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The objective of this study was to obtain genetic marker information in the Gyr breed by analyzing bGH and Pit-1 gene polymorphisms and to verify their association with milk production traits. One sample including 40 Gyr bulls were genotyped at two bGH gene restriction sites (bGH- AluI and bGH-MspI) and at one restriction site in the Pit-1 gene (Pit-1 HinfI). The bGH-MspI(-) allele was favorable for fat milk percentage. The heterozigous Pit-1 HinfI (+/-) bulls were superior for fat milk production, in relation to homozigous Pit-1 HinfI (+/+). The Pit-1 and bGH genes are strong candidates in the dairy cattle QTL search, and zebuine populations are promising samples for this purpose.

O objetivo deste trabalho foi estimar as tendências genéticas para a produção de leite e gordura nos rebanhos da raça Holandesa do Estado de Minas Gerais. As análises estatísticas foram realizadas por intermédio da metodologia dos modelos mistos, usando-se máxima verossimilhança restrita e modelo animal. Os efeitos fixos considerados no modelo foram: rebanho-ano-estação de parto e os efeitos linear e quadrático da idade da vaca ao parto. Os efeitos aleatórios foram animal e erro. A tendência genética foi calculada pela regressão do valor genético sobre o ano de nascimento da vaca; nesta análise, foram usados 18.482 registros de produção de leite e gordura de vacas de primeira cria, nascidas de 1986 a 1996. As estimativas de ganho genético para produção de leite e gordura foram 18,4 e 0,6 kg, respectivamente. A média de produção de leite e gordura aumentou anualmente, variando de 5083 ± 50 a 6876 ± 51 kg, e de 172,6 ± 1,7 a 228,5 ± 1,7 kg, respectivamente. Os valores genéticos estimados foram negativos em 1986 e 1987 e positivos após este período, com aumento acentuado nos últimos anos, indicando que os produtores de leite de Minas Gerais vêm adotando critérios para aumentar a capacidade de produção de seus rebanhos.

O objetivo deste trabalho foi verificar se é necessário o ajustamento da produção de leite até 305 dias para produção no início da lactação, ao se determinar o efeito do período de serviço sobre a produção de leite. As produções no início da lactação foram representadas pelas produções acumuladas até 80, 100 e 120 dias e pela produção no pico. As análises foram realizadas para as primeiras e lactações seguintes, por meio de modelos em que se consideraram os períodos de serviço corrente e anterior, respectivamente. Em um modelo animal para produção de leite até 305 dias, incluíram-se os efeitos fixos de rebanho-ano-estação de parto, composição racial, idade e período de serviço em classes e produção no início da lactação e os efeitos aleatórios de animal, meio permanente e erro. Quando foi considerada a produção inicial para primeiras lactações, as diferenças relativas entre cada classe de período de serviço, por exemplo, 160 dias, permaneceram praticamente inalteradas. Por exemplo, quando as produções iniciais foram de até 80 e 100 dias, as diferenças entre produções de vacas com 40 e 160 dias de período de serviço corrente foram 675 e 677 kg, respectivamente. Esta diferença foi de 672 kg, quando a produção inicial foi ignorada. Para vacas de lactações seguintes, não houve influência da variação da duração da produção inicial sobre a relação entre produção e período de serviço anterior. O valor estimado da heritabilidade da produção de leite, quando não houve ajustamento para produção inicial, foi de 0,24, reduzindo-se para 0,15, quando o pico foi considerado. Concluiu-se que não é necessário considerar o efeito da produção inicial, ao se ajustarem as produções para período de serviço.

Genetic redundancy in bacteria plays a crucial role in enhancing adaptability and accelerating evolution in response to selective pressures, particularly those associated with rapid environmental changes. Aminopenicillins like ampicillin are important therapeutic options for Enterococcus infections in both humans and animals, with resistance mostly associated with pbp5 gene mutations or overexpression. While the occurrence of redundant pbp5 genes has been occasionally reported, the advantages for the host bacteria have not been explored in detail. During a whole-genome sequencing project of Enterococcus faecium from bacteremic patients, we identified an ST592 strain (Efm57) with redundant pbp5 genes. This presented an opportunity to investigate the prevalence and implications of multiple pbp5 acquisitions in diverse Enterococcus species across various sources, geographical regions, and timeframes. The analysis of 618 complete Enterococcus genomes from public databases revealed that 3.2 % harbored redundant pbp5 genes, located on chromosomes or plasmids across different species from diverse epidemiological backgrounds. The proteins encoded by these genes showed homologies ranging from 51.1 % to 97.5 % compared to native copies. Phylogenetic analysis grouped redundant PBP5 amino acid sequences into three distinct clades, with insertion sequences (mostly IS6-like) facilitating their recent spread to diverse plasmids with varying genetic backbones. The presence of multiple antibiotic resistance genes on pbp5-plasmids, including those conferring resistance to linezolid, underscores their involvement in co-selection and recombination events with other clinically-relevant antibiotics. Conjugation experiments confirmed the transferability of a specific 24 kb pbp5-plasmid from the Efm57 strain. This plasmid was associated with higher minimum inhibitory concentrations of ampicillin and conferred bacteria growth advantages at 22 °C. In conclusion, the widespread distribution of redundant pbp5 genes among Enterococcus spp. highlights the complex interplay between genetic mobility, environmental factors, and multidrug resistance in overlapping ecosystems emphasizing the importance of understanding these dynamics to mitigate antibiotic resistance spread within the One Health framework., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Enterococcus spp. are opportunistic human pathogens colonizing the human gut and a significant reservoir for the continuous adaptