Browsing by Author "Henriques, Adriano O."
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- Characterization of Clostridium difficile 027 strains from an outbreak in a Portuguese hospitalPublication . Antunes, Wilson; Serrano, Mónica; Santos, Andrea; Rodrigues, João; Pereira, Fátima; Oleastro, Mónica; Henriques, Adriano O.C. difficile infection (CDI) is the cause of an intestinal disease mediated by two potent cytotoxins, TcdA and TcdB. Symptoms of CDI can range from asymptomatic colonization or mild diarrhea, to life-threatening inflammatory lesions such as pseudomembraneous colitis, toxic megacolon or bowel perforation. In part because of the recent emergence of so-called hypervirulent strains, especially (but not exclusively) those belonging to ribotype 027, C. difficile is now considered a main nosocomial enteric pathogen. Hypervirulent epidemic strains have been associated with more severe disease conditions, with higher relapse rates and increased mortality. Health care-associated CDI develops in hospitalized patients undergoing antibiotic treatment because C. difficile can colonize the gut if the normal intestinal microbiota is disturbed. However, C. difficile is also emerging as an important pathogen in the community, as well as in animal husbandry. The organism is an obligate anaerobe, and has the ability to form spores. Spores are extremely resilient and can accumulate and remain viable in the environment or in the host for long periods of time. Spores that remain latent in the gut are responsible for the recurrence of C. difficile-associated disease (CDAD) when antibiotic therapy is stopped. At least some of the hypervirulent epidemic strains show a greater sporulation capacity in vitro, as well as robust toxin production. The first detection of C. difficile 027 hypervirulent epidemic strains implicated in a hospital outbreak in Portugal dates from January 2012, involving 12 patients, with a crude mortality rate of 50%. Here we report on the genetic characterization of those strains as well as the antibiotic resistance profile, toxin production, and rate and efficiency of spore formation. In parallel, C. difficile 027 non-outbreak strains isolated from other Portuguese health care facilities are also investigated.
- An endoribonuclease of the YicC-like family delays sporulation via sRNA degradation in Clostridioides difficilePublication . Martins, Diogo; Salgueiro, Bruno; Sobral, Daniel; Gragera, Marcos; Hensel, Zach; Henriques, Adriano O.; Romão, Célia V.; Serrano, MónicaClostridioides difficile CD25890 is a YicC-like endoribonuclease involved in regulating sporulation initiation, a process critical for the host-host transmission of this anaerobic pathogen. Using comparative transcriptomics we identified a small RNA, SQ528, that accumulates at higher levels in a CD25890 deletion mutant and we show that purified CD25890 cleaves SQ528 in a metal-dependent manner. Moreover, the overexpression of SQ528 increases sporulation under certain nutritional conditions phenocopying a CD25890 deletion mutant. CD25890 is an hexamer in solution and in vivo. An N-terminal domain, which self-interacts as assessed by size exclusion chromatography and a two hybrid assay, is essential for oligomerization of CD25890. A C-terminal domain harbours residues H230, E254, and E258, conserved among orthologues, important for catalysis. AlphaFold2 modelling and cryo-EM suggest an elongated barrel-like structure with an internal cavity lined with basic residues that may aid in RNA binding. We show that CD25890 forms a complex with polynucleotide phosphorylase which combines the endoribonuclease activity of the first with the exonucleolytic activity of the latter and leads to the complete degradation of SQ528. This study identifies a native substrate for the YicC-family of ribonucleases and advances our understanding of the role of CD25890 in sporulation initiation in C. difficile.
