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      Transmission Dynamics of Extended-Spectrum β-lactamase–Producing Enterobacteriaceae in the Tertiary Care Hospital and the Household Setting

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          Abstract

          Transmission of extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae in households outweighs nosocomial dissemination in the non-outbreak setting. Importation of ESBL producers into the hospitals is as frequent as transmission during hospital stay. ESBL– Klebsiella pneumoniae might be more efficiently transmitted within the hospital than ESBL– Escherichia coli.

          Abstract

          Background . Studies about transmission rates of extended-spectrum β-lactamase (ESBL)–producing Enterobacteriaceae in hospitals and households are scarce.

          Methods . Eighty-two index patients with new carriage of ESBL-producing Escherichia coli (ESBL- Ec; n = 72) or ESBL-producing Klebsiella pneumoniae (ESBL- Kp; n = 10) and their hospital (n = 112) and household (n = 96) contacts were studied prospectively from May 2008 through September 2010. Isolates were phenotypically and molecularly characterized (sequencing of bla genes, repetitive extragenic palindromic polymerase chain reaction, pulse-field gel electrophoresis, and multilocus sequence typing). Transmission was defined as carriage of a clonally-related ESBL producer with identical bla ESBL gene(s) in the index patient and his or her contact(s).

          Results . CTX-M-15 was the most prevalent ESBL in ESBL- Ec (58%) and ESBL- Kp (70%) in the index patients. Twenty (28%) ESBL- Ec isolates were of the hyperepidemic clone ST131. In the hospital, transmission rates were 4.5% (ESBL- Ec) and 8.3% (ESBL- Kp) and the incidences of transmissions were 5.6 ( Ec) and 13.9 ( Kp) per 1000 exposure days, respectively. Incidence of ESBL- Kp hospital transmission was significantly higher than that of ESBL- Ec ( < .0001), despite implementation of infection control measures in 75% of ESBL- Kp index patients but only 22% of ESBL- Ec index patients. Detection of ESBL producers not linked to an index patient was as frequent (ESBL- Ec, 5.7%; ESBL- Kp, 16.7%) as nosocomial transmission events. In households, transmission rates were 23% for ESBL- Ec and 25% for ESBL- Kp.

          Conclusions . Household outweighs nosocomial transmission of ESBL producers. The effect of hospital infection control measures may differ between different species and clones of ESBL producers.

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          Most cited references 23

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          Dissemination of Clonally Related Escherichia coli Strains Expressing Extended-Spectrum β-Lactamase CTX-M-15

