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Journal of Medical Microbiology (2012), 61, 780–785 DOI 10.1099/jmm.0.041087-0 High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile Verena Pecavar,1 Marion Blaschitz,1 Peter Hufnagl,1 Josef Zeinzinger,2 Anita Fiedler,1 Franz Allerberger,3 Matthias Maass4 and Alexander Indra3 Correspondence Alexander Indra [email protected] 1 Austrian Agency for Health and Food Safety (AGES), Institute for Medical Microbiology and Hygiene, Waehringerstrasse 25a, A-1090 Vienna, Austria 2 Austrian Agency for Health and Food Safety, Institute for Medical Microbiology and Hygiene, Spargelfeldstrasse 191, A-1220 Vienna, Austria 3 Paracelsus Medical University, Institute for Medical Microbiology, Hygiene and Infectious Diseases, Muellner Hauptstrasse 48, A-5020 Salzburg, Austria 4 Institut für Laboratoriumsmedizin, Mikrobiologie und Umwelthygiene, Klinikum Augsburg, Stenglinstrasse 2, D-86156 Augsburg, Germany Received 25 November 2011 Accepted 17 February 2012 Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the b-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates. INTRODUCTION Clostridium difficile, a spore-forming, Gram-positive bacterium, is the most frequently identified causative agent of hospital-acquired diarrhoea and pseudomembranous colitis (Wiström et al., 2001). Numerous C. difficile outbreaks have been caused by strains of PCR ribotype 027, REA Abbreviations: CDI, C. difficile infections; CP, curve profiles; HRM, high-resolution melting; SNP, single nucleotide polymorphisms; ST, sequence-types. 780 (restriction endonuclease analysis) type BI and NAP1 (North American pulsed-field type 1), as well as toxinotype III, which is said to lead to more severe disease and higher mortality rate compared to other C. difficile strains (Kuijper et al., 2006). The main antibiotics used for treatment of C. difficile infection (CDI) are metronidazole and vancomycin (Kuijper et al., 2006). In addition, a promising antibiotic for the treatment of CDI is rifaximin, a rifamycin derivative. The mechanism of action of rifamycin is to inhibit the elongation of 2 or 3 nt RNA transcripts at the 59 end by Downloaded from www.microbiologyresearch.org by 041087 G 2012 SGM IP: 88.99.165.207 On: Sun, 30 Apr 2017 01:51:04 Printed in Great Britain SNP profiling for rifaximin resistance in C. difficile binding the RNA polymerase holoenzyme (Campbell et al., 2001). Recently, four working groups have reported C. difficile strains with reduced susceptibility to rifaximin due to SNPs within the encoding gene for the b-subunit of the RNA polymerase (rpoB) (O’Connor et al., 2008; Curry et al., 2009; Huang et al., 2010; Huhulescu et al., 2011). An accurate method for complete genotyping of most single nucleotide polymorphisms (SNPs) is high-resolution melting (HRM) analysis (Liew et al., 2004) where DNA amplification takes place in the presence of a fluorescent dye that attaches only to dsDNA (Wittwer et al., 2003). During a subsequent melting step, denaturation of dsDNA occurs accompanied by reduction of the emitted fluorescence, resulting in characteristic differences measured by the LightCycler 480 software (Roche Diagnostics). The aim of the present study was to develop an assay for the fast identification of SNPs in a proposed hot-spot region of the rpoB gene, leading to reduced rifaximin susceptibility in C. difficile by applying HRM. METHODS Strains used in this study. A total of 348 C. difficile isolates of different PCR-ribotypes (Table 1) were obtained from the reference strain collection of the Austrian national C. difficile reference centre, which included 25 strains from the ECDC strain collection, 50 strains from the Leeds University Hospital (UK), two strains from the Public Health Laboratory Maribor (Slovenia), 242 clinical isolates (129 of which were previously described by Indra et al., 2008), and 29 strains from the CDIFFGEN strain collection. Rifaximin susceptibility testing