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- Potential pathogenic and opportunistic oral bacteria in early life: the role of maternal factors in a portuguese populationPublication . Fernandes, Mariana; Azevedo, Maria João; Campos, Carla; Ferreira, Ana Filipa; Azevedo, Álvaro; Falcão-Pires, Inês; Zaura, Egija; Ramalho, Carla; Campos, Joana; Sampaio-Maia, BeneditaIn early life, maternal factors are of the utmost relevance for oral microbiome acquisition and maturation. Therefore, our study explored the impact of maternal factors, such as saliva and breastmilk colonization, cardiovascular risk factors (CRF), type of delivery, oral health, and caregiving habits on the prevalence of potential pathogenic and opportunistic oral bacteria in early life. A total of 26 healthy mothers, 23 mothers with CRF, and their 50 children were included and samples (child’s oral swabs, mother’s saliva, and breastmilk) were collected 4 to 12 weeks after delivery and inoculated in selective and differential media for detection of non-fastidious Gram-negative and Gram-positive bacteria to isolate potential pathogenic and opportunistic bacteria identified by MALDI-TOF MS (414 isolates). Within mother–child dyads, the same species were identified in 86% of the pairs and potential pathogenic microorganisms from the Staphylococcaceae and Enterobacteriaceae families were found to be statistically significantly concordant between mother–child samples, particularly in the healthy group. Staphylococcus saprophyticus and Stenotrophomonas maltophilia oral colonization in mother–child pairs were associated with the presence of CRF. Breastfeeding was related to the early life oral colonization of Staphylococcus epidermidis in children from healthy mothers and C-section was associated with higher diversity of pathogens, independent of cardiovascular status (p = 0.05). This study reveals the presence of potential oral opportunistic and pathogenic bacteria in early life and highlights the importance of maternal factors in its acquisition.
- Unveiling the relevance of the oral cavity as a staphylococcus aureus colonization site and potential source of antimicrobial resistancePublication . Campos, Joana; Faria Pires, Mariana; Sousa, Marta; Campos, Carla; Fernandes Ferreira Alves da Costa, Carolina; Sampaio-Maia, BeneditaStaphylococcus aureus is both a human commensal and a pathogen, that causes serious nosocomial and community-acquired infections. Despite nostrils being considered its preferred host habitat, the oral cavity has been demonstrated to be an ideal starting point for auto-infection and transmission. The antibiotic resistance assessment of S. aureus is a priority and is often reported in clinical settings. This study aimed to explore the prevalence and antimicrobial susceptibility of S. aureus in the oral and nasal cavities of healthy individuals. The participants (n = 101) were subjected to a demographic and clinical background survey, a caries evaluation, and to oral and nasal swabbing. Swabs were cultured in differential/selective media and S. aureus isolates were identified (MALDI-TOF MS) and tested for antibiotic susceptibility (EUCAST/CLSI). Similar S. aureus prevalence was found exclusively on nasal (13.9%) or oral (12.0%) habitats, whereas 9.9% of the population were simultaneous nasal and oral carriers. In oro-nasal cavities, similar antibiotic resistance rates (83.3–81.5%), including MDR (20.8–29.6%), were observed. Notably, 60% (6/10) of the simultaneous nasal and oral carriers exhibited different antibiotic resistance profiles between cavities. This study demonstrates the relevance of the oral cavity as an independent colonization site for S. aureus and as a potential source of antimicrobial resistance, a role which has been widely neglected so far.
- Vertical transmission and antifungal susceptibility profile of yeast isolates from the oral cavity, gut, and breastmilk of mother–child pairs in early lifePublication . Azevedo, Maria João; Araújo, Ricardo; Campos, Joana; Campos, Carla; Ferreira, Ana Filipa; Falcão-Pires, Inês; Ramalho, Carla; Zaura, Egija; Pinto, Eugénia; Sampaio-Maia, BeneditaYeast acquisition begins at birth; however, the contribution of the mother on yeast trans mission to the offspring and associated resistance is yet to be clarified. The aim of this study was to explore the vertical transmission of yeasts and their antifungal susceptibility profile in early life. Oral, fecal, and breastmilk samples were collected from 73 mother–child pairs four to twelve weeks after delivery and cultured on Sabouraud dextrose agar with chloramphenicol. The isolates were identified by MALDI-TOF MS. The vertical transmission was studied by microsatellite genotyping. Antifungal susceptibility was determined for fluconazole, voriconazole, miconazole, anidulafungin, and nystatin by broth microdilution assay, following CLSI–M60 guidelines. A total of 129 isolates were identified from 53% mother–child pairs. We verified the vertical transmission of Candida albicans (n = three mother–child pairs) and Candida parapsilosis (n = one mother–child pair) strains, includ ing an antifungal resistant strain transmitted from breastmilk to the gut of a child. Most isolates were susceptible to the tested antifungals, with the exception of four C. albicans isolates and one R. mucilaginosa isolate. The vertical transmission of yeasts happens in early life. This is the first work that demonstrated the role of the mother as a source of transmission of antifungal-resistant yeasts to the child.
- Study of staphylococcus aureus in the nasal and oral cavities of dentistry studentsPublication . Pires, Mariana Faria; Campos, Joana; Campos, Carla; Costa, Carolina Fernandes Ferreira Alves da; Maia, Maria Benedita Almeida Garrett de SampaioStaphylococcus aureus permanently and asymptomatically colonizes 30% of the population, causing most nosocomial and community-acquired infections. The nostrils are considered its preferred host habitat and the main reservoir for transmission. However, the oral cavity has also demonstrated to be an ideal starting point for auto-infection and transmission.
- Nasal colonization by Staphylococcus aureusin Health Sciences students and analysis of risk fac-tors under a One Health perspectivePublication . Gomes-Sampaio, L. M.; Cláudia-Ferreira, A.; Prata, J. C.; Azevedo, R. M. S.; Pacheco, P.; Campos, Carla; Novais, C.; Peixe, L.; Dinis-Oliveira, R. J.; Coelho, C.; Miranda, C.; Quinteira, S.; Freitas, A. R.Staphylococcus aureusis the leading bacterial cause of death globally. Nasal carriage of S. aureusincreases the risk of invasive infections, including by methicillin-resistant S. aureus(MRSA) strains, but studies including Portuguese university students (PUS) are scarce. To analyse the prevalence of methicillin-susceptible S. aureus(MSSA) and MRSA among PUS enrolled in different courses/years (1st-4th) at IUCS-CESPU, characterize their antibiotic resistance profiles, and assess the potential risk factors. Swabs collected during March-December 2022 from anterior nares of 156 volunteers (median 22-years) were processed in mannitol-salt agar and, in parallel, enriched in brain-heart broth with NaCl 6.5% further plated onto ChromID® MRSA SMART. Typical colonies were stored for species identification (MALDITOF-MS) and antibiotic susceptibility testing (disk diffusion; EU-CAST/CLSI guidelines). Each student completed a questionnaire comprising demographic/clinical/social parameters. Statistical analysis was conducted in IBM-SPSS Statistics 26 using binary logistic regression applying a backward stepwise (likelihood ratio) method, with α=0.05, selecting variables using Chi-square tests and Mann-Whitney U tests for which p≤0.20, >10 occurrences, not biologicallycorrelated. Prevalence of MSSA and MRSA (cefoxitin screening) were 28.8% and 1.9%, respectively. From the 45 positive samples, 9% were multidrug-resistant, 38% were resistant to penicillin, 40% to erythromycin, 40% to clindamycin (inducible),7% to cefoxitin, 2% to tetracycline, and 2% to rifampicin. Self-reported frequent contact with animals (OR=3.44, CI 95%: 1.10–10.66) were positively associated with S. aureus, while regular sports participation presented a negative association (OR=0.36, CI 95%: 0.17–0.77). Sports participation was not correlated with self-reported excellent health (χ2=0.680, p=0.409). This is one of the first studies assessing MSSA/MRSA rates in PUS after the COVID-19 pandemics imposing higher self-protection/hygienization. While PUS-MSSA rates are similar to that previously observed, PUS-MRSA rates are slightly higher. Additional samples are being processed to explore future trends and other potential One Health factors influencing MSSA/MRSA colonization.
- Antimicrobial susceptibility of Staphylococcus aureus and coagulase-negative staphylococci in oral and nasal cavitiesPublication . Sousa, Marta; Sampaio Maia, Benedita; Costa, Carolina; Campos, Carla; Campos, JoanaStaphylococcus infections are becoming increasingly difficult to treat due to the growing number of resistant strains to old and new antibiotics. Staphylococcus aureus is referenced as the most clinically relevant but other species, known as coagulase-negative staphylococci (CoNS), have emerged as major causes of serious infections. Although nostrils are considered Staphylococcus preferred host habitat, the oral cavity has also been appointed as a relevant reservoir. Thus, this study aimed to evaluate the antibiotic susceptibility (AS) of nasal and oral staphylococci isolates. Therefore, AS was determined to 102 nasal and 90 oral isolates, previously recovered from dentistry students, by disc diffusion agar for amoxicillin, cefoxitin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, gentamicin, quinupristin-dalfopristin, tetracycline, and trimethoprim+sulfamethoxazole following the EUCAST/CLSI guidelines. In either nasal (N) or oral (O) cavities, the Staphylococcus isolates included 22% of S. aureus and 78% of CoNS isolates, namely S. epidermidis (41%-N,37%-O), S. warneri (15%-N,16%-O), S. capitis (10%-N,4%-O), S. saprophyticus (9%-N,6%-O), S. pasteuri (7%-O), S. lugdunensis (3%-O), and other (S. haemolyticus, S. hominis, S. sciuri, S. cohnii, and S. condimenti/S. carnosus). All S. aureus isolates presented resistance to at least one antibiotic, and 36% in oral and 25% in nasal cavities presented multidrug resistance (MDR). Among CoNS 65% in oral and 49% in nasal cavities presented resistance to at least one antibiotic, and ~5% presented MDR, namely isolates of S. epidermidis, S. pasteuri, S. haemolyticus, and S. hominis. In conclusion, this study revealed the relevance of the oral cavity as a colonization site for Staphylococcus, including S. aureus, and as a potential source/reservoir of antibiotic resistance, which has been widely neglected so far, highlighting the need for oral cavity inclusion in the Staphylococcus carriage and antibiotic screening.