          Plasmid-mediated CTX-M type expanded-spectrum β-lactamases (ESBLs), which have been extensively reported for the past 10 years, are detected mostly in community-acquired pathogens and are associated mainly with Escherichia coli. These β-lactamases compromise the efficacy of all β-lactams, except carbapenems and cephamycins, and are associated with many non–β-lactam resistance markers because of their locations on plasmids. Therefore, they may constitute a real threat for treating community-acquired E. coli–mediated urinary tract infections ( 1 , 2 ). Different variants of CTX-M ESBLs are grouped in 5 clusters, although their distribution varies greatly depending on the geographic area (www.lahey.org/studies/webt.htm). CTX-M-15, which was first detected in isolates from India in 2001 ( 3 ), is now recognized as the most widely distributed CTX-M enzyme. It is derived from CTX-M-3 by 1 amino acid substitution at position 240 (Asp-240 → Gly), which apparently confers an increased catalytic activity to ceftazidime ( 4 ). Clonal outbreaks of CTX-M-15–producing Enterobacteriaceae have been reported in France, Italy, Spain, Portugal, Austria, Norway, the United Kingdom, Tunisia, South Korea, and Canada, and E. coli is the most frequently involved species. Within E. coli, CTX-M-15–producing strains of the B2 phylogenetic group are commonly found and frequently harbor multidrug resistance and virulence determinants ( 5 – 18 ). Plasmids encoding bla CTX-M-15 have been isolated from clinical isolates in France, Spain, Portugal, the United Kingdom, Canada, India, Pakistan, South Korea, Taiwan, the United Arabic Emirates, and Honduras ( 5 – 8 , 10 , 11 , 15 , 19 , 20 ). Plasmid characterization, which has only been accomplished for those plasmids from Canada, France, Spain, and the United Kingdom, classified most of them as members of incompatibility group FII ( 5 , 7 , 8 , 17 , 19 ). Lack of detailed studies on isolates expressing particular CTX-Ms from different geographic areas has precluded identification of factors involved in recent and worldwide spread of specific CTX-M variants. In this article, through analysis of the population biology of CTX-M-15–producing isolates from 7 countries and characterization of their genetic elements, we provide a comprehensive picture of elements involved in international spread of a particularly widespread mechanism of antimicrobial drug resistance. Materials and Methods Bacterial Strains, Production of ESBL, and Susceptibility Testing We studied 43 CTX-M-15–producing E. coli clinical isolates from France (n = 17), Kuwait (n = 9), Switzerland (n = 7), Canada (n = 4), Portugal (n = 3) and Spain (n = 3), and 6 CTX-M-15 plasmids from India ( 3 ), all obtained from 2000 through 2006. These strains and plasmids were considered representative of these areas because they either caused outbreaks or were the first isolates recovered in those countries ( 3 , 11 , 16 , 19 , 21 – 23 ). Samples were isolated from urine (n = 33/43, 77%), wounds (n = 4/43, 9.%), respiratory tract infections (n = 3/43, 7%) and other sites (1 from feces, 1 from an intravenous catheter, and 1 from blood) in hospitalized patients. ESBL production was confirmed by a standard double-disk synergy test, and bla genes were characterized by PCR and additional sequencing as described ( 19 ). Susceptibility patterns to 13 non–β-lactam antimicrobial drugs were determined by the standard disk diffusion method following published standards ( 24 ). Strains with intermediate susceptibility were considered resistant. Clonal Relationships Clonal relationships were established by pulsed-field gel electrophoresis (PFGE) of XbaI-digested genomic DNA (New England Biolabs, Ipswich, MA, USA) as described ( 25 ). Assignment of E. coli phylogenetic groups was conducted by using a multiplex PCR assay described by Clermont et al. ( 26 ). All E. coli isolates belonging to phylogroups B2 and D were characterized by multilocus sequence typing (MLST) using the standard 7 housekeeping loci (www.mlst.net). All fumC sequences from E. coli isolates belonging to phylogroup D were analyzed for a C288T single nucleotide polymorphism. This polymorphism is specific for a globally disseminated E. coli strain arbitrarily designated as E. coli clonal group A (CgA) that is associated with community-acquired urinary tract infections ( 27 , 28 ). Transferability and Location of bla CTX-M-15 Transferability was tested by broth and filter mating assays using E. coli K12 strain BM21R (resistant to nalidixic acid and rifampin, positive for lactose fermentation, and free of plasmids) as recipient at a 1:2 donor: recipient ratio. Transconjugants were selected on Luria-Bertani agar plates containing cefotaxime (1 mg/L) and rifampin (100 mg/L) and incubated at 37°C