of the 348 C. difficile samples was performed in a previous study using a rifaximin broth microdilution test (Huhulescu et al., 2011). Due to the lack of CLSI or EUCAST MIC breakpoints for rifaximin against C. difficile we interpreted samples showing an MIC .32 mg ml21 as having reduced in vitro susceptibility as suggested by Huhulescu et al. (2011). We investigated the 70 samples displaying reduced rifaximin susceptibility and the 278 samples that were rifaximin susceptible by using sequence analysis and HRM analysis for the detection of SNPs within the rpoB gene. Capillary gel electrophoresis-based ribotyping of C. difficile isolates. DNA was extracted from cultures using the MagNA Pure Compact system (Roche Diagnostics) according to the manufacturer’s instructions to a final volume of 50 ml. For capillary gel electrophoresisbased PCR ribotyping, primers 16S (59-GTGCGGCTGGATCACCTCCT-39) and 23S (59-CCCTGCACCCTTAATAACTTGACC-39) were used. The 16S primer was labelled at the 59 end with either carboxyfluorescein (FAM), hexachlorofluorescein (HEX) or tetrachlorofluorescein (TET). Reactions contained 1.5 ml sample DNA, 25 ml HotStarTaq Master Mix (Qiagen), 0.3 ml (10 pmol ml21) each primer and 22.9 ml of PCR-grade water. PCR was performed in a standard thermocycler (Analytic Jena) using the following programme: 95 uC for 15 min, followed by 17 cycles of 95 uC for 1 min, 61 uC for 1 min and 72 uC for 1 min, and a final step at 72 uC for 30 min. PCR products were sequenced by using an ABI 310 Genetic Analyzer with a 41 cm capillary loaded with POP4 gel (Applied Biosystems). The samples were injected at 5 kV over 5 s with a total running time of 30 min at 15 kV run voltage. As an internal marker for each sample, a 50–625 bp TAMRA ladder (Chimerx) was used. Data analysis was performed by using Peak Scanner software 1.0 (Applied Biosystems). http://jmm.sgmjournals.org Sequencing of the partial rpoB. All C. difficile samples were investigated for the occurrence of SNPs within a 325 bp fragment of the rpoB gene (amino acid positions 458–565) by performing PCRs in a standard thermocycler and sequence analysis using an ABI 3130 Genetic Analyzer as described previously (Huhulescu et al., 2011). Briefly, DNA was isolated using the MagNA Pure Compact system (Roche Diagnostics) according to the manufacturer’s instructions and eluted to a final volume of 50 ml. The primer pair RifFOR (59CAAGATATGGAAGCTATAAC-39) and RifREVlang (59-GTGATTCTATAAATCCAAATTC-39) was used in PCRs containing 25 ml HotStar Taq Master Mix (Qiagen), 5 ml (5 pmol ml21) each primer, 13 ml PCR grade water and 2 ml sample DNA. DNA amplification was performed in a standard thermocycler using the following programme: 96 uC for 15 min, followed by 30 cycles of 94 uC for 1 min, 52 uC for 1 min and 72 uC for 1 min, with a final step at 72 uC 10 min. PCR products were purified with exonuclease I and shrimp alkaline phosphatase (Fermentas) according to the supplier’s instructions. Sequencing PCRs were performed using a BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems), each reaction containing 2 ml Terminator Ready Reaction Mix, 1 ml sequencing buffer (Applied Biosystems), 4 ml PCR grade water, 1 ml RifFOR or RifREVlang primer (10 pmol21) and 2 ml DNA, in a standard thermocycler with the following programme: 96 uC for 1 min, followed by 30 cycles of 96 uC for 20 s, 50 uC for 20 s and 60 uC for 4 min. For dye terminator cleanup, PCR products were purified with Centri Sep 96-well plates or Centri Sep 8 well strips (Applied Biosystems) according to the manufacturer’s instructions. Samples were analysed in an ABI 3130 Genetic Analyzer (Applied Biosystems) with 36 cm capillary loaded with POP7 gel (Applied Biosystems). Data analysis was performed by using Kodon (Applied Maths) version 3.5. Sequences were aligned to the rpoB sequence of a reference wild-type C. difficile strain (CD630; GenBank accession number NC_009089), which was obtained from the NCBI database. HRM