Band sizes of DNA ranged between 220–3054 base pairs (bp). There were bands that were more densely stained than others, but all bands were treated identically. Four outgroup strains that were in the same family as H. parasuis but Selleck Nirogacestat from different genera were included in the analysis. Fingerprints of DNA were unique for each outgroup isolate and different from

the fingerprint of H. parasuis for each primer (Selleck ISRIB Figure 2A). Figure 1 RAPD analysis of H. parasuis strains using primer 2 (panel A), primer 7 (panel B), and primer 12 (panel C). Reference strains A-O are described in Table 1. Reference strains were obtained Oligomycin A price between 1978 and 1990. Field strains 1–31 are described in Table 2. Field strains 1–24, 25–29, 30–31 were obtained in 2004, 1999, and 1984, respectively. Each lane was loaded with 10 μl of PCR amplification product containing approximately 30 ng of DNA. A DNA control (no cells) was included in lanes marked “No”. The Standard (Std) was a 1 kb DNA ladder. Table 1

Description of H. parasuis reference strains a # Serovar Strain Country Isolation Site Diagnosis Virulenceb A 1 No. 4 Japan Nose Healthy H B 2 SW140 Japan Nose Healthy L+ C 3 SW114 Japan Nose Healthy A D 4 SW124 Japan Nose Healthy L+ E 5 Nagasaki Japan Meninges Meningitis, H           septicemia   F 6 131 Switzerland Nose Healthy A G 7 Y27632 174 Switzerland Nose Healthy A H 8 C5 Sweden

Unknown Unknown L- I 9 D74 Sweden Unknown Unknown A J 10 H367c Germany Unknown Unknown H K 11 H465 Germany Trachea Pneumonia A L 12 H425 Germany Lung Polyserositis H M 13 84-17975 United States Lung Unknown H N 14 84-22113 United States Joint Septicemia H O 15 84-15995 United States Lung Pneumonia L+ aoriginally published by Kielstein and Rapp-Gabrielson (1992) and adapted by Zehr and Tabatabai (2011). bH, Highly virulent, death of pig within 96 h post-inoculation; L+, Polyserositis and arthritis at necropsy; L-, Mild clinical symptoms; A, Avirulent, no clinical symptoms at necropsy as described by Kielstein and Rapp-Gabrielson (1992). cH367 (serovar 10) is a field strain with the same characteristics as the original H555. Reference strain H555 was lost during culture passage prior to our acquisition of the reference strains above. Table 2 Description of H. parasuis field isolates a # Serovar Strain U.S.

95-72.96 79.34-81.11 5.645/0.3 0.53 EF 3 70.8-72.62 78.46-79.71 5.645/0.3 0.62 HIS 3 68.65-69.82 77.42-78.56 5.645/0.3 0.68 Multiplex       0.77 For sequencing, amplicons were treated with ExoSap –IT (GE Health Care, Madrid, Spain) following the manufacturer’s instructions. Sequencing reactions were performed in a GeneAmp PCR system 9700 (Applied Biosystems). Sequences were analyzed

in triplicate. The numerical index of discriminatory power for each marker and for the multiplex analysis was calculated in both genotyping analysis using the Simpson biodiversity index (D) [31]. The percentage of heterozygosis has been calculated by the ratio number of heterozygous genotypes/ total Selleckchem ACY-1215 number of genotypes. Results Antifungal susceptibility testing Antifungal susceptibility results are shown in Table 1. At first, isolates

were susceptible to all antifungal agents tested; however, in August 2006 an isolate showed an azole-resistant phenotype and subsequently isolates susceptible and resistant to azoles appeared at random. Between March 2006 and June 2007 all strains tested were azole-resistant but this pattern changed again between July and November 2007. The latest azole resistant strain recovered was from March 2008. Fluconazole resistance selection Ten colonies of each of the nine isolates genotyped were tested for fluconazole resistance at 8 and 16 mg/l final concentration. From five out of the 9 strains we were able to select resistant and susceptible isolates. SAHA HDAC cell line On the other hand, from one strain all colonies were resistant and from the remaining three strains all checked colonies

