Our data showed that cd5, cd6, and cd7 loci did not decrease the congruency with PCR-ribotyping (Table 2; Additional File 2). The result may be due to that the 16S-23S intergenic spacer region, on which the PCR-ribotyping based on, was not as conserved as a housekeeping gene

that is used to construct the phylogenic tree [9, 38]. However, the variations from these incomplete repeat loci should be detected in our follow-up surveillance. PCR ribotyping is a standard technique used worldwide for epidemic clone detection, but the ambiguous Histone Methyltransferase inhibitor & PRMT inhibitor data generated by this technique is difficult for assessing inter-laboratory efficacy. MLVA is a fast and easy-to-use method, and its numerical profile output is more transferable than the standard PCR ribotyping technique. In our laboratory setting, the cost of PCR ribotyping, MLVA10, and TRST per isolate was $0.87, $2.53, and $13.60, respectively, and the cost of the most recent MLST is $24.65 according to Griffiths’ estimation [21]. In the current study, the cost of

MLVA10 was slightly higher than that of PCR ribotyping, but was still significantly less expensive than the TRST and MLST Lazertinib price sequence-based typing techniques. Moreover, when analyzing a large number of isolates, it is simpler to perform one genotyping technique than multiple techniques. Taken together, the MLVA10 is recommended for the detection of C. difficile PCR-ribotype groups and for use in combination with the MLVA panel designed for the detection of outbreak strains. Future studies

will involve evaluation of MLVA10 for c-Met inhibitor its phylogenetic information by comparison to MLST typing. Conclusions For the classification of C. difficile strains, the MLVA technique can result in a distinguishable data set that is more useful for comparison and is highly congruent with PCR-ribotype results. The MLVA10 panel may be used either to detect the PCR-ribotype groups or to overcome the drawbacks of the PCR ribotyping technique. In addition, the MLVA4 can be used to detect closely-related strains. These two MLVA panels can be combined and used for epidemiological studies of C. difficile. Methods Bacterial strains A total of 142 C. difficile strains that were either toxigenic or non-toxigenic Amobarbital were used in this study. Five reference strains (NCTC11204, NCTC13366, NCTC13287, NCTC13404, and NCTC13307) were purchased from the National Collection of Type Cultures (NCTC, London, UK) and three reference strains (BCRC17900, BCRC17702, and BCRC17678) were purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). One strain (NAP1/027) was kindly provided by Dr. Brandi Limbago from the United States Centers for Disease Control and Prevention (CDC), and 133 strains were isolated from clinical laboratory specimens in Taiwan.

Other genes in cluster 9 involved in energy production are ATP synthase subunits (atpABEF, gbs 0875–7 and 9). Interestingly, cluster 9 contains a IWP-2 in vitro transcript of putative catabolite control protein A (ccpA), and the amount grows steadily to increase about three-fold in S phase in comparison with ML (Table 1). CcpA is a major mediator of carbon catabolite repression – the control mechanism of nutrient utilization. In GAS, CcpA has recently been shown to be a critical direct link between carbohydrate utilization and virulence [21]. Function of CcpA in GBS has been

not experimentally confirmed yet. Based on the consensus CcpA binding selleck inhibitor site (cre sequence), we detected that genome of NEM316 strain contains multiple putative cre sites in promoter sequences of multiple genes (Table 2), what might be correlated with changes in expression of genes involved in arginine and carbohydrate metabolism (see below). The STA-9090 transcript encoding HPr carrier protein, another element of the CcpA regulatory pathway in Gram-positive bacteria, also belongs to cluster 9. HPr kinase, however, is an S phase-related gene (see below). Table 1 Fold changes in transcript levels of GBS genes. Gene Fold change in S phase (S/ML ratio) Putative function S phase related genes hrcA, grpE, dnaK (gbs0094–96), +4 to +7.5 Stress

response clpE, and clpL (gbs0535 and gbs1376) +4.5 and +7.5 Chaperones gbs1202/1204, gbs1721, gbs1772 + 30 to +64 Putative stress response proteins from Gls24 and universal stress response families gbs2083–2085 +350 to over +1000 arginine/ornithine antiporter, carbamate kinase, ornithine carbamoyltransferase gbs2122–2126 +55 to +150 arginine deiminase ornithine carbamoyltransferase, arginine/ornithine antiporter carbamate kinase glpKDF (gbs0263–5) +45 to +63 putative operon responsible for glycerol uptake and utilization. Nutrient utilization

