Percorrer por autor "Igrejas, Gilberto"
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- Antimicrobial Resistance and Molecular Epidemiology of Staphylococcus aureus from Hunters and Hunting DogsPublication . Silva, Vanessa; Caniça, Manuela; Manageiro, Vera; Vieira-Pinto, Madalena; Pereira, José Eduardo; Maltez, Luís; Poeta, Patrícia; Igrejas, GilbertoSeveral studies have showed that a dog-to-human transmission of Staphylococcus aureus occurs. Hunting dogs do not have as much contact with their owners as dogs that live in the same household as the owners; however, these dogs have contact with their owners during hunting activities as well as when hunting game; therefore, we aimed to isolate S. aureus from hunters and their hunting dogs to investigate a possible S. aureus transmission. Nose and mouth samples were collected from 30 hunters and their 78 hunting dogs for staphylococcal isolation. The species identification was performed using MALDI-TOF. The antimicrobial susceptibility profiles were accessed using the Kirby-Bauer method and respective antimicrobial resistance genes were investigated by PCR. Multilocus sequence typing (MLST) and spa- and agr-typing was performed in all S. aureus isolates. S. aureus were detected in 10 (30%) human samples and in 11 (15.4%) dog samples of which 11 and 5 were methicillin-resistant S. aureus (MRSA). Other staphylococci were identified, particularly, S. pseudintermedius. Most S. aureus isolates were resistant to penicillin, erythromycin, and tetracycline. Evidence of a possible transmission of S. aureus between human and dogs was detected in three hunters and their dogs. S. aureus isolates were ascribed to 10 STs and 9 spa-types. A moderate colonization of S. aureus in hunting dogs and their owners was detected in this study. A few dog-to-dog and dog-to-human possible transmissions were identified.
- Antimicrobial resistance determinants in Staphylococcus spp. recovered from birds of prey in PortugalPublication . Sousa, Margarida; Silva, Nuno; Igrejas, Gilberto; Silva, Filipe; Sargo, Roberto; Alegria, Nuno; Benito, Daniel; Gómez, Paula; Lozano, Carmen; Gómez-Sanz, Elena; Torres, Carmen; Caniça, Manuela; Poeta, PatríciaAntibiotic resistance among wild animals represent an emerging public health concern. The objective of this study was to analyze the staphylococcal nasal microbiota in birds of prey and their content in antimicrobial resistance determinants. Nasal samples from 16 birds of prey were collected, swabs were dipped and incubated into BHI broth [6.5% NaCl] and later seeded on manitol salt agar and oxacillin-resistance screening agar base media. Staphylococcal colonies were isolated from both media and were identified by biochemical and molecular methods. Susceptibility testing to 18 antimicrobial agents was performed by disk-diffusion method. Six of the 16 tested animals carried staphylococci (37.5%) and 7 isolates of the following species were recovered: Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus sciuri rodentium, Staphylococcus cohnii urealitycum, and Staphylococcus gallinarum. The S. aureus isolate was penicillin-resistant (with blaZ gene) but methicillin-susceptible and was ascribed to spa-type t012, sequence-type ST30 and agr-type III. The S. epidermidis isolate carried blaZ, mecA, mrs(A/B), mphC, tet(K), drfA, and fusC genes, ica operon, and was typed as ST35. The genes ant6′-Ia, tet(K), tet(L), dfrG, cat221, cat194, and cat223 were detected in S. saprophyticus or S. gallinarum isolates. Birds of prey seem to be a natural reservoir of S. aureus and coagulase-negative staphylococci resistant to multiple antibiotics. Due to the convergence between habitats, the contact between wildlife, other animals and humans is now more common and this involves an increased possibility of interchange of these microorganisms in the different ecosystems.
