João Rodrigues

Livestock faeces constitute an important environmental interface within the One Health framework, as bacteria shed by animals are directly exposed to external environmental conditions, where they may persist and subsequently disseminate across the human, animal, and environmental sectors. The ability of these microorganisms to tolerate environmental stresses is therefore critical for their persistence and ecological distribution. In this study, the responses of 5 Pseudomonas putida, 15 Pseudomonas fulva, and 12 Alcaligenes faecalis isolates recovered from livestock faeces to different environmental stress conditions were investigated. Bacterial growth was evaluated under different temperatures, increasing NaCl concentrations, and carbon starvation, while biofilm formation was assessed using the microtiter plate assay. Growth was markedly affected by all stress conditions, although substantial variability was observed among isolates of the same species. Despite reduced growth under the most adverse conditions, all three species remained capable of growing across a wide range of environmental stresses. A. faecalis exhibited greater salt tolerance and biofilm-forming capacity than Pseudomonas spp., with biofilm formation detected in almost all isolates of both species. Furthermore, no statistically significant correlations were detected among the evaluated stress tolerance traits. These findings demonstrate that livestock-associated P. putida, P. fulva, and A. faecalis possess physiological characteristics that promote persistence under diverse environmental conditions, highlighting the potential role of livestock faeces as environmental reservoirs of opportunistic bacteria within the One Health context.

Background: Foodborne diseases are a serious public health concern, and food-producing animals are a major source of contamination. Methods: The present study analysed Escherichia coli and Salmonella spp. isolated from faecal samples of 100 fattening pigs and from 52 samples of pork meat. Results: The results showed that the majority of the analysed meat samples were considered satisfactory in terms of microbiological quality (92.3% for E. coli and 94.2% for Salmonella spp.). Salmonella spp. was identified in 5.8% of the meat samples, whereas E. coli was detected in 89.5% of all samples (69.2% in meat and 100% in faecal samples). Furthermore, 1.9% of the faecal samples contained Shiga-toxin-producing E. coli and 3.9% contained enterotoxigenic E. coli. All sequenced isolates presented virulence genes for extraintestinal pathogenic E. coli. Moreover, 75.0% of E. coli isolates from meat and 71.8% from faeces samples showed antibiotic resistance, with 40.7% and 51.4%, respectively, being multidrug-resistant (MDR). The most prevalent resistances were to tetracycline, ampicillin, and sulfamethoxazole, and one E. coli isolate showed resistance to extended-spectrum β-lactamase. Conclusions: This study highlights the role of pigs as a potential source of human contamination and the importance of a One Health approach to ensure food safety and to promote public health.

Aeromonas spp. are environmental bacteria able to infect animals and humans. Here, we aim to evaluate the role of biofilms in Aeromonas persistence in freshwater. Aeromonas were isolated from water and biofilm samples and identified by Vitek-MS and 16S rRNA sequencing. Antibiotic susceptibility profiles were determined according to EUCAST, and a crystal violet assay was used to assess biofilm assembly. MTT and the enumeration of colony-forming units were used to evaluate biofilm and planktonic Aeromonas susceptibility to chlorination, respectively. Identification at the species level was challenging, suggesting the need to improve the used methodologies. Five different Aeromonas species (A. salmonicida, A. hydrophila, A. media, A. popoffii and A. veronii) were identified from water, and one species was identified from biofilms (A. veronii). A. veronnii and A. salmonicida presented resistance to different antibiotics, whith the highest resistance rate observed for A. salmonicida (multiple antibiotic resistance index of 0.25). Of the 21 isolates, 11 were biofilm producers, and 10 of them were strong biofilm producers (SBPs). The SBPs presented increased tolerance to chlorine disinfection when compared with their planktonic counterparts. In order to elucidate the mechanisms underlying biofilm tolerance to chlorine and support the importance of preventing biofilm assembly in water reservoirs, further research is required.

This study extensively analyzed campylobacteriosis surveillance in Portugal from 2009 to 2021, aiming to investigate demographic shifts, seasonal variations, and antimicrobial resistance (AMR) within Campylobacter isolates. Surveillance network and sentinel laboratory-based system data revealed a substantial under-notification of campylobacteriosis cases, suggesting an underestimated disease burden. Notification rates exhibited a paradigm shift, with a notable prevalence among the pediatric population, particularly in children aged 1–4 years, diverging from European reports. Additionally, an emerging trend of Campylobacter infections in younger adults (15–44 years) was observed. The study unveiled a unique seasonal distribution of cases, defying typical summer peaks seen elsewhere. AMR analysis revealed high resistance to ciprofloxacin and tetracycline, in both C. jejuni (93.7% and 79.2%, respectively) and C. coli (96.5% and 93.2%, respectively), stable throughout the studied period (2013–2021). C. coli exhibited significantly higher resistance to erythromycin, gentamicin, ampicillin and ertapenem compared to C. jejuni (p < 0.001). Multilocus Sequence Typing (MLST) data demonstrated the distribution of resistance markers across diverse sequence types, challenging the notion of a clonal origin for multidrug-resistant isolates. In conclusion, the study highlights the need for enhanced surveillance and raises concerns about alarming AMR levels, recommending the implementation of whole-genome sequencing (WGS)-based surveillance for a deeper comprehension of disease patterns and an evolving AMR landscape.

This study conducted an extensive analysis of campylobacteriosis surveillance in Portugal from 2009 to 2021, aiming to investigate demographic shifts, seasonal variations, and antimicrobial resistance (AMR) within Campylobacter isolates. Surveillance network and sentinel laboratory-based system data revealed a substantial under-notification of campylobacteriosis cases, suggesting an underestimated disease burden. A paradigm shift was observed in notification rates, with a notable prevalence among the pediatric population, particularly in children aged 1-4 years, diverging from European reports. However, the study also highlighted an emerging trend of Campylobacter infections in younger adults (15-44 years). The study unveiled a unique seasonal distribution of cases, defying typical summer peaks seen elsewhere. AMR analysis revealed extremely to very high-level resistance to ciprofloxacin and tetracycline, both in C. jejuni (93.7% and 79.2%, respectively) and in C. coli (96.5% and 93.2%, respectively), stable throughout the studied period (2013-2021). C. coli also exhibited significantly higher resistance to erythromycin, gentamicin, ampicillin, amoxicillin-clavulanic acid (decreased resistance), and ertapenem compared to C. jejuni (P<0.001). In conclusion, the study highlights the need for enhanced surveillance efforts and emphasizes worrying AMR levels. The implementation of WGS-based surveillance is recommended to attain a more profound comprehension of disease patterns and the evolving landscape of AMR.

Category: Ankle; Sports Introduction/Purpose: A ligament-centered analysis is currently incorporated in the assessment of joint stability in malleolar fractures. Although several imaging tests are available, in the setting of acute isolated non-displaced posterior malleolar fractures Bartoníček/Rammelt types II and IV diagnosing syndesmotic instability remains challenging. The objective of this multi-center cross- sectional study was to evaluate the syndesmotic and fracture instability using conventional ankle CT with external rotation and dorsiflexion in the setting of acute, isolated non-displaced Bartoníček and Rammelt type II posterior malleolar fractures. Methods: Between March 2018 and September 2022, a consecutive sample of 123 individuals with an ankle sprain were assessed for eligibility. In total, 33 participants met the inclusion criteria. All participants underwent a CT scan (CTSM), comprising a first phase with the ankles in a neutral position, then a stress phase with the ankles in external rotation and dorsiflexion and semiflexed knees. Investigators used the patterns of ligament tear found at MRI and instability at CTSM to classify all participants into West Point grades I, IIA, IIB, or III. Mann-Whitney test was used to test the differences in the numerical variables between injured and uninjured syndesmoses. The Spearman correlation tested the strength of the association between the tibial joint surface involved in posterior malleolus fracture and syndesmotic instability. Results: In MRI reading parameters two patterns of syndesmotic ligament injury predominated. A completely torn AITF and IO ligaments and a completely torn AITF was combined with a partially torn IO. Regarding the deep layer of the deltoid ligament, participants were classified as normal, strained and partially torn. In CT scan reading parameters in the neutral phase, the median difference of 0.2 mm in d measurements between injured and uninjured syndesmoses was not statistically significant (P = 0.057). During the stress phase, the injured and uninjured syndesmoses had distinct behavior, and the 2.3 mm median difference for d measurement was statistically significant (P < 0.0001). Conclusion: The conventional computed tomography with external rotation and dorsiflexion represent a reproducible and accurate diagnostic option for the detection of syndesmosis instability and fracture instability in acute isolated posterior malleolar fractures Bartonícek and Rammelt type II.

Category: Ankle; Sports Introduction/Purpose: Syndesmotic injury in an athletic population is associated with a prolonged ankle disability after an ankle sprain and often requires a longer recovery than a lateral collateral ligament injury. Although several imaging tests are available, diagnosing syndesmotic instability remains challenging. The main objective was to determine the diagnostic accuracy of conventional ankle computed tomography (CT) scans with the joint in external rotation and dorsiflexion and compare it with that of conventional ankle CT scans in a neutral position. Methods: Between September 2018 and April 2021, this prospective study consecutively included adults visiting the foot and ankle outpatient clinic with a positive orthopedic examination for acute syndesmotic injury. Participants underwent 3 CT scan tests. First, ankles were scanned in a neutral position. Second, ankles were scanned with 45° of external rotation, dorsiflexion, and extended knees. Third, ankles were scanned with 45° of external rotation, dorsiflexion, and flexed knees. Three measurements, comprising rotation (measurement a), lateral translation (measurement c), and anteroposterior translation (measurement f) of the fibula concerning the tibia, were used to diagnose syndesmotic instability in the 3 CT scans. Magnetic resonance imaging was used as a reference standard. The area under the curve (AUC) was used to compare the diagnostic accuracy, and Youden’s J index was calculated to determine the ideal cutoff point. Results: Images obtained in 68 participants (mean age, 36.5 years; range, 18-69 years) were analyzed, comprising 36 syndesmotic injuries and 32 lateral collateral ligament injuries. The best diagnostic accuracy occurred with the rotational measurement a, in which the second and third CT scans with stress maneuvers presented greater AUCs (0.97 and 0.99) than the first CT scan in a neutral position (0.62). The ideal cutoff point for the stress maneuvers was 1.0 mm in the rotational measurement and reached a sensitivity and specificity of 83% and 97% for the second CT scan with extended knees and 86% and 100% for the third CT scan with flexed knees, respectively. Conclusion: Conventional ankle CT with stress maneuvers has excellent performance for diagnosing subtle syndesmotic rotational instability, as it shows a greater AUC and enhanced sensitivity at the ideal cutoff point compared with ankle CT in the neutral position.

Biofilm-associated infections are a public health concern especially in the context of healthcare-associated infections such as catheter-related bloodstream infections (CRBSIs). We evaluated the biofilm formation and antimicrobials resistance (AMR) of Enterobacter cloacae complex and Candida parapsilosis co-isolated from a CRBSI patient. Antimicrobial susceptibility of central venous catheters (CVCs) and hemoculture (HC) isolates was evaluated, including whole genome sequencing (WGS) resistome analysis and evaluation of gene expression to obtain insight into their AMR determinants. Crystal violet assay was used to assess dual biofilm biomass and microscopy was used to elucidate a microorganism’s distribution within biofilms assembled on different materials. Bacteria were multidrug-resistant including resistance to colistin and beta-lactams, likely linked to the mcr-9-like phosphoethanolamine transferase and to an ACT family cephalosporin-hydrolyzing class C beta-lactamase, respectively. The R398I and Y132F mutations in the ERG11 gene and its differential expression might account for C. parapsilosis resistance to fluconazole. The phenotype of dual biofilms assembled on glass, polystyrene and polyurethane depends on the material and how biofilms were initiated by one or both pathogens. Biofilms assembled on polyurethane were denser and richer in the extracellular polymeric matrix, and microorganisms were differently distributed on the inner/outer surface of the CVC.