for 24–48 h. Transformation was performed for a subset of isolates by using conditions reported ( 3 ). Chromosomal or plasmid location of bla CTX-M-15 genes was assessed by hybridization of I-CeuI–digested genomic DNA with bla CTX-M-15 and 16S rDNA probes and electrophoresis (5–25 s for 23 h and 60–120 s for 10 h at 14°C and 6 V/cm2) ( 25 ). Transfer and hybridization were performed by using standard procedures. Labeling and detection were conducted by using enhanced chemiluminescence (Amersham Life Sciences, Uppsala, Sweden) following manufacturer’s instructions. Plasmid Characterization Plasmid DNA was obtained by using different midiprep plasmid purification kits (QIAGEN, Hilden, Germany, and Marlingen Biosciences, Ijamsville, MD, USA). Plasmids were classified according to their incompatibility group by a PCR-based replicon-typing scheme ( 29 ). Determination of plasmid size and confirmation of replicon content was established for transconjugants (or wild-type strains in the absence of transfer) by hybridization of S1 nuclease–digested genomic DNA with probes specific for bla CTX-M-15 and for different F replicons (FII, FIA, FIB), which were obtained by PCR as described ( 19 ). Relationships among plasmids were determined by comparison of EcoRI and HpaI digested DNA patterns and comparison of repFII sequences. Genescan software (Applied Biosystems, Foster City, CA, USA) was used for collection of gel images. Data of a subset of representative patterns were exported into Fingerprinting II Informatix version 3.0 software (Bio-Rad Laboratories, Hercules, CA, USA) for further interpretation. Cluster analysis was conducted by using the unweighted pair group method with arithmetic averages (optimization 0.5%, tolerance 1.00%). Presence of genes previously associated with plasmids encoding CTX-M-15 as bla OXA-1, bla TEM-1, and aac(6′)-Ib-cr was screened by PCR by using primers bla OXA-1 (oxa1 FW: 5′-TTT TCT GTT GTT TGG GTT TT-3′ and oxa1 RV: 5′-TTT CTT GGC TTT TAT GCT TG-3′), bla TEM-1 (TEM-F: 5′-ATG AGT ATT CAA CAT TTC CG-3′ and TEM-R: 5′-CTG ACA GTT ACC AAT GCT TA-3′), and aac(6′)-Ib-cr (aac-cr-F: 5′-TTG CGA TGC TCT ATG AGT GG-3′ and aac-cr-R: 5′-GCG TGT TCG CTC GAA TGC C-3′) ( 11 , 19 , 30 ). Additional sequencing was necessary to identify the corresponding genes. Results Epidemiologic Background Most CTX-M-15–producing E. coli isolates belonged to phylogroups B2 (50%) and D (25%), which are known to be associated with the hospital setting and extraintestinal pathogenic E. coli. Phylogroups A (18%) and B1 (7%), which are associated with animal or human commensal strains, were less frequently represented. All isolates of phylogroups B2, A, and D corresponded to subgroups B23, A1, and D1, respectively, which are the most common ones within each phylogenetic group ( 31 ). The 43 clinical isolates were classified into 32 PFGE types (B23, 13; D1, 10; A1, 6; and B1, 3). Among B23 strains, 10 PFGE types (18 isolates from France, Canada, Spain, Portugal, Kuwait, and Switzerland) were possibly related according to criteria of Tenover et al. ( 32 ) (difference 80% similarity) and were assigned to the sequence type (ST) ST131. The 4 unrelated B2 strains were classified within ST695 (1 from France), ST28 (1 from Switzerland), ST354 (1 from Portugal and Spain) and ST405 (1 from Portugal). All isolates of phylogroup D1 were clonally unrelated by PFGE (difference >6 bands), although MLST studies indicated that 4 PFGE types (5 isolates) from Kuwait, Switzerland, and Spain corresponded to ST405. The fumC sequences of the remaining 6 E. coli D strains were highly diverse (alleles 4, 13, 26, 88, and 132). None of the strains had the C288T single nucleotide polymorphism specific for E. coli strain CgA ( 28 ). All 3 B1 isolates were found in France. Among B2 E. coli isolates, all but 4 were isolated from urine and all but 2 belonged to ST131. These strains correspond to 2 isolates recovered from wounds and identified as ST28 and ST354 and 2 ST131 isolates from respiratory and fecal samples, respectively. CTX-M-15 clinical strains were considered resistant to different antimicrobial drugs: amoxicillin-clavulanate (98%), tobramycin (89%), kanamycin (87%), tetracycline (84%), gentamicin (82%), nalidixic acid (74%), streptomycin (68%), sulfonamides (61%), ciprofloxacin (61%), trimethoprim (58%), chloramphenicol (21%), nitrofurantoin (12%), and amikacin (11%). All CTX-M-15 transconjugants expressed resistance to aminoglycosides, tetracycline, or trimethoprim. All but 2 strains contained bla OXA-1 and aac(6′)-Ib-cr; 1 contained only aac(6′)-Ib-cr, and 1 contained bla OXA-1 and aacA4, which confers reduced susceptibility to amikacin and kanamycin. Location and Transferability of bla CTX-M-15 The bla CTX-M-15 gene was located on plasmids in all