Characterization of the virulence of three novel clade 2 Clostridioides (Clostridium) difficile strains and a two-year screening in animals and humans in Brazil

Abstract

Clostridioides (Clostridium) difficile infection (CDI) is an evolving global healthcare problem, and owing to the diverse and dynamic molecular epidemiology of C. difficile, new strains continue to emerge. In Brazil, only two cases of CDI due to the so called hypervirulent PCR ribotype (RT) 027 belonging to clade 2 have ever been reported, whereas incidence of CDI due to another “hypervirulent” RT078 (clade 5) has not yet been reported. In contrast, novel clade 2 strains have been identified in different hospitals. To better understand the epidemiology of CDIs in Brazil, this study aimed to genotypically and phenotypically characterize three novel Brazilian clade 2 strains (RT883, 884, and 885) isolated from patients with confirmed CDI. In addition, to better understand the circulating RTs, a two-year sampling was conducted in patients from the same hospital and in several domestic and wild animal species. The three strains examined showed lower production of A/B toxins than the control RT027, although two of these strains harbored a truncated tcdC gene. All strains showed swimming motility similar to that of RT027, while RT883 showed higher spore production than the reference strain. In the in vivo hamster model, the lethality of all strains was found to be similar to that of RT027. Both cgMLST and cgMLSA analyses revealed a high genetic similarity among the three-novel clade 2 isolates. In the two-year survey in animals and humans, RT883, 884, and 885 were not detected; however, three new RTs (RT988, RT989, and RT990) were isolated, two of which were genetically related to the three previously reported clade 2 strains. RT106 and RT126 were most frequently detected in humans (47.9%) and animals (57.9%), respectively. Furthermore, RT027 and RT078 were not detected in humans. The results of this study suggest that these novel clade 2 strains have virulence potential and that new strains from clade 2 continue to emerge in our setting, indicating the need for long-term local surveillance.

Use of tcdC gene sequencing to prevent misidentification of Clostridioides difficile ribotype 176 and 027

“…As a result, the interaction between active site of lysozyme and acetyl groups of peptidoglycane is not possible (17). However, a combination of lysozyme (10 mg/ml) and proteinase K (20 mg/ml) to isolate DNA from C. difficile was used in other studies: in the first step, cells are incubated with lysozyme solution at 37 °C overnight, then proteinase K is added and incubated at 56 °C for 30 minutes (18). These findings do not correlate with information about lysozyme resistance of C. difficile, but prolonging the lysis process during the DNA extraction was sufficient in some samples also in our process of DNA extraction and it could be the way how to improve the yield of the DNA extraction.…”

Clostridioides difficile, a New “Superbug”

“…Evidence that pets can play an important role in C. difficile dissemination is the fact that in companion animals, the researchers detected similar RTs as in humans. The isolates of ribotype 106, RT078 and 014/020 have been isolated from humans and also from cats and dogs [73,80,87,88]. A bigger challenge may be the fact that some of the isolated strains have shown resistance to metronidazole [78,89,90], which could pose a serious threat to human health.…”

Prevalence of Clostridioides difficile in dogs (Canis familiaris) with gastrointestinal disorders in Rio de Janeiro

“…Strains susceptible to the antibiotics according to the following measurements: VAN ≥19 mm; MTZ ≥23 mm; MXF ≥20 mm; ERY ≥20 mm; RIF ≥30 mm. The C. difficile R20291 strain (027 ribotype) was used as a control [41,42].…”