were susceptible to fluconazole in a final concentration of 8 mg/l. When fluconazole concentration was increased to 16 mg/l, the number of resistant colonies was reduced PRKACG (Table 2). Genotyping studies Microsatellite length genotyping Microsatellite markers were used to NF-��B inhibitor genotype the nine strains recovered from the patient. Each PCR product was assigned to an allele [14] so each strain was characterized by 6 alleles that were differently coupled (Table 3). Strains from the patient showed the same microsatellite pattern for the three markers and they were different from the control population (Table 3). All the isolates recovered from the patient were homozygous for CDC 3 and HIS 3 markers while they showed a heterozygous genotype for EF 3 (Table 5). Table 5 Characteristics of the microsatellite loci analyzed by capillary electrophoresis Microsatellite Marker No. of alleles No. of genotypes No. of heterozygotic genotypes DP % heterozygosity CDC 3 5 8 4 0.81 50.00 EF 3 10 11 7 0.86 63.63 HIS 3 14 15 11 0.88 53.30 Multiplex       0.92   The D value for EF3 was 0.86, similar to that previously reported [14, 15], for CDC 3 it was 0.81, and for HIS 3 it was 0.87. The combination of three markers yielded a discriminatory power of 0.92 (Table 5).

Table 3 Distribution of the proteins identified by CMAT and 2D-PAGE across phage genomes Gene Other Stx phages carrying the proteins in the study (identity) Accession number Other phages Accession number CM1 Stx2 JPH203 manufacturer converting phage II (99%) YP_003828920.1       phage VT2-Sakai (99%) NP_050557.1       phage 933 W (99%) NP_049519.1       Stx1 converting phage (99%) YP_003848832       phage BP-933 W (99%) YP_003848832.1       phage VT2phi_272 (99%) ADU03741.1       phage Min27(100%) ADU03741     CM2 Stx2 converting phage II (100%) BAC78116       phage VT2-Sakai (100%) NP_050531.1       phage Min27

(100%) YP_001648926       phage HK97 (99%) AAF31137       phage Lahn2 (99%) CAJ26400       phage Lahn3 (98%) CAC95062.1       phage 2851 (99%) CAQ82016       phage CP-1639(99%) selleck compound CAC83142       prophage CP-933 V(99%) AAG57233       Phage Nil2 (99%)(99%) CAC95095       Stx1

converting phage (99%) YP_003848889.1       Phage CP-1639 (99%) CAC83142.1       Phage YYZ-2088 (99%) YP_002274170.1       Stx2-converting phage 1717 (99%) YP_002274244.1     CM5 phage Min27 (100%) YP_001648966.1       Stx2 converting phage II(100%) YP_00388933.1       Stx2 converting phage I(100%) NP_612929.1       phage VT2-Sakai (100%) NP_050570.1       phage 933 W (100%) NP_049532.1       phage VT2phi_272 (100%) ADU03756     CM7 phage VT2-Sakai (99%) NP_050570       Stx1 converting phage (99%) BAC77866.1       Phage VT2phi_272 (97%) ADU03756.1       Phage 933 W (97%) NP_049532.1       Stx2 converting phage I (97%) NP_612929.1       Stx2 converting phage II(97%) BAC78032.1       Phage BP-933 W (97%) AAG55616.1       Stx2 converting phage 86 (91%) YP_794082.1       Phage Min27 Epigenetics inhibitor (97%) YP_001648966.1     CM18 phage VT2-Sakai (100%) NP_050564.1       Stx1 converting phage Resminostat (100%) YP_003848839.1

      Phage 933 W (100%) NP_049526.1       Stx2 converting phage I (100%) ZP_02785836.1       Stx2 converting phage II (100%) YP_003828926.1       Phage BP-933 W (100%) NP_286999.1       Stx2 converting phage 86 (97%) YP_794076.1       Phage Min27 (100%) YP_001648959.1     P1 Stx2 converting phage II (99%) YP_003828937.1 Phage phiV10 (78%) YP_512303.1   Stx2 converting phage I (99%) NP_612952.1       Phage 933 W (99%) NP_049538.1       Phage BP-933 W (99%) AAG55619.1       phage VT2-Sakai (99%) NP_050575.1       Phage Min27 (96%) YP_001648901.1       Stx2-converting phage 86 (96%) YP_794094.1       Phage BP-4795 (96%) YP_001449244.1       phage CP-1639 (74%) CAC83133.1     P2 Stx2 converting phage I (100%) NP_612997.1 Salmonella enteric YP_002455860.1   Phage 933 W (100%) NP_049484.1 bacteriophage SE1 (86%)     Phage BP-933 W (100%) AAG55573.1 Salmonella phage ST160 (86%) YP_004123782.1   Phage Min27 (100%) ABY49878.1       Stx2-converting phage 86 (100%) YP_794109.1     P3 Stx2 converting phage I (100%) NP_612995.1       Phage 933 W (100%) NP_049483.1       Stx2-converting phage 86 (100%) YP_794108.1       Phage Min27 (100%) YP_001648915.

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