analysis of the SNP hot-spot region in rpoB. For HRM analysis a 151 bp fragment of rpoB (amino acid positions 469–518) was amplified using the primers RifFORlang (59-GTTAACATAAGACCAGTTTC-39) and RifREV (59-CTCTTTCTCTTGAAAGACC-39). Each HRM reaction mixture contained 2.4 ml MgCl2 (25 mM), 10 ml LightCycler 480 HRM Master mix (Roche Diagnostics), 2 ml each primer (10 pmol ml21) and 0.6 ml PCR-grade water. To generate artificial heterozygotes, wild-type and sample DNA were mixed in a ratio of 1 : 6 (0.5 ml wild-type DNA and 2.5 ml sample DNA) and PCR grade water was used as a negative control. PCR and melting-curve analysis was performed using the LightCycler 480 System (Roche Diagnostics) with following conditions: preincubation at 95 uC for 10 min, followed by 45 cycles of 95 uC for 10 s, 52 uC for 20 s and 72 uC for 20 s. After amplification, the melting curve was produced by heating samples up to 95 uC for 1 min and subsequent cooling to 40 uC for 1 min. HRM data were obtained at a rate of 25 acquisitions per uC. Data were analysed with the LightCycler 480 gene scanning software version 1.5 with default settings for sensitivity set to 0.30 and temperature shift set to threshold of 5. The pre-melt normalization ranged from 73.16 to 74.12 and the post-melt normalization ranged from 81.44 to 82.70. All samples were tested in triplicate and wildtype samples were used as the baseline in difference plots. RESULTS Sequence analysis RpoB sequencing (Table 1) of the 348 C. difficile strains resulted in the detection of 246 (70.68 %) samples displaying Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 01:51:04 781 V. Pecavar and others Table 1. Amino acid substitutions in the rpoB gene detected in 348 C. difficile strains based on sequencing results Amino acid substitutions located in the proposed hot-spot region are written in bold. PCR ST HRM CP RpoB amino acid substitution Reduced rifaximin susceptibility Ribotype (RT) (number of isolates) 1 2 1 2 Wild-type R505K No Yes 3 3 T501T+L506L+G510G+G512G+F521F+ E541E+K556K No 4 4 H502N+R505K Yes 5 1 A555A No 6 7 5 6 H502L H502N Yes Yes 8 9 7 8 Yes Yes 10 11 12 13 14 15 16 9 10 11 6 2 1 1 H502Y S475S+F481F+D492D+T501T+A508A+ G510G+T539T+K556K+S575A L487F+H502Y D492N D492V H502N+A555A R505K+I548M S550F S550Y Different RTs (246) RT027 (46) RT072 (1) RT027 (7) RT126 (4) RT078 (3) RT033 (2) RT078 (2) RT579 (2) RT045 (1) RT053 (1) RT063 (1) RT012 (5) RT027 (4) RT023 (3) RT027 (3) RT115 (1) RT063 (1) RT053 (1) RT027 (1) RT027 (2) RT047 (1) RT027 (2) RT539 (1) the wild-type sequence (PCR ST-1) of the rpoB gene. Fifteen different PCR sequence types (excluding PCR ST-1) were determined by 23 different SNPs, four of which (L487F, H502L, S550F and S575A) had not been previously described (Table 2) and were detected in 102 of the 348 (29.31 %) C. difficile samples. Nine of the 15 PCR sequencetypes had only one point mutation within the 325 bp fragment of the rpoB gene and six PCR sequence-types showed additional SNPs (Table 1). The most common PCR sequence-type was PCR ST-2 which displayed amino acid substitution R505K detected in 47 of the 102 (46.07 %) rpoBmutated isolates. The second most common sequence-type was PCR ST-3, containing seven synonymous point mutations (T501T+L506L+G510G+G512G+F521F+E541E+K556K) within the 325 bp amplicon of the rpoB gene. This sequencetype was detected in 23 of the 102 (22.54 %) isolates tested. PCR ST-4, characterized by point mutations H502N+ R505K, and PCR ST-5, characterized by A555A, were both detected in nine of the 102 (8.82 %) samples tested as shown 782 Yes Yes Yes Yes Yes Yes Yes RT027 RT027 RT053 RT058 RT027 RT027 RT027 (1) (1) (1) (1) (1) (1) (1) in Table 1. Sequence analysis revealed point mutations H502N (PCR ST-7) and H502Y (PCR ST-8) in three (2.94 %) and two (1.96 %) of the 102 C. difficile isolates tested, respectively. Furthermore, PCR ST-6 (H502L), PCR ST-9 (S475S+F 481F+D492D+T501T+A508A+G510G+T539T+K556K +S575A), PCR ST-10 (L487F+H502Y), PCR ST-11 (D492N), PCR ST-12 (D492V), PCR ST-13 (H502N+A555A), PCR ST14 (R505K+I548M), PCR ST-15 (S550F) and PCR ST-16 (S550Y) were found infrequently in the rpoB genes of the C. difficile strains tested. Frequency of occurrence and allocation to respective ribotypes are shown in Table 1. HRM analysis Gene scanning was performed on DNA of 348 C. difficile isolates (spiked with wild-type DNA for the production of artificial heteroduplexes) representing 16 different PCR STs Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 01:51:04 Journal of Medical Microbiology 61 SNP profiling for rifaximin resistance in C. difficile of the rpoB gene (Table 1). After normalization, shifting and difference plotting of the obtained melting curves, 11 different HRM curve profiles (CP) were detected as demonstrated in Fig. 1. Table 2. SNPs detected in the 325 bp rpoB amplicon by HRM analysis SNPs located in the proposed hot-spot region are written in bold. Numbering of nucleotide position starts with the A of the start codon of the rpoB gene of C. difficile (CD630, NC_009089). Amino acid substitution S475S F481F L487F* D492N D492V D492D T501T H502N H502Y H502L* R505K L506L A508A G510G G512G F521F T539T E541E I548M S550F* S550Y A555A K556K S575A*D SNP Located in rpoB amplicon A1425T T1443C A1461C G1474A A1475T T1476C G1503A C1504A C1504T A1505T G1514A T1516C C1524T G1530A A1536G C1563T T1617G G1623A A1644G C1649T C1649A A1665G A1668G T1723G Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No No No The HRM CP 8, characteristic of PCR sequence-type 9 (S475S+F481F+D492D+T501T+A508A+G510G+T539T+ K556K+S575A), was generated by the presence of six out of nine SNPs located within the 151 bp HRM amplicon (Tables 1, 2), which displayed a unique melting curve shape in the difference plot. Furthermore, HRM CP-3 (PCR ST-3), which showed seven synonymous SNPs (resulting in the following amino acid substitutions: T501T+L506L+G510G+ G512G+F521F+E541E+K556K) within the 325 bp rpoB fragment in sequencing analysis, produced a clearly distinguishable melting curve in the difference plot based on the presence of four of the seven SNPs within the shorter HRM amplicon (Table 2). Five rpoB sequence types could not be clearly differentiated by HRM analysis. Sequence-types characterized by a common CP (HRM CP-1) were: the rpoB wild-type PCR ST-1 and the three sequence types PCR ST-5 (A555A), PCR ST-15 (S550F) and PCR ST-16 (S550Y) as demonstrated in Table 1. Furthermore, PCR ST-13 (H502N+A555A) showed the same HRM CP (HRM CP-6) as PCR ST-7 (H502N). PCR ST-14 (R505K+I548M) shared a common HRM CP (HRM CP-2) with PCR ST-2 (R505K). DISCUSSION Rifaximin was reported to be a promising drug used for a follow-up therapy after vancomycin treatment for preventing recurrent CDI (Johnson et al., 2007). Thereby, the rapid detection of point mutations causing reduced rifaximin susceptibility in C. difficile strains is of high importance for the appropriate treatment of CDI and to ensure rapid therapeutic response. Previous studies showed that amino *Mutations leading to amino acid substitutions not previously described for C. difficile. DS575A is not located within the 325 bp rpoB amplicon. 30 Relative signal difference 25 HRM CP-4 (PCR ST-4) H502N + R505K 20 HRM CP-6 (PCR ST-7) H502N HRM CP-9 (PCR ST-10) L487F + H502Y HRM CP-2 (PCR ST-2) R505K HRM CP-5 (PCR ST-6) H502L HRM CP-7 (PCR ST-8) H502Y HRM CP-10 (PCR ST-11) D492N HRM CP-11 (PCR ST-12) D492V HRM CP-3 (PCR ST-3) T501T + L506L + G510G + 15 G512G +F521F + E541E + K556K 10 HRM CP-8 (PCR ST-9) S475S + F481F + D492D + T501T +A508A + G510G + T539T + K556K 5 0 72 73 74 75 76 77 –5 Temperature C http://jmm.sgmjournals.org 80 78 79 HRM CP-1 (PCR ST-1) Wild type 81 82 Fig. 1. Difference plot of 11 HRM curve profile standards and correlated PCR sequencetypes. Amino acid substitutions located in the proposed hot-spot region are written in bold. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 01:51:04 783 V. Pecavar and others Table 3. Frequency of amino acid substitutions in the rpoB gene, previously published and newly described in this study Amino acid substitution Frequency O’Connor et al. (2008) L487F*+H502Y S488P S488T+R505K D492N* D492N+R505K D492V* S498T+R505K H502L* H502N+R505K H502N H502N+A555A H502R H502Y R505K R505K+I548M S550F*D S550Y*D S475S+F481F+ D492D+T501T+ A508A+G510G+ T539TD+K556KD+ S575A*D Number of strains Detectable by HRM analysis Curry et al. (2009) Miller et al. (2011) This study 1 (0.28 %) 1 (0.55 %) 1 (1.25 %) 8 (7.84 %) 1 (0.28 %) 1 (1.25 %) 1 (0.28 %) 6 (7.50 %) 1 1 3 1 (1.25 %) (1.25 %) (3.75 %) (1.25 %) n580 5 (4.90 %) 3 (1.66 %) 8 (7.84 %) 4 (3.92 %) 7 (3.88 %) 2 (1.11 %) 38 (37.25 %) 21 (20.58 %) 6 (3.33 %) n5102 n5180 1 9 3 1 (0.28 %) (2.58 %) (0.86 %) (0.28 %) 2 47 1 1 1 1 (0.57 %) (13.50 %) (0.28 %) (0.28 %) (0.28 %) (0.28 %) Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes n5348 *Non-synonymous mutations not previously described for C. difficile. DMutations located outside of the 325 bp rpoB amplicon. acid substitutions in the rpoB gene, compared with wild-type sequences, are a reliable indicator of reduced rifaximin susceptibility in C. difficile (O’Connor et al., 2008; Curry et al., 2009; Huang et al., 2010; Huhulescu et al., 2011; Miller et al., 2011). Due to the lack of an official MIC breakpoint for rifaximin against C. difficile we suggest that samples showing an MIC .32 mg ml21 are interpreted as reduced in vitro susceptible (Huhulescu et al., 2011). Based on our results and previously described amino acid substitutions, we established an HRM assay using the LightCycler 480 system for the detection of SNPs within a 151 bp amplicon enclosing the proposed SNP hot-spot region of the rpoB gene in C. difficile. Aberrations in HRM CPs of unknown samples, different from that of the wild-type (HRM CP-1), clearly indicate the presence of point mutations within the investigated DNA region, suggesting a reduced rifaximin susceptibility of the isolates. Comparison of these samples with the 11 different HRM CP standards defined for this assay provided an indication of reduced rifaximin susceptibility by the identification of the given sequence types (Fig. 1). During this study, all samples containing SNPs within the investigated DNA region were correctly identified as sequences deviated from wild-type independent from non-synonymous or synonymous SNPs, these mutations can only be ascertained by addition of HRM CP standards. 784 Overall, 16 sequence-types were identified in a 325 bp region of the rpoB gene during this study, 11 of which were also identified by the described HRM assay (Table 1). The HRM assay design was based on the location of the most important point mutations within the rpoB gene (hot-spot region) (O’Connor et al., 2008; Curry et al., 2009; Huhulescu et al., 2011; Miller et al., 2011) and the size limitation of the HRM amplicon length (Liew et al., 2004), leading to the exclusion of seldom found point mutations located outside of the HRM amplicon (Table 1). Thereby, two of the 13 STs, PCR ST-15 and PCR ST-16, were not recognized by the HRM assay of the present study (Table 1). Nevertheless, the nonsynonymous PCR sequence-type PCR ST-14 (R505K+ I548M) and PCR ST-2 (R505K) shared a common HRM CP (HRM CP-2) and could be identified, since mutation I548M was located outside of the HRM amplicon. Interestingly, ST-9 showed reduced rifaximin susceptibility (Huhulescu et al., 2011) despite displaying only synonymous point mutations in the hot-spot region (Tables 1 and 2); this remarkable finding could be explained by a nonsynonymous mutation at position S575A in the flanking region of the investigated rpoB fragment (data not shown). The HRM assay presented here is suitable to detect all of the previously published amino acid substitutions (O’Connor et al., 2008; Curry et al., 2009; Huang et al., 2010; Huhulescu Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sun, 30 Apr 2017 01:51:04 Journal of Medical Microbiology 61 SNP profiling for rifaximin resistance in C. difficile et al., 2011; Miller et al., 2011), including four new substitutions (L487F, H502L, S550F and S575A) found during the course of this study (Table 3). Only the rare mutation types S550F and S550Y at amino acid position 550, which were only identified in one sample each during this study, are located outside of the HRM amplicon. Interestingly, position 502 seems to be a locus of high frequency mutation (H502N, H502R and H502Y). With the amino-acid-substitution H502L we detected an additional SNP at this locus. Our data demonstrate that PCR ribotype 027 (n571) is associated with 11 of 13 detected sequence types (Table 1). The majority of the 71 isolates (n546) belonged to PCR ST2 (R505K), suggesting that PCR ribotype 027 is predominantly associated with this respective sequence-type. Further analysis revealed that 42 of these 46 isolates were associated with a multicentre outbreak in three Viennese hospitals sharing similar MLVA (multiple locus variable-number tandem-repeat analysis) patterns (data not shown) (Indra et al., 2009). These findings underpin the idea that SNP analysis is a valuable tool for additional epidemiological investigations (Mutreja et al., 2011). Further studies will be needed to clarify the correlation between different PCR ribotypes of C. difficile and the acquisition of reduced rifaximin susceptibility. In conclusion, the HRM assay of the present study is a rapid and specific detection method for the most common mutations in the SNP hot-spot region of the rpoB gene in C. difficile and is comparable with sequencing of the respective rpoB section, which may help eliminate the need for MIC resistance testing for non-wild-type samples. ACKNOWLEDGEMENTS Clostridium difficile strains. Anaerobe 16, 633–635. Huhulescu, S., Sagel, U., Fiedler, A., Pecavar, V., Blaschitz, M., Wewalka, G., Allerberger, F. & Indra, A. (2011). Rifaximin disc diffusion test for in vitro susceptibility testing of Clostridium difficile. J Med Microbiol 60, 1206–1212. Indra, A., Schmid, D., Huhulescu, S., Hell, M., Gattringer, R., Hasenberger, P., Fiedler, A., Wewalka, G. & Allerberger, F. (2008). Characterization of clinical Clostridium difficile isolates by PCR ribotyping and detection of toxin genes in Austria, 2006–2007. J Med Microbiol 57, 702–708. Indra, A., Huhulescu, S., Fiedler, A., Kernbicherler, S., Blaschitz, M. & Allerberger, F. (2009). Outbreak of Clostridium difficile 027 infection in Vienna, Austria 2008–2009. Euro Surveill 30, pii19186. Johnson, S., Schriever, C., Galang, M., Kelly, C. P. & Gerding, D. N. (2007). Interruption of recurrent Clostridium difficile-associated diarrhea episodes by serial therapy with vancomycin and rifaximin. Clin Infect Dis 44, 846–848. Kuijper, E. J., Coignard, B., Tüll, P., ESCMID Study Group for Clostridium difficile, EU Member States & European Centre for Disease Prevention and Control (2006). Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect 12 (Suppl. 6), 2–18. Liew, M., Pryor, R., Palais, R., Meadows, C., Erali, M., Lyon, E. & Wittwer, C. (2004). Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons. Clin Chem 50, 1156– 1164. Miller, M. A., Blanchette, R., Spigaglia, P., Barbanti, F. & Mastrantonio, P. (2011). Divergent rifamycin susceptibilities of Clostridium difficile strains in Canada and Italy and predictive accuracy of rifampin Etest for rifamycin resistance. J Clin Microbiol 49, 4319–4321. Mutreja, A., Kim, D. W., Thomson, N. R., Connor, T. R., Lee, J. H., Kariuki, S., Croucher, N. J., Choi, S. Y., Harris, S. R. & other authors (2011). Evidence for several waves of global transmission in the seventh cholera pandemic. Nature 477, 462–465. This work was supported by the ERA-NET project CDIFFGEN. O’Connor, J. R., Galang, M. A., Sambol, S. P., Hecht, D. W., Vedantam, G., Gerding, D. N. & Johnson, S. (2008). 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