Bottom: Mean biofilm values (BU) for the populations formed by isolates showing hemolytic activity or absence of hemolysis. MK 8931 supplier Figure 6 Transcriptional levels of sarA determined by using ΔΔC T comparative method. (1) USA400-related isolates 08–008 (agr-dysfunctional) and (2) 96/05 (agr-functional). selleck products (3) BMB9393 was used as a control and (4) RN6390B as calibrator. RQ: Relative quantity. Animal model The naturally agr-dysfunctional MRSA was able to colonize and grow on the surface of implanted catheter fragment, as well as to accumulate an increased amount of biofilm (2-log CFU/mL) when compared with the agr-functional isolate (Figure 7, top). The stability of the agr expression in the agr-dysfunctional
MRSA was examined by observing the hemolytic activity of individual colonies. No hemolytic halo was detected before and after passages in mice (Figure 7, bottom). Figure 7 In vivo biofilm accumulation and stability of agr inhibition.
Top: For the foreign body animal model, data were transformed in percentage considering the CFU/mL of the isolate 08–008 as the reference value (100%). Bottom: The stability of agr inhibition was tested by examining the hemolytic activity of individual colonies of the isolates 08–008 before (left) and after (right) passage in the animal. JAK inhibitor Expression of agr-regulated genes Total RNA obtained from isolates with significant differences (p<0.001) in the RNAIII transcription level (08–008; RQ=0.0001±0.16 and 96/05; RQ=0.53±0.13) was used to analyze the expression of genes that are well known to be regulated by agr. As expected, the agr-up-regulated hla was less expressed (p<0.01) in the isolate 08–008 (Figure 8) when compared with the isolate 96/05 (RQ=0.05±0.01 and RQ=0.33±0.05, respectively). Similar pattern of expression was found for another agr-up-regulated gene, Reverse transcriptase psmα (RQ96/05=75.90±0.10 and RQ08-008=0.005±0.12; p<0.001), except that in this case we also observed a very high expression of psmα for 96/05 (Figure 8). To verify if this amplified expression was a characteristic of this MRSA
clone, other agr-functional isolates were randomly selected for testing. High level of psmα transcripts was also detected for the isolates 07–035, 07–059 and 08–068 (RQ07 035=35.71±0.06; RQ07-059=48.90±0.07; RQ08-068=31.30±0.07). For all virulence genes tested, the expression of the agr-functional isolate BMB9393 was higher than that of USA400-related isolates, except for psmα gene (Figure 8). Accordingly, the RNAIII-down-regulated spa gene showed a very significant lower expression (p<0.001) in the agr-functional 96/05 (RQ=0.8±0.20) compared with the agr-dysfunctional isolate 08–008 (RQ= 52.8±0.17; Figure 8). Figure 8 Transcriptional levels of virulence-associated genes determined by RT-qPCR, using ΔΔC T comparative method. (1) USA400-related isolates 08–008 (agr-dysfunctional) and (2) 96/05 (agr-functional).