IHMT: MM - Documentos de conferências internacionais
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- Development of multidrug resistance in staphylococci driven by effluxPublication . Costa, SS; Rosato, Adriana E.; Viveiros, M; Couto, I; Instituto de Higiene e Medicina Tropical (IHMT); Global Health and Tropical Medicine (GHTM); TB, HIV and opportunistic diseases and pathogens (THOP)Aim: To study the efflux driven response of two major staphylococcal pathogens, S. aureus and S. epi-dermidis to the challenge by non-antibiotic drugs. Methods: We adapted three reference strains to ethidium bromide (EtBr), a broad substrate of bacte-rial efflux pumps. The parental strains, S. aureus ATCC25923, S. epidermidis ATCC12228 and S. epider-midis RP62A were cultured in varying concentrations of EtBr, to obtain their EtBr-adapted derivatives; ATCC25923_EtBr; ATCC12228_EtBr and RP62A_EtBr. Susceptibility of parental and adapted strains to 10 antibiotics and 6 biocides was evaluated by microdilution MIC determination with or without efflux inhibitors. Efflux activity was established by fluorometric assays and the relative expression of the genes coding for the main efflux pumps (EPs) of each species quantified by RT-PCR.Results: For each strain tested, exposure to EtBr resulted in the development of a multidrug resistance (MDR) phenotype, which included resistance to fluoroquinolones and decreased susceptibility to bio-cides, including cetrimide, benzalkonium chloride and tetraphenylphosphonium bromide. Efflux inhib-itors such as verapamil reduced these resistance levels. The EtBr-adapted cultures showed increased efflux activity, which was accompanied by over-expression of distinct EP genes, in a temporal pattern. Conclusion: These results show that both S. aureus and S. epidermidis have the potential to develop efflux driven MDR phenotypes when exposed to a non-antibiotic substrate of multidrug EPs, which can be mediated by distinct efflux pumps, depending on the drug and the bacterial genetic background.
- Biocide susceptibility in Staphylococcus epidermidis causing infection in petsPublication . Costa, SS; Rosa, Mafalda; Belas, Adriana; Viveiros, M; Pomba, Constança; Couto, I; Instituto de Higiene e Medicina Tropical (IHMT); Global Health and Tropical Medicine (GHTM); TB, HIV and opportunistic diseases and pathogens (THOP)Aim: To characterize biocide susceptibility of Staphylococcus epidermidis isolates causing infection in pets and evaluate proposed biocide epidemiological cut-off (tentative ECOFF) values. Methods: The study comprised a collection of 17 S. epidermidis isolates collected from several infection sources in cats and dogs from 2001 to 2016. Susceptibility to benzalkonium chloride (BAC), cetrim-ide, chlorhexidine digluconate, tetraphenylphosphonium bromide (TPP), triclosan (TCL) and ethidium bromide (EtBr) was evaluated by microdilution MIC determination. The efflux pump genes qacA/B, qacG, qacJ and smr, associated with biocide resistance, and the triclosan resistance gene sh-fabI were screened by PCR.Results: The MIC distributions for all biocides and EtBr were analyzed against the tentative ECOFF val-ues proposed for these compounds (see other poster by Costa et al, ISSSI 2018). Non-wild-type (NWT) populations were detected towards BAC (6/17), TPP (8/17) and TCL (4/17) and EtBr (9/17). The NWT populations towards BAC, TPP and EtBr were associated with the presence of either qacA or smr plas-mid-encoded genes, whereas the TCL NWT population was linked to sh-fabI carriage. One isolate carry-ing the smr gene was only detected taking into consideration the proposed EtBr ECOFF.Conclusion: This study illustrates the frequently reduced susceptibility towards relevant biocides of S. epidermidis infecting pets. It also supports the tentative ECOFF values suggested for BAC, TPP and TCL, as criteria to detect S. epidermidis harboring biocide resistance genes. The establishment of such crite-ria would enable early detection of S. epidermidis isolates with a non-wild-type phenotype and improve therapeutics in veterinary medicine.
- Emergence of non-wild-type populations towards biocides in Staphylococcus epidermidis colonizing veterinary staffPublication . Costa, SS; Rosa, Mafalda; Holtreman, Frederico; Rodrigues, Ana Catarina; Viveiros, M; Pomba, Constança; Couto, I; Instituto de Higiene e Medicina Tropical (IHMT); Global Health and Tropical Medicine (GHTM); TB, HIV and opportunistic diseases and pathogens (THOP)Aims: To characterize biocide susceptibility of Staphylococcus epidermidis colonizing veterinary staff; propose epidemiological cut-off (ECOFF) values; assess relationships between biocide and antibiotic resistance phenotypes.Methods: The susceptibility profiles of 112 S. epidermidis isolates from nasal colonization of veter-inary staff towards six biocides (including benzalkonium chloride, chlorhexidine and triclosan) and ethidium bromide (EtBr), was determined by microdilution MIC determination. The MIC distribu-tions were analyzed by the normalized resistance interpretation method and the iterative statistical method to determine ECOFF values. Antibiotic susceptibility was determined by disc diffusion and antimicrobial resistance determinants screened by PCR.Results: Analysis ofMIC distributions enabled the detection of non-wild-type (NWT) populations towards most biocides and to the efflux marker EtBr and the proposal of tentative ECOFF values for the compounds tested. The NWT populations were correlated with carriage of the plasmid-en-coded efflux pump genes qacA/B and/or smr. NWT populations to triclosan were correlated with sh-fabI gene. We observed high frequencies of methicillin resistance(mecA+, 61 %) as well as of multidrug resistance phenotypes (50 %). Several determinants were associated with resistance phenotypes, including blaZ, erm, vga and fus genes. Carriage of qacA/B and/or smr was statistically associated with MDR phenotypes. Conclusions: This study discloses the first tentative ECOFF values of several biocides for S. epider-midis. It also reveals a high prevalence of biocide and antibiotic resistant S. epidermidis colonizing humans in close contact with animals, suggesting that these bacteria can be reservoirs for antimi-crobial resistance.
