Truncation in the tcdC region of the Clostridium difficile PathLoc of clinical isolates does not predict increased biological activity of Toxin B or Toxin A

Abstract

Background: The increased severity of disease associated with the NAP1 strain of Clostridium difficile has been attributed to mutations to the tcdC gene which codes for a negative regulator of toxin production. To assess the role of hyper-production of Toxins A and B in clinical isolates of Clostridium difficile, two NAP1-related and five NAP1 non-related strains were compared.

Methods: Sequencing was performed on tcdC, tcdR, and tcdE to determine if there were differences that might account for hyper-production of Toxin A and Toxin B in NAP1-related strains. Biological activity of Toxin B was evaluated using the HFF cell CPE assay and Toxin A biological activity was assessed using the Caco-2 Trans-membrane resistance assay.

Results: Our results confirm that Toxin A and Toxin B production in NAP1-related strains and ATCC 43255 occurs earlier in the exponential growth phase compared to most NAP1-nonrelated clinical isolates. Despite the hyper-production observed in ATCC 43255 it had no mutations in tcdC, tcdR or tcdE. Analysis of the other clinical isolates indicated that the kinetics and ultimate final concentration of Toxin A and B did not correlate with the presence or lack of alterations in tcdC, tcdR or tcdE.

Conclusion: Our data do not support a direct role for alterations in the tcdC gene as a predictor of hyperproduction of Toxin A and B in NAP1-related strains.

Figure 1

Characteristics of Clostridium difficile strains. All strains had tcdA and tcdB as the appropriate size of A3 and B1 amplicon was detected.[27] To assess NAP1 relatedness, PFGE analysis was performed using Sma1 as outlined by[32].

Figure 2

Toxin B production over time for various strains of C. difficile. The viable count (line) and Toxin B production (black bars) were determined over 60 hours for isolates grown in BHI broth. The strains evaluated included; ATCC 43255 (A), ATCC 43594 (B), NAP1 clinical strain 57A (C), NAP1 clinical strain 83 (D), historical clinical strains 79A292 (E), 81A330 (F) and 1083 (G).

Figure 3

Toxin A activity of C. difficile strains grown in BHI broth culture. The Toxin A activity for 48 hour culture supernatants was determined using the trans-membrane resistance assay. The positive control consisted of purified Toxin A (460 ng/ml) and the negative control was BHI broth. Strain 700057 is a strain that in a non-toxin producing strain.

Figure 4

Sporulation activity of C. difficile strains grown in BHI broth culture. Spore production by strains of C. difficile grown in BHI broth for 24 (A), 48 (B) and 72 hours (C). The total viable count of C. difficile (□) was determined as well as an assessment using alcohol shock (as described in the Materials and Methods) to determine what portion was in the spore form (■). Each bar represents the average of triplicate testing. The 24 hour levels for strain 43255 were tested in triplicate on two separate occasions to verify that spore levels were consistently < 1 Log₁₀.

Figure 5

Amino acid sequence alignment from tcdC gene of various strains of C. difficile. Amino acids identical to the ATCC 43225 sequence (shown) are indicated by periods, differences are shown, and the region of the six amino acid deletions (dashes) in strains 79A292 and 81A330 are boxed. The transmembrane helical regions (TMH) are indicated by a line.