- Assessment of Antibiotic Resistance Among Isolates of Klebsiella spp. and Raoultella spp. in Wildlife and Their Environment from Portugal: A Positive Epidemiologic OutcomePublication . Sabença, Carolina; de la Rivière, Rani; Barros, Paulo; Cabral, João Alexandre; Sargo, Roberto; Sousa, Luís; Dapkevicius, Maria de Lurdes Enes; Silva, Filipe; Lopes, Filipa; Abrantes, Ana Carolina; Vieira-Pinto, Madalena; Caniça, Manuela; Igrejas, Gilberto; Torres, Carmen; Poeta, PatríciaOne of the significant challenges facing modern medicine is the rising rate of antibiotic resistance, which impacts public health, animal health, and environmental preservation. Evaluating antibiotic resistance in wildlife and their environments is crucial, as it offers essential insights into the dynamics of resistance patterns and promotes strategies for monitoring, prevention, and intervention. and genera isolates were recovered from fecal samples of wild animals and environmental samples using media without antibiotic supplementation. Antibiograms were performed for 15 antibiotics to determine the phenotypic resistance profile in these isolates. Extended-spectrum β-lactamase (ESBL) production was tested by the double-disc synergy test, and one ESBL-producing isolate was screened by PCR and whole-genome sequencing. Biofilm production was analyzed using the microtiter plate method. A total of 23 spp. and 3 spp. isolates were obtained from 312 fecal samples from wild animals, 9 spp. and 4 spp. isolates were obtained from 18 river and stream water samples, and 4 spp. and 3 spp. isolates from 48 soil samples. Regarding antibiotic resistance, only one isolate of from soil samples was an ESBL-producer and showed resistance to six antibiotics. This isolate harbored multiple β-lactams genes (, , , and ), as well as genes of resistance to quinolones, sulfonamides, tetracycline, aminoglycosides, and chloramphenicol, and belonged to the lineage ST307. Most of the spp. and spp. isolates were biofilm producers (except for one isolate), and 45.6% were weak biofilm producers, with the remaining being moderate to strong biofilm producers. We can conclude that antibiotic resistance is not widespread in these environment-associated isolates, which is a positive epidemiological outcome. However, identifying a single ESBL- isolate should serve as a warning of potential hotspots of resistance emergence.
- Bats as Hosts of Antimicrobial-Resistant Mammaliicoccus lentus and Staphylococcus epidermidis with Zoonotic RelevancePublication . Silva, Vanessa; Caniça, Manuela; de la Rivière, Rani; Barros, Paulo; Cabral, João Alexandre; Poeta, Patrícia; Igrejas, GilbertoBats are increasingly recognized as reservoirs for antimicrobial-resistant bacteria, playing a potential role in the dissemination of resistance genes across species and regions. In this study, 105 bats from 19 species in Portugal were sampled to investigate the presence, antimicrobial resistance, and genetic characteristics of Mammaliicoccus and Staphylococcus isolates. Thirteen Mammaliicoccus lentus and Staphylococcus epidermidis were recovered. Antimicrobial susceptibility testing revealed multidrug resistance in three isolates, with S. epidermidis carrying mph(C), msr(A), and dfrC genes, and M. lentus harboring salB, tet(K), and str. Notably, qacA was detected in S. epidermidis, highlighting its plasmid-associated potential for horizontal gene transfer to more pathogenic bacteria. Heavy metal resistance genes (arsB and cadD) were also identified, suggesting the role of environmental factors in co-selecting antimicrobial resistance. Molecular typing revealed the S. epidermidis strain as ST297, a clone associated with both healthy humans and invasive infections. These findings emphasize the need for monitoring bats as reservoirs of resistance determinants, particularly in the context of zoonotic and environmental health. The presence of mobile genetic elements and plasmids further underscores the potential for the dissemination of resistance. This study reinforces the importance of adopting a One Health approach to mitigate the risks associated with antimicrobial resistance.
- Bioinformatics study of expression from genomes of epidemiologically related MRSA CC398 isolates from human and wild animal samplesPublication . Ribeiro, Miguel; Sousa, Margarida; Borges, Vítor; Gomes, João Paulo; Duarte, Sílvia; Isidro, Joana; Vieira, Luís; Torres, Carmen; Santos, Hugo; Capelo, José Luís; Poeta, Patrícia; Igrejas, GilbertoOne of the most important livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) genetic lineages is the clonal complex (CC) 398, which can cause typical S. aureus-associated infections in people. In this work, whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics were applied to study the genetic characteristics of three MRSA CC398 isolates recovered from humans (strains C5621 and C9017), and from an animal (strain OR418). Of the three strains, C9017 presented the broadest resistance genotype, including resistance to fluroquinolone, clindamycin, tiamulin, macrolide and aminoglycoside antimicrobial classes. The scn, sak, and chp genes of the immune evasion cluster system were solely detected in OR418. Pangenome analysis showed a total of 288 strain-specific genes, most of which are hypothetical or phage-related proteins. OR418 had the most pronounced genetic differences. RNAIII (δ-hemolysin) gene was clearly the most expressed gene in OR418 and C5621, but it was not detected in C9017. Significant differences in the proteome profiles were found between strains. For example, the immunoglobulin-binding protein Sbi was more abundant in OR418. Considering that Sbi is a multifunctional immune evasion factor in S. aureus, the results point to OR418 strain having high zoonotic potential. Overall, multiomics biomarker signatures can assume an important role to advance precision medicine in the years to come. SIGNIFICANCE: MRSA is one of the most representative drug-resistant pathogens and its dissemination is increasing due to MRSA capability of establishing new reservoirs. LA-MRSA is considered an emerging problem worldwide and CC398 is one of the most important genetic lineages. In this study, three MRSA CC398 isolates recovered from humans and from a wild animal were analyzed through whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics in order to gather systems-wide omics data and better understand the genetic characteristics of this lineage to identify distinctive markers and genomic features of relevance to public health.
