donkova Markovska, MD, PhD Department of Medical Microbiology Faculty of Medicine Medical University of Sofia 2, Zdrave Street 1431 Sofia Bulgaria E-mail:
[email protected]