and energy metabolism fba gbs0125 +2.2 fructose-bisphosphate aldolase click here plr gbs1811 +3.1 glyceraldehyde 3P-dehydrogenase pgk gbs1809 +2.8 phosphoglycerate kinase eno gbs0608 +2.5 enolase acoAB (gbs 0895–0896) +4 pyruvate dehydrogenase ldh gbs0947 +2.8 L-lactate dehydrogenase Regulators and signal transduction systems gbs 1671/2 -2 TCS CovR/S gbs1908/9 +10/14 TCS, homolog of GAS Spy1106/7 (SF370) gbs1934/5 +5/+5 TCS, homolog of Spy1061/2 (SF370) gbs2081/2 -2.3/-1.7 TCS, putative arginine utilization regulator gbs2086/7 2.5/2.6 TCS, putative arginine utilization regulator gbs1834/5 -7.5/-11.7 TCS gbs1397/8 -7/-5.8 TCS gbs0597/8 -5/-8.5 TCS gbs0121/2 -2/1 TCS gbs0298/9 -3/-1.8 TCS gbs0309/10 -3.3/-3 TCS gbs0429/30 -2.4/-1.6 TCS gbs0963/4 +1.7/+2 TCS gbs1019/20 -1.9/-1.9 TCS gbs1947/8 -3/-2.4 TCS gbs1943/4 -2.1/-2.7 TCS gbs0680 +3.

IGS type I was found selleck products in the nodules of only Omondaw, type II in both Omondaw and Bechuana white, type III in all the genotypes except Omondaw and Bechuana white, type IV in IT82D-889 only, type V in all genotypes except Omondaw, type VI in Glenda, Brown eye and Fahari, type VII in Omondaw, IT82D-889, Bechuana white and

Glenda, type VIII in all the genotypes except Glenda, types IX, X, XI and XII in only Glenda, type XIII in only Fahari and Apagbaala, type XIV in only Apagbaala, types XV, XVI and XVII in only Fahari, and type XVIII in only Apagbaala (Table 4). Nodules from Fahari contained the highest number (8) of IGS types, followed by Apagbaala with 6, IT82D-889 with 5, Omondaw, Bechuana white and Brown eye each with 4, and ITH98-46 and Mamlaka each with 3 IGS types (Table 4). Table 4 Percent Belnacasan manufacturer nodule occupancy by different IGS types

in 9 cowpea genotypes grown in Ghana, Botswana and South Africa   Percent Ipatasertib purchase nodule occupancy per cowpea variety IGS Type Omondaw IT82D-889 Bechuana white Glenda ITH98-46 Brown eye Mamlaka Fahari Apagbaala I 33.3 0 0 0 0 0 0 0 0 II 44.4 0 15.8 0 0 0 0 0 0 III 0 28 0 16 68.2 83.3 15.8 13.3 28.6 IV 0 11 0 0 0 0 0 0 0 V 0 25 57.9 36 26.3 16.7 5.3 6.7 28.6 VI 0 0 0 8 0 0 0 6.7 0 VII 11.1 4 10.5 4 0 0 0 0 0 VIII 11.2 32 15.8 0 5.5 0 78.9 46.6 16.6 IX 0 0 0 16 0 0 0 0 0 X 0 0 0 4 0 0 0 0 0 XI 0 0 0 4 0 0 0 0 0 XII 0 0 0 4 0 0 0 0 0 XIII 0 0 0 0 0 0 0 13.3 16.6 XIV 0 0 0 0 0 0 0 0 4.8 XV 0 0 0 0 0 0 0 6.7 0 XVI 0 0 0 0 0 0 0 6.7 0 XVII 0 0 0 8 0 0 0 0 0 XVIII 0 0 0 0 0 0 0 0 4.8 Values (Mean ± SE)

with dissimilar letters in a column are statistically significant at p ≤ 0.001 (***); p ≤ 0.01 (**) The per-country data for nodule occupancy by each strain (or IGS type) are shown in Table 5. IGS types I, IV, IX, X, XI, XIII, XIV, XVI, XVII and XVIII were only found in the root nodules of cowpea plants SSR128129E grown at Taung, South Africa (but not in those from Ghana and Botswana), while XV and XIX were exclusively found in nodules from Glenvalley in Botswana, and IGS type XII was unique to nodules from Ghana. Table 5 Percent nodule occupancy by different IGS types per country PCR-RFLP IGS type Sample no. of IGS types selected for gene sequencing Percent nodule occupancy per country     South Africa Botswana Ghana I 5 100 0 0 II 8 25 0 75 III 116 71.4 18.6 0 IV 22 100 0 0 V 68 78.6 9.4 12 VI 103 85.7 14.3 0 VII 27 60 0 40 VIII 36 94.2 0 5.8 IX 104 100 0 0 X 115 100 0 0 XI 117 100 0 0 XII 201 0 0 100 XIII 91 100 0 0 XIV 106 100 0 0 XV 7/116 0 100 0 XVI 146 100 0 0 XVII 150 100 0 0 XVIII 153 100 0 0 Strain IGS type diversity from PCR-RFLP analysis When DNA from each nodule was amplified with the two primers, FGPL 132-38 and FGPS 1490-72, a PCR product of about 900 bp was found that corresponded to the size of 16S-23S IGS region.