but 6 strains and was positively transferred by conjugation or transformation in 37% of the strains tested. In 8 clinical isolates corresponding to 7 PFGE types, the probe for bla CTX-M-15 hybridized in chromosomal bands (2 belonging to B23 ST131, 2 to D1, 1 to D1 ST405, and 1 to A1). In 2 other strains, the bla CTX-M-15 probe hybridized both with plasmid and chromosomal bands (1 strain from D ST405 and 1 from phylogroup B1). Plasmids Encoding CTX-M-15 Plasmids positive for the bla CTX-M-15 gene showed variable sizes (85–160 kb), belonged to the narrow host range incompatibility group IncF, and had replicon FII alone or in association with the FIA or FIB replicons (Appendix Table). Many restriction fragment length polymorphism (RFLP) patterns were observed, with overrepresentation of 3 profiles corresponding to 3 plasmids arbitrarily designated as plasmid A (85 kb), plasmid B (120 kb), and plasmid C (85 kb). Plasmid A, which was isolated from B2 E. coli strains from 4 countries (India, France, Portugal, and Spain), was associated with different STs (ST131, ST354, or ST405). Plasmid C was also detected in clonally unrelated E. coli of phylogroups B2 and D from Switzerland, Canada and France. Plasmid B, which was only associated with E. coli ST131, was widely disseminated in all countries studied. Sequence analysis of the replicons showed 4 repFII types: repFII(1), which was identical to that of plasmids R100, NR1, or pC15–1a, and was the most represented and identified in 23 plasmids; repFII(2), which had 99%–100% homology with plasmid pRSB107 (GenBank accession no. AJ851089), was identified in 6 plasmids; and repFII(3) and repFII(4), which were detected in 2 and 7 plasmids, respectively, and showed >93% homology with repFII(1). All repFIA and repFIB sequences were 99% and 100% homologous, respectively, with that of pRSB107 (GenBank accession no. AJ851089). Computer analysis of representative RFLP patterns and repFII sequences grouped CTX-M-15 plasmids within 3 major clusters with similarity >70%. Cluster I comprises most plasmids, including plasmids A and B, most containing repFII(1) and showing variable replicon content. Cluster II comprised only plasmid C derivatives showing slightly different repFII sequences, and cluster III included 2 plasmids carrying repFII(2), FIA, and FIB replicons (Figure). Figure Computer analysis of a subset of representative HpaI restriction profiles of IncF CTX-M-15 plasmids from Escherichia coli isolates in the Appendix Table. Cluster analysis was done by using Fingerprinting II in Informatix software version 3.0 (Bio-Rad Laboratories, Hercules, CA, USA) and applying the unweighted pair group method with arithmetic averages (optimization 0.5%, tolerance 1.00%). Ph, phylogenetic; ST, sequence type; RFLP, restriction fragment length polymorphism; Can, Canada; Fra, France; Por, Portugal; Swi, Switzerland; Ind, India; Kuw, Kuwait; Sp, Spain. In the 8 strains with chromosomal location of bla CTX-M-15, repFII plasmids were identified but these plasmids were negative for the bla CTX-M-15 gene. Several strains that were also positive for additional plasmids and negative for the bla CTX-M-15 gene were assigned to different incompatibility groups or were untypeable by the PCR-based replicon typing scheme used. Discussion Our study indicates that current worldwide spread of the bla CTX-M-15 gene is driven mainly by 2 epidemic E. coli strains belonging to phylogroups B2 (ST131) and D (ST405) and by its location on IncF plasmids harboring multiple antimicrobial drug–resistance determinants, including the recently described aac(6′)-Ib-cr gene. The presence of bla CTX-M-15 has previously been associated with E. coli strains of phylogroups B2 and D, and in some instances, with specific PFGE types ( 9 – 12 , 16 ). We detected an emerging and globally disseminated CTX-M-15 phylogroup B2 E. coli strain corresponding to the ST131 that was responsible for clonal outbreaks in Canada, France, Spain, and Portugal ( 11 , 14 , 16 , 23 ). Other CTX-M-15 B2 strains belong to clonal complexes ST695, ST405, ST354, or ST28, which have previously been detected in different geographic areas among isolates that do not express CTX-M-15 (Appendix Figure). Globally disseminated E. coli strains associated with acute, uncomplicated, community-acquired cystitis and pyelonephritis, designated in community patients as clone CgA (ST69), have only been occasionally associated with CTX-M-15 production in Canada ( 16 , 27 , 28 ). Although the isolates in our study do not belong to clone CgA, they were isolated mainly from urine samples, and an association of ST131 E. coli isolates with urinary tract infections might be inferred. Although most CTX-M-15 isolates studied were recovered from hospitalized patients, these microorganisms