Browsing by Author "Menezes, Juliana"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
- 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.
- Molecular epidemiology and antibiotic resistance of Clostridium difficile: focus on toxin A-negative/toxin B-positive strains isolated in PortugalPublication . Menezes, Juliana; Oleastro, Mónica; Tenreiro, AnaClostridium difficile affects patients in hospitals and communities worldwide, is responsible for significant annual mortalities and represents a considerable economic burden on healthcare systems. This bacterium might also be carried asymptomatically in the gut, potentially leading to ‘silent’ onward transmission. Treatment has always been difficult, because the disease is both caused and resolved by antibiotic intake. The two main C. difficile virulence factors are toxins A and B, both of which are pro-inflammatory and enterotoxic in human intestine. Clinically relevant toxin A-negative/toxin B-positive strains that cause diarrhoea and colitis in humans have been isolated with increasing frequency worldwide, namely the multidrug resistant PCR-ribotype (RT) 017. Previous studies documented changes in C. difficile infection (CDI) epidemiology associated with the rapid emergence of antibiotic-resistant strains, highlighting the importance of antimicrobial susceptibility surveillance. The present work describes epidemiological and antimicrobial susceptibility data of C. difficile strains isolated in Portugal. A total of 378 C. difficile strains from 11 Portuguese hospital centres were characterized regarding toxin profile and RT, and part of these strains were also evaluated for its susceptibility to moxifloxacin, vancomycin, metronidazole, rifampicin and imipenem and determinants of antimicrobial resistance. Multilocus variable tandem repeat analysis (MLVA) and whole genome sequencing (WGS) analysis was also performed in a subgroup of epidemic, multidrug isolates. Most of the isolates were toxigenic (91.3%), of which 94.2% had both toxins A and B and 25.8% of them also had a binary toxin. Seventy-five different RTs were identified. RT027, RT014, RT106 and RT017 were the most frequently isolated. There was no evidence of resistance to vancomycin among the 183 tested strains, and reduced susceptibility to metronidazole was rare (2.2%). Resistance to moxifloxacin was evident in multiple RTs, and were mainly from RTs positive for the three toxins, RT027 (18/18), RT126 (8/9) and RT078 (6/12), except the RT017 (19/19), which is toxin A-negative/toxin B-positive. All moxifloxacin-resistant strains exhibited a known mutation in GyrA (Thr82Ile). Rifampicin resistance was found in 11.5% of the 183 strains tested, most from RT017 (19/19) but also in one strain from RT241 and other from RT043. Most rifampicin-resistant strains harbour the previously described mutations in RpoB (His502Asn and Arg505Lys), although one mutation, Ser507Leu, found alone in a resistant strain was not previously described. Of the 181 strains belonging to 57 RTs tested for imipenem susceptibility, only strains from RT017 showing high level of resistance to this antibiotic (MIC > 32 mg/L). The resistance determinants, ermB and tetM genes were present in 34 (10.1%) and in 63 (20.12%) strains, respectively, being that 22 (6.5%) contained both genes. Twenty strains were toxin A-negative/toxin B-positive, 19 of them belonging to the well-known emerging RT017, 11 from hospital A isolated in a short period of time suggesting that an outbreak have occurred, the remaining eight from hospital B, isolated between 2016 and 2017, where this RT seems to be endemic. Overall, these strains were multiresistant, presenting resistance to six of the 10 antibiotics tested: moxifloxacin, rifampicin, imipenem, tetracycline, clindamycin and erythromycin (these two belonging to the MLSB group), with high level of resistance. PCR screening of the resistance determinants showed that all strains harboured the tetM gene, but only the eight strains from hospital B were positive for ermB. Analysis by WGS revealed the presence of the ermG gene in the ermB-negative/MLSB-resistant strains. This gene was found to be in a putative mobile element of 63 kb exclusive of the hospital A clonal cluster. Transformation of a susceptible strain (C. difficile 630Δerm), with a plasmid containing the ermG gene, proved that the presence of this gene provides high resistance to clindamycin and erythromycin in C. difficile. Mutations in penicillin-binding proteins were also observed in all imipenem resistant strains. Phylogenetic analysis of single nucleotide polymorphisms (SNPs) of RT017 isolates collected from 2012 to 2017 revealed three clusters, each from a single hospital. Subtyping MLVA was also applied to detect the clonal spread of C. difficile belonging to toxin A-negative/toxin B-positive, and the results were overall similar with the WGS analysis. In addition, 74 SNPs variations were found among RT017 strains, namely in proteins involved in antimicrobial resistance and in hypothetical proteins. The current work gives a contribution to the knowledge of the molecular epidemiology and resistance patterns of C. difficile in Portugal. The results presented herein alert to the presence of multidrug resistant strains of RT017 in Portuguese hospitals in endemic and outbreak situations, and indicates the need for adequate use of antimicrobial agents, especially carbapenems, whose resistance was only observed among the strains of this emerging RTs. The lineage of strains from RT017 appears to be constantly evolving, acquiring new resistance determinants, which highlights the need for continued epidemiological and antimicrobial surveillance. The wide variety of RTs found suggests that there are other routes of transmission beyond nosocomial transmission, raising concern about the epidemiological change in this pathogen. As such, other potential sources, particularly in animals, which may also act as a reservoir for C. difficile and antimicrobial resistance determinants, should be investigated in the future. Finally, this study provides the basis for investigating important factors for the spread and persistence of toxin A-negative/toxin B-positive strains, such as studies on the importance of proteins that distinguish these strains, namely the hypothetical proteins.