- Genomic Study of a Clostridium difficile Multidrug Resistant Outbreak-Related Clone Reveals Novel Determinants of ResistancePublication . Isidro, Joana; Menezes, Juliana; Serrano, Mónica; Borges, Vítor; Paixão, Pedro; Mimoso, Margarida; Martins, Filomena; Toscano, Cristina; Santos, Andrea; Henriques, Adriano O.; Oleastro, MónicaBackground: Clostridium difficile infection (CDI) is prevalent in healthcare settings. The emergence of hypervirulent and antibiotic resistant strains has led to an increase in CDI incidence and frequent outbreaks. While the main virulence factors are the TcdA and TcdB toxins, antibiotic resistance is thought to play a key role in the infection by and dissemination of C. difficile. Methods: A CDI outbreak involving 12 patients was detected in a tertiary care hospital, in Lisbon, which extended from January to July, with a peak in February, in 2016. The C. difficile isolates, obtained from anaerobic culture of stool samples, were subjected to antimicrobial susceptibility testing with Etest®strips against 11 antibiotics, determination of toxin genes profile, PCR-ribotyping, multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS). Results: Of the 12 CDI cases detected, 11 isolates from 11 patients were characterized. All isolates were tcdA -/tcdB + and belonged to ribotype 017, and showed high level resistance to clindamycin, erythromycin, gentamicin, imipenem, moxifloxacin, rifampicin and tetracycline. The isolates belonged to four genetically related MLVA types, with six isolates forming a clonal cluster. Three outbreak isolates, each from a different MLVA type, were selected for WGS. Bioinformatics analysis showed the presence of several antibiotic resistance determinants, including the Thr82Ile substitution in gyrA, conferring moxifloxacin resistance, the substitutions His502Asn and Arg505Lys in rpoB for rifampicin resistance, the tetM gene, associated with tetracycline resistance, and two genes encoding putative aminoglycoside-modifying enzymes, aadE and aac(6')-aph(2″). Furthermore, a not previously described 61.3 kb putative mobile element was identified, presenting a mosaic structure and containing the genes ermG, mefA/msrD and vat, associated with macrolide, lincosamide and streptogramins resistance. A substitution found in a class B penicillin-binding protein, Cys721Ser, is thought to contribute to imipenem resistance. Conclusion: We describe an epidemic, tcdA -/tcdB +, multidrug resistant clone of C. difficile from ribotype 017 associated with a hospital outbreak, providing further evidence that the lack of TcdA does not impair the infectious potential of these strains. We identified several determinants of antimicrobial resistance, including new ones located in mobile elements, highlighting the importance of horizontal gene transfer in the pathogenicity and epidemiological success of C. difficile.
- Imipenem Resistance in Clostridium difficile Ribotype 017, PortugalPublication . Isidro, Joana; Santos, Andrea; Nunes, Alexandra; Borges, Vítor; Silva, Catarina; Vieira, Luís; Mendes, Aristides L.; Serrano, Mónica; Henriques, Adriano O.; Gomes, João Paulo; Oleastro, MónicaWe describe imipenem-resistant and imipenem-susceptible clinical isolates of Clostridium difficile ribotype 017 in Portugal. All ribotype 017 isolates carried an extra penicillin-binding protein gene, pbp5, and the imipenem-resistant isolates had additional substitutions near the transpeptidase active sites of pbp1 and pbp3. These clones could disseminate and contribute to imipenem resistance.
- Overview of Clostridium difficile Infection: Life Cycle, Epidemiology, Antimicrobial Resistance and TreatmentPublication . Isidro, Joana; Mendes, Aristides L.; Serrano, Mónica; Henriques, Adriano O.; Oleastro, MónicaThe use of antimicrobial agents and acquired resistances explains in part the emergence and spreading of epidemic strains of Clostridium difficile. Continued use of antimicrobial therapy still represents an acute danger in triggering the emergence and spreading of new resistant and multiresistant strains including against first line antibiotics. We examine the pathway of peptidoglycan synthesis in this organism and associated resistances, as well as resistance to other classes of antibiotics. The life-cycle of C. difficile involves growth, spore formation and germination. Spores endow the organism with a formidable capacity of persistence in the environment and in the host, resistance, dissemination and infectious potential. Highly resistant spores produced by antibiotic resistant/multiresistant strains may be one of the most serious challenges we face in what concerns the containment of C. difficile. Finally, we review recent developments in treatment and prevention of C. difficile infection.