- BlaGES-6 producing Pseudomonas aeruginosa ST235 is involved in resistance to different β-lactamsPublication . de Sousa, Telma; Machado, Sandro; Carvalho, Márcia; Caniça, Manuela; Ramos, Miguel J.N.; Santos, Daniela; Beyrouthy, Racha; Bonnet, Richard; Hébraud, Michel; Gomes, João Paulo; Igrejas, Gilberto; Poeta, PatríciaMultidrug resistance in Pseudomonas aeruginosa, particularly resistance to carbapenem, represents a major challenge for public health. This study investigated resistance mechanisms in three P. aeruginosa isolates: HU63 (blaGES-6 carbapenemase-positive), HU141 (carbapenem-resistant without carbapenemase), and PAO1 (control). Genomic analysis revealed distinct sequence types (ST235 for HU63, ST253 for HU141) and chromosomal integration of resistance genes. HU63 harbored diverse resistance mechanisms, including β-lactamases (bla, bla, bla) and efflux pumps. Minimum inhibitory concentration assays demonstrated HU63's resistance to all β-lactams tested (meropenem, imipenem-cilastatin, ceftazidime, piperacillin-tazobactam), while HU141 remained susceptible except to cefoxitin and cloxacillin. Time-kill assays revealed tolerance phenotypes, with HU63 showing regrowth after 8-24 h despite initial reductions in bacterial density. Gene expression varied significantlydepending on the antibiotic and the isolate. The HU63 isolate (GES-6 positive) stands out for its marked induction of bla in all the antibiotics tested, contributing to its resistance to carbapenems and broad-spectrum cephalosporins. These expression profiles corroborate the classic molecular mechanisms of resistance: regulation of entry pores (oprD), activation of efflux pumps (mexA) and production of β-lactamases (bla, ampC) adapted to each situation. These findings underscore the multifactorial nature of resistance in Carbapenem-resistant Pseudomonas aeruginosa (CRPA), combining enzymatic inactivation, efflux, and genetic adaptability. The study emphasizes the urgent need for genomic surveillance to track high-risk clones and develop therapies targeting tolerance mechanisms alongside traditional resistance.
- Comprehensive typing and genetic analysis of L. monocytogenes isolates: implication for food safety and antibiotic resistance surveillancePublication . Silva, Adriana; Silva, Vanessa; Borges, Vítor; Coelho, Anabela; Batista, Rita; Esteves, Alexandra; Igrejas, Gilberto; Saraiva, Cristina; Gomes, João Paulo; Poeta, PatríciaListeria species are commonly found in various environments and contaminated food, with livestock serving as a significant source of foodborne pathogens. Among these species, Listeria monocytogenes (L. monocytogenes) is particularly noteworthy as it can affect both livestock and humans. Antibiotics are frequently used in food animals for disease treatment and prevention on a large scale. This practice can lead to the selection of antibiotic-resistant bacterial strains, which can then spread to humans through the food chain. Consequently, L. monocytogenes, a ubiquitous foodborne pathogen, has been associated with global outbreaks of foodborne illnesses. To address this concern, the aim of the study was to conduct comprehensive typing and genetic analysis of 13 L. monocytogenes isolates obtained from food and food-processing environments.Among the 13 L. monocytogenes isolates, eight sequence types (ST) were identified: two isolates were identified as belonging to ST9; one as ST155; four as ST3, two as ST121, one as ST8; one as ST87; one as ST1; and one new ST belonging to CC121. Core-genome clustering analysis of L. monocytogenes was made to assess the genetic relatedness among the isolates. The core genome Multilocus Sequence Typing (cgMLST) analysis revealed three genetic clusters of high closely related isolates (≤7 allelic differences (ADs)): cluster 1. Regarding L. monocytogenes typing, ST3 was the most prevalent among the isolates, found in 4 isolates, followed by ST9 and ST121. Some of these isolates, like ST1, ST9 and ST87, were previously associated with human clinical cases. We used Whole Genome Sequencing (WGS) alongside epidemiological data to link strains to human illnesses and potential food sources. Through cgMLST analysis, we identified genetic clusters of closely related isolates, all linked to the same producers. This approach helped us pinpoint common sources of contamination and gain insights into the transmission dynamics of L. monocytogenes in the context of food safety and public health. The escalating antibiotic resistance in Listeria species, particularly in L. monocytogenes, emphasizes the need for heightened surveillance and improved hygiene practices in the food industry to curb the spread of antibiotic resistance and ensure food safety.