- Frequent biofilm production by Staphylococcus epidermidis causing infection in pets or colonizing veterinary staffPublication . Costa, SS; Santos, Cláudia Mineiro; Viveiros, M; Pomba, Constança; Instituto de Higiene e Medicina Tropical (IHMT); Global Health and Tropical Medicine (GHTM); TB, HIV and opportunistic diseases and pathogens (THOP)Aim: To characterize biofilm production by Staphylococcus epidermidis isolates causing infection in pets or colonizing veterinary staff. Methods: The study collection comprised 129 S. epidermidis isolates, from nasal colonization of vet-erinary staff (n = 112) and causing infection in cats and dogs (n = 17). The capacity to produce biofilm was evaluated by the Crystal Violet (CV) method, performed in flat-bottom 96-well polystyrene plates and biofilm production was categorized (as strong, moderate or weak) according to specific ranges of 570 nm optical density. The biofilm producer S. epidermidis RP62A was used as control in each assay. Biofilm-associated genes icaAD and aap were screened by PCR.Results: Amongst thehumancommensal isolates, the CV method enabled the identification of 66/112 (58.9 %) biofilm producers, of which 34/66 (51.5 %) were strong or moderate producers. In the group of pet infection isolates, 16/17 isolates (94.1 %) produced biofilm, of which 12/16 (75.0%) were strong or moderate producers. The majority of the isolates carried icaAD and/or aap genes. The genotype icaAD-aap- was associated with non-biofilm production whereas genotype icaAD+aap+ was linked to strong biofilm production. No correlation was found between any biofilm producing phenotype and the genotypes icaAD+aap-icaAD-aap+.Conclusion: These results evidence a high frequency of biofilm production by S. epidermidis either causing infection in pets or colonizing humans in close contact with them. The findings also highlight the importance of biofilm-associated genes other than ica in the biofilm phenotype.
- Complete sequences of two Staphylococcus aureus plasmids carrying genes for resistance to antibiotics, heavy-metals, biocides and/or virulencePublication . Costa, SS; Kadlec, Kristina; Viveiros, M; Melo-Cristino, Josè; Schwarz, Stefan; Couto, I; Instituto de Higiene e Medicina Tropical (IHMT); Global Health and Tropical Medicine (GHTM); TB, HIV and opportunistic diseases and pathogens (THOP)Aim: To gain detailed insight into the genetic organization of two multidrug resistance plasmids, pSM31 and pSM39, from clinical S. aureus isolates collected in a hospital in Lisbon, Portugal, and to identify factors that may explain their selection and persistence. Methods: Plasmid pSM31 was sequenced by Sanger sequencing using cloning and primer walking whereas pSM39 was sequenced by next generation sequencing. Sequence analysis was carried out using the BlastN and BlastP programs and the ORF Finder software from NCBI.Results: The two plasmids fully characterized in this work were non-conjugative multiresistance plas-mids. pSM31 (27,424 bp) is closely related to the family of globally dispersed plasmids from the pGSA23 group harbouring determinants for resistance to β-lactams and cadmium as well several enterotoxin genes. pSM39 (26,036 bp) is a newly described member of the pGSA11 group that carries genes con-ferring resistance to β-lactams, biocides, cadmium, zinc and mercury and comprises a region that may have been acquired by recombination with a S. epidermidis plasmid. Conclusions: The sequence analysis of these two plasmids confirmed their involvement in antimicrobial resistance among S. aureus strains isolated from a clinical setting. It also provided genetic evidence for the potential risk of co-selection of antibiotic resistance genes and/or virulence genes by selective pressure of antibiotics, biocides and/or heavy metals.
- Characterization of reduced susceptibility to biocides, dyes, and quinolones in a Staphylococcus aureus strain exposed to ethidium bromidePublication . Costa, SS; Couto, I; Viveiros, M; Martins, Marta; Leandro, Clara; Amaral, Leonard; Instituto de Higiene e Medicina Tropical (IHMT); CREM - Centro de Recursos Microbiológicos; Unidade de Parasitologia e Microbiologia Médicas (UPMM)