Identification of Clostridium innocuum hypothetical protein that is cross-reactive with C. difficile anti-toxin antibodies.

OBJECTIVES: Previously considered solely an opportunistic pathogen, Clostridium innocuum (CI) was recently reported in Taiwan to be an emerging cause of antibiotic-associated diarrhea and clinically indistinguishable from Clostridioides difficile (CD) infection. We previously identified CI culture supernatant being cross-reactive with commercial CD toxin enzyme immunoassays. We aimed to identify and characterize the cross-reacting protein and determine whether it functioned as a human toxin. METHODS: We performed western blots using CI culture supernatants and CD anti-toxin antibodies and identified interacting bands. We identified protein(s) using tandem mass spectrometry and evaluated them by cytotoxicity assays. RESULTS: CI, but not CD, was isolated from stool of 12 children and adults with diarrhea. Culture supernatant from 6/12 CI isolates, and an ATCC reference strain, tested positive for CD toxins (total 7/13 isolates) by commercial EIA. Using two of these isolates, we identified two ∼40 kDa hypothetical proteins, CI_01447 and CI_01448, and confirmed cross-reactivity with CD anti-toxin antibodies by enzyme immunoassay and Western blot. Whole-genome sequencing confirmed all 13 isolates contained both genes, which were highly conserved. We observed no cytopathic or cytotoxic effects to HeLa cells when treated with these proteins. We identified amino acid sequence similarity to the NlpC/P60 family of proteins. CONCLUSIONS: Our findings do not suggest CI proteins CI_01448 and CI_01447, which cross-react with antibodies against CD toxins A and B, are toxic to HeLa cells. Further studies are needed to determine the function of these cross-reacting proteins and the potential virulence factors that could be responsible for CI diarrheal disease.

Staphylococcus aureus spa type t267, clonal ancestor of bovine subclinical mastitis in India.

AIM: To evaluate the virulence determinants and genetic diversity of Staphylococcus aureus from bovine subclinical mastitis milk. METHODS AND RESULTS: PCR detection of virulence genes was performed for 173 Staph. aureus from bovine subclinical mastitis milk. Further, genetic diversity was analysed by agr and spa typing followed by pulsed field gel electrophoresis (PFGE) of selected isolates. Screening of virulence genes (n = 19) showed the adherence genes viz. fnbA, clfA, fnbB and cna in 98·8, 97·1, 68·8 and 28·3 percentage of isolates, respectively, and 80 strains (46·24%) positive for enterotoxin genes were distributed as 23 toxinotypes, of which, 5 genotypes contained a single gene and the rest comprised of multiple toxin genes. Out of agr type-1 (87·3%), 74·2 per cent belonged to the three predominant spa types. Of 27 spa types, 11 were identified for the first time. The predominant spa types were t267 (N =44), t359 (N = 42) and t6877 (N =29), which together accounts to 66·5 per cent of isolates. PFGE analysis of isolates (N = 45) covering all the spa types revealed mostly similar or closely related pulsotypes. Local emergence of spa type t6877 in herd-dependant manner was observed. spa sequence-based phylogenetic analysis suggested t267 as the ancestral clone of t359, t6877 and other spa types except two. CONCLUSION: Heterogenous virulence profile of the isolates had no significant association with the genotype. High prevalence of agr group I reaffirms their association with persistent subclinical mastitis. The spa type t267 appears to be the ancestral clone endemic in the region causing subclinical mastitis. In addition, few new spa types have emerged in the geographic region. SIGNIFICANCE AND IMPACT OF STUDY: Gives an insight into the genetic and evolutionary behaviour of Staph. aureus associated with bovine subclinical mastitis in India. The study would pave the way for devising effective control strategy for bovine mastitis in Indian context.