are now widely spread in the community setting, including long-term care facilities in the countries from which isolates included in this study originated ( 2 , 5 , 14 , 33 ). Our study has increased knowledge of the number of epidemic E. coli clonal complexes causing urinary tract infections. All plasmids carrying bla CTX-M-15 included in this study corresponded to incompatibility group F, and all had the FII replicon, which was assorted mainly in multireplicon plasmids with additional replicons of the FIA and FIB types. Association of the bla CTX-M-15 gene with IncFII replicons has been described in studies conducted in Canada, France, Spain, and the United Kingdom ( 5 , 7 , 8 , 17 , 19 ). Although we observed intercontinental dissemination of 3 major IncFII plasmid scaffolds (A, B, and C) carrying bla CTX-M-15, similarity >70% among all variants studied and presence of genes also found in pC15–1a, a CTX-M-15 plasmid (GenBank accession no. AY458016) that has a 28.4-kb multidrug resistance region containing bla TEM-1, bla OXA-1, the aac(6′)-Ib-cr gene (aminoglycoside 6′-N-acetyltransferase type Ib-cr variant responsible for reduced susceptibility to both aminoglycosides and certain fluoroquinolones), and genetic determinants coding for resistance to tetracycline and aminoglycosides ( 5 , 30 ), suggest a common origin or a common particular plasmid scaffold involved in the dissemination of CTX-M-15. Because IncF plasmids are a heterogeneous and largely diffused family of plasmids in E. coli, they could acquire the bla CTX-M-15 gene. IncF plasmids negative for the bla CTX-M-15 gene in strains with this gene at a chromosomal location also suggest dynamic horizontal exchanges between the chromosome and resident plasmids. Extensive recombination events among IncF plasmids are frequent and may have contributed to their apparent high diversity (variable rep content, plasmid size, transferability, antimicrobial drug–resistance genes), driving their evolution and enabling them to persist in diverse E. coli populations ( 34 , 35 ). Such recombination events among plasmids of the same incompatibility group within the same cell occur frequently ( 34 , 35 ). This hypothesis is supported by the results of Lavollay et al. ( 17 ), who described mosaicism in a CTX-M-15 plasmid isolated in France that contained genes from 2 different IncFII plasmids, pC15–1a and pRSB107 (from IncFII plasmids first isolated from persons in Canada and activated sludge bacteria from a wastewater treatment plant in Germany, respectively) ( 5 , 36 ). Spread and maintenance of conjugative plasmids across bacterial populations have been intensively studied from a theoretical point of view, but data from natural populations are scarce ( 34 , 37 , 38 ). Recovery of related plasmids from clonally unrelated B2 strains might reflect efficient transfer of these elements among different B2 E. coli populations. Sharing the same environment, successive immigrant B2 strains might sweep through the population, enabling plasmid hitchhiking at a high frequency in each selective sweep. However, we lack detailed information on the specificity and stability of different plasmid groups in specific hosts. An evolutionary convergent relationship among B2 genetic background and IncFII plasmids cannot be ruled out and should be studied because it might explain successful dissemination of CTX-M-15 plasmids within this E. coli lineage. In addition, our study is one of the few that have identified bla ESBL genes in the chromosome, which might respond either to plasmid integration or transposition driven by ISEcp1 located upstream from the bla CTX-M-15 gene ( 25 , 39 , 40 ). In conclusion, worldwide dissemination of bla CTX-M-15 is driven by B2 or D E. coli clones associated mainly with urinary tract infections or IncFII plasmids containing a multiple antimicrobial drug–resistance platform that contributes to spread of CTX-M-15. Further studies to test the stability/variability and fitness of particular plasmids among different bacterial hosts will be relevant in developing additional strategies to control dissemination of antimicrobial drug resistance. Supplementary Material Appendix Table Epidemiologic data of CTX-M-15-producing Escherichia coli isolates from 7 countries* Appendix Figure Geographic distribution of widely disseminated Escherichia coli clonal complexes associated with CTX-M-15. Data from strains lacking blaCTX-M-15 are from published studies (17,27,28; http://web.mpiib-berlin.mpg.de/mlst/dbs/Ecoli). E. coli clonal group A (CgA) has been identified as different sequence types (STs), most belonging to ST69 (27).
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            Emergence of Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs) in the community.