- Diversity and genetic lineages of environmental staphylococci: a surface water overviewPublication . Silva, Vanessa; Caniça, Manuela; Capelo, José L.; Igrejas, Gilberto; Poeta, PatríciaAntimicrobial resistance in the environmental dimension is one of the greatest challenges and emerging threats. The presence of resistant bacteria and resistance genes in the environment, especially in aquatic systems, has been a matter of growing concern in the past decade. Monitoring the presence of antimicrobial resistance species, in this particular case, Staphylococcus spp., in natural water environments could lead to a better understanding of the epidemiology of staphylococci infections. Thus, the investigation of natural waters as a potential reservoir and vehicle for transmission of these bacteria is imperative. Only a few studies have investigated the prevalence, antimicrobial resistance and genetic lineages of staphylococci in natural waters. Those studies reported a high diversity of staphylococci species and lineages in surface waters. Methicillin-resistant S. aureus were relatively prevalent in surface waters and, as expected, often presented a multidrug-resistant profile. There was a high diversity of S. aureus lineages in surface waters. The presence of S. aureus CC8 and CC5 suggests a human origin. Among the coagulase-negative staphylococci, the most frequently found in natural waters was S. warneri and S. epidermidis. These studies are extremely important to estimate the contribution of the aquatic environment in the spread of pathogenic bacteria.
- Diversity of methicillin-resistant staphylococci among wild Lepus granatensis: first detection of mecA-MRSA in haresPublication . Silva, Vanessa; Pereira, José Eduardo; Maltez, Luís; Ferreira, Eugénia; Manageiro, Vera; Caniça, Manuela; Capelo, José L.; Igrejas, Gilberto; Poeta, PatríciaMRSA in humans, pets and livestock have been widely investigated, nevertheless, there is still little information of MRSA in wild animals. Therefore, this study aimed to investigate the occurrence and antimicrobial resistance profiles of methicillin-resistant staphylococci (MRS) in wild Iberian hares and to characterize their genetic lineages. Samples from 83 wild hares (Lepus granatensis) were collected during the hunting season. Isolation of MRS was accomplish using Oxacillin Resistant Screening Agar medium with 2 mg/L of oxacillin. The susceptibility of the isolates was tested by the Kirby-Bauer disc diffusion method. The presence of resistance and virulence genes was studied by PCR. S. aureus strains were further characterized by multilocus sequence typing, agr, spa and SCCmec typing. From the 83 samples, 12 (14.45%) coagulase-negative staphylococci and 3 (3.6%) MRSA strains were isolated. Nine coagulase-negative isolates showed resistance to at least one antibiotic. One MRSA isolate showed a multidrug-resistant profile with resistances to ß-lactams, aminoglycosides, macrolides and lincosamides. All MRSA strains were ascribed to ST2855, t1190 and SCCmec type III. The frequency of MRSA strains in wild hares was low, nevertheless, the presence of MRSA in game animals is considered a public health problem and may represent a route of transmission between animals and humans.
- Emergence of community-acquired methicillin-resistant Staphylococcus aureus EMRSA-15 clone as the predominant cause of diabetic foot ulcer infections in PortugalPublication . Silva, Vanessa; Almeida, Francisco; Carvalho, José António; Castro, Ana Paula; Ferreira, Eugénia; Manageiro, Vera; Tejedor-Junco, María Teresa; Caniça, Manuela; Igrejas, Gilberto; Poeta, PatríciaMethicillin-resistant Staphylococcus aureus (MRSA) are often found in infected diabetic foot ulcers, in which the prevalence may reach 40%. These complications are one of the main causes of morbidity in diabetic patients. The objectives of this study were to investigate the prevalence and antimicrobial resistance of MRSA strains in infected diabetic foot ulcers and to characterize their genetic lineages. Samples collected from 42 type 2 diabetic patients, presenting infected foot ulcers, were seeded onto ORSAB plates with 2 mg/L of oxacillin for MRSA isolation. Susceptibility to 14 antimicrobial agents was tested by the Kirby-Bauer disk diffusion method. The presence of resistance genes, virulence factors, and the immune evasion cluster system was studied by PCR. All isolates were characterized by MLST, accessory gene regulator (agr), spa, and staphylococcal chromosomal cassette mec (SCCmec) typing. Twenty-five MRSA strains were isolated. All isolates showed resistance to penicillin and cefoxitin. Sixteen isolates showed phenotypic resistance to erythromycin being 7 co-resistant to clindamycin. Resistance to trimethoprim-sulfamethoxazole was found in 2 isolates harboring the dfrA and dfrG genes. The IEC genes were detected in 80% of isolates, 16 of which were ascribed to IEC-type B. Isolates were assigned to 12 different spa types. The MLST analysis grouped the isolates into 7 sequence types being the majority (68%) ascribed to SCCmec type IV. In this study, there was a high prevalence of the EMRSA-15 clone presenting multiple resistances in diabetic foot ulcers making these infections complicated to treat leading to a higher morbidity and mortality in diabetic patients.