- Mapping of key bacterial species for Postmorteminterval calculationPublication . Ferreira, Ana Cláudia; Barbosa, Daniel J.; Campos, Carla; Oliveira, Ricardo Jorge Dinis; Freitas, Ana R.Estimation of the PostmortemInterval (PMI), the time elapsed since death, is one of the most challenging issues in forensic sciences. Most studies focus on extensive bacterial sequencing, but culture-based experiments for higher taxonomic resolution remain scarce. We aimed to analyze total bacterial counts and map Enterococcus faecalis, Staphylococcus aureusand Escherichia coliin different organs and timepoints. Male C57BL/6J SPF mice underwent three independent assays during 11 postmortemtimepoints. Feces and organs (n=10: intestine/stomach/skeletical muscle/liver/spleen/kidney/bladder/lungs/ brain/heart) were collected and resuspended in buffered peptone water, then plated onto enriched nonselective and selective culture media (n=4). Following routine aerobic incubation, Colony Forming Units (CFU) per gram/tissue or per mL/sample were quantified for total/individual bacterial loads. Species were identified by MALDI-TOF MS and statistics were done in GraphPad-Prism v.10.0.1. Species (n=44) from 13 families and 3 phyla were identified, with notable consistency in the presence of Staphylococcus xylosus, E. faecalis, and E. coliacross all experiments. Particular families were consistently identified across all organs, including Enterococcaceae and Enterobacteriaceae mostly in the later stages of decomposition, and Bacillaceae resisting often until the last timepoint, whereas Staphylococcaceae was variably detected. The early and substantial contamination observed in skeletal muscle, stomach, and intestine, makes them unsuitable for PMI calculations. E. faecalisappeared promising as a potential biomarker for kidney, liver, and, possibly, brain invasion at later timepoints, whereas E. faecalisand E. coliin the bladder, and E. coliin the spleen and heart, warrant further investigation for similar biomarker potential. This is one of the first quantitative cultural studies assessing how time elapsing postmortem affects the growth/evolution of key bacterial species, with E. faecalisand E.coliemerging as promising traceable biomarkers in real postmortem contexts. While recognizing the limitations of not considering the complex microbiota network, our pilot study brings an easy species-specific approach and offers a baseline for future human-oriented investigations.
- High occurrence of multidrug-resistant Esche-richia colifrom Holstein-Friesian cattle in Northern Portugal: a One Health challengePublication . Quinteira, S.; Dias, A.B.; Pinho, L.; Brito, N.V.; Campos, Carla; Freitas, A.R.; Miranda, C.Escherichia coli is currently a leading pathogen for deaths associated with antimicrobial resistance (AMR). AMR related to food-producing animals is a public health risk requiring a One Health approach. Animals are major reservoirs of antibiotic resistant E. coli which can easily reach humans through the food chain, direct contact, or the environment. To analyze the AMR profiles of E. coli in fecal samples from Holstein-Friesian cattle, as information on AMR from dairy farming in Portugal is scarce. Samples (n=112) collected from 7 different farms at Northern Portugal were pooled during February-March 2023 based on age group (8 calves and 8 cows per farm). Characteristic E. coli colonies were selected from MacConkey Agar supplemented with or without antibiotics (4 µg/ml cefotaxime; 3 µg/ml colistin), for confirmation by MALDI-TOF mass spectrometry and antimicrobial susceptibility testing (AST), according to EUCAST/CLSI guidelines. ESBL phenotype were searched using the Double-Disk Synergy Test. Presumptive E. coli isolates (n=110) representing different farming production systems were obtained and 51 representatives were identified by MALDI-TOF and tested by AST. From both calves and cows, 95% and 63% of the E. coli isolates exhibited resistance to ≥1 antimicrobial classes and multidrug resistance (MDR, resistance to ≥3 antimicrobial classes), respectively. Isolates were mostly resistant to gentamycin (88%), ampicillin (72%), amoxicillin plus clavulanic acid (63%), tetracycline (63%) and cefotaxime (53%). Only two isolates showed susceptibility two all tested antibiotics. ESBL activity was observed in 21% of E. coli isolates. All analyzed pools were classified as MDR. This is one of the first studies on AMR rates of E. coli from dairy cattle in Northern Portugal. It reveals a high prevalence of MDR E. coli, which is a worrying finding and emphasize the need of a multisectoral One Health approach to minimize its impact.