            Enterobacteriaceae, especially Klebsiella spp. producing extended-spectrum beta-lactamases (ESBLs) such as SHV and TEM types, have been established since the 1980s as a major cause of hospital-acquired infections. Appropriate infection control practices have largely prevented the dissemination of these bacteria within many hospitals, although outbreaks have been reported. However, during the late 1990s and 2000s, Enterobacteriaceae (mostly Escherichia coli) producing novel ESBLs, the CTX-M enzymes, have been identified predominantly from the community as a cause of urinary tract infections. Resistance to other classes of antibiotics, especially the fluoroquinolones, is often associated with ESBL-producing organisms. Many clinical laboratories are still not aware of the importance of screening for ESBL-producing Enterobacteriaceae originating from the community. A heightened awareness of these organisms by clinicians and enhanced testing by laboratories, including molecular surveillance studies, is required to reduce treatment failures, to limit their introduction into hospitals and to prevent the spread of these emerging pathogens within the community.
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              Escherichia coli sequence type ST131 as the major cause of serious multidrug-resistant E. coli infections in the United States.

              Escherichia coli sequence type ST131 (O25:H4), associated with the CTX-M-15 extended-spectrum beta-lactamase, has emerged internationally as a multidrug-resistant pathogen but has received little attention in the United States. From the SENTRY and Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) surveillance programs, 127 E. coli clinical isolates from hospitalized patients across the United States in 2007, stratified by extended-spectrum cephalosporin and fluoroquinolone phenotype and bla(CTX-M-15) genotype, were assessed for phylogenetic group, ST131 status, susceptibility profile, virulence genotype, gyrA and parC sequence, and pulsed-field gel electrophoresis profile. The 54 identified ST131 isolates (all fluoroquinolone resistant) accounted for an estimated 17% of the source populations, including 67%-69% of isolates resistant to extended-spectrum cephalosporins or fluoroquinolones, 55% of those resistant to both fluoroquinolones and trimethoprim-sulfamethoxazole, and 52% of multidrug-resistant isolates. Their distinctive virulence profiles were more extensive compared with other antimicrobial-resistant isolates but similarly extensive compared with antimicrobial-susceptible isolates. Pulsed-field profiling suggested ongoing dissemination among locales, with concentration of bla(CTX-M-15) within specific ST131 lineages. A historical ST131 isolate lacked the 2007 ST131 isolates' conserved fluoroquinolone resistance-associated single-nucleotide polymorphisms in gyrA and parC. A single E. coli clonal group, ST131, probably caused the most significantly antimicrobial-resistant E. coli infections in the United States in 2007, thereby constituting an important new public health threat. Enhanced virulence and/or antimicrobial resistance compared with other E. coli, plus ongoing dissemination among locales, may underlie ST131's success. Urgent investigation of the sources and transmission pathways of ST131 is needed to inform mitigation efforts.
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                Author and article information

                Journal
                Clin Infect Dis
                Clin. Infect. Dis
                cid
                cid
                Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America
                Oxford University Press
                1058-4838
                1537-6591
                1 October 2012
                20 June 2012
                20 June 2012
                : 55
                : 7
                : 967-975
                Affiliations
                [1 ]Institute for Infectious Diseases, University of Bern
                [2 ]Department of Infectious Diseases, University Hospital of Bern , Switzerland
                Author notes
                [a]

                M. H. and B. Y. B. contributed equally to this work.

                [b]

                Present affiliation/address: Swissmedic, Hallerstrasse 7, 3000, Bern, Switzerland (K. B.-S.); Division of Infectious Diseases, University of California, San Francisco, USA (N. H.).

                Correspondence: Kathrin Mühlemann, MD, PhD, Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, CH-3010 Bern, Switzerland ( kathrin.muehlemann@ 123456ifik.unibe.ch ).
                Article
                cis581
                10.1093/cid/cis581
                3436924
                22718774
                © The Author 2012. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please email: journals.permissions@ 123456oup.com .

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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