S. Huhulescu and others Table 2. Distribution of ribotypes among 898 clinical C. difficile isolates from 2009 RESULTS No. of strains Percentage 22 9.0 8.1 4.0 3.1 2.8 17.8 33.2 Ribotype 053 014/0 027 002/2 001 005 ‘Infrequent’ ribotypes (n510–25)* ‘Rare’ ribotypes (n5,10)D 198 81 73 35 28 26 161 296 *010, 012, 018, 020, 029, 078, 241, 408, 600. D003, 009, 014/5, 015, 017, 019, 023, 025, 026, 031, 043, 045, 046, 049, 054, 056, 066, 070, 080, 081, 087, 126, 153, 176, 203, 205, 206, 207, 208, 209, 211, 212, 220, 232, 236, 237, 239, 250, 403, 404, 405, 411, 413, 415, 419, 425, 429, 430, 431, 432, 434, 438, 439, 440, 441, 442, 448, 449, 451, 453, 457, 470, 472, 477, 481, 483, 484, 486, 492, 495, 496, 498, 499, 500, 501, 502, 503, 504, 505, 507, 508, 510, 512, 514, 515, 516, 518, 519, 520, 523, 525, 526, 530, 531, 532, 535, 537, 542, 548, 549, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 655, 656, 657, 658. diameter breakpoints for rifaximin susceptibility testing by disc (40 mg) diffusion was performed according to the recommendations of Turnidge & Paterson (2007). Statistical analyses were performed with STATA/IC 10.1 (StataCorp). Detection of single-nucleotide polymorphisms (SNPs) within the rpoB gene. DNA was extracted from cultures using the MagNA Pure Compact (Roche Diagnostics) according to the producer’s manual to a final volume of 50 ml. Primers RifFOR (59-CAAGATA- TGGAAGCTATAAC-39) and RifREVlang (59-GTGATTCTATAAAT- CCAAATTC-39) were used in PCRs containing 25 ml HotStar Taq Master Mix (Qiagen), 5 ml (5 pmol ml21) of each primer, 13 ml water and 2 ml DNA. Amplification was performed in a PCR thermocycler (15 min 96 uC, 30 cycles of 1 min 94 uC, 1 min 52 uC and 1 min 72 uC, and finally 10 min 72 uC). PCR products were cleaned up with Escherichia coli exonuclease I, and shrimp alkaline phosphatase (Fermentas) according to the manufacturer’s instructions. Sequencing PCR containing 2 ml Big-Dye-Mix (Applied Biosystems), 1 ml Sequencing Buffer (Applied Biosystems) 4 ml water, 1 ml RifFOR or RifREVlang primer (10 pmol21) and 2 ml DNA was performed in a commercial PCR thermocycler (1 min 96 uC, 30 cycles of 20 s 96 uC, 20 s 50 uC and 4 min 60 uC). The amplified products were cleaned up with Centri Sep 96-well plates or Centri Sep 8 well strips (Applied Biosystems) for dye terminator clean-up according to the manufac- turer’s manual. Samples were analysed in an ABI 3130 genetic analyser (Applied Biosystems) with 36 cm capillary loaded with a POP7 gel (Applied Biosystems). Sequences were analysed for the presence of SNPs within the rpoB gene using Kodon (Applied Maths) version 3.5 by aligning the samples to the rpoB gene sequence of a reference wild-type C. difficile strain (CD630; NC_009089) downloaded from the NCBI database. 1208 Results for reference strains Rifaximin disc (40 mg) diffusion testing of the 184 reference strains yielded inhibition zone diameters ranging from 6 mm to 74 mm. Rifaximin broth microdilution tests of the 184 reference strains yielded rifaximin MICs ranging from 0.001 (n51) to ¢1024 mg ml21 (n561); 62 strains showed reduced susceptibility with an MIC of at least 32 mg ml21. The MIC50 of rifaximin was 0.032 mg ml21 and the MIC90 was ¢1024 mg ml21; the inhibition diameters in compar- ison to the results obtained by broth microdilution test are summarized in Fig. 1. The most common rpoB mutation found within this group was R505K (n546); other mutations were H502N+R505K (n56), H502Y (n52), H502N (n52) and one each of H502L, H502N+A555A, L487F+H502Y, R505K+I548M, D492V and S550F. For 11 arbitrarily chosen strains with rifaximin MICs of .1024 mg ml21, rifaximin end-point MICs were deter- mined by broth dilution: 4096 mg ml21 (n52: strains 2228 and 2639), 8192 mg ml21 (n56: strains 2236, 2285, 2312, 2383, 2816 and 3025), 16 384 mg ml21 (n52: strains 3091 and 910016) and 32 678 mg ml21 (n51: strain 3098). These 11 strains yielded the following mutations in their rpoB genes: R505K (strains 2228, 2236, 2285, 2312, 2383, 2639, 3091 and 910016), L487F+H502Y (strain 2816), H502N+ R505K (strain 3025), R505K+I548M (strain 3098). Rifampicin Etests on these 184 C. difficile strains yielded MICs ranging from ¡0.002 (n5117) to ¢32 mg ml21 (n559); 59 strains had reduced susceptibility (MIC ¢32 mg ml21). The MIC50 of rifampicin was ¡0.002 mg ml21 and the MIC90 was ¢32 mg ml21; all 59 strains showed mutations in the rpoB region (R505K (n546), H502N+R505K (n56), H502Y (n52) and one each of H502L, S550F, H502N+A555A, L487F+H502Y, Fig. 1. Rifaximin disc (40 mg) diffusion test results of 184 C. difficile reference strains compared to MICs obtained by the rifaximin broth microdilution test. All measured ‘sensitive’ C. difficile strains (n5122) are grouped in quadrant A, in contrast to the 62 strains identified as ‘reduced susceptibility’ in quadrant B. Journal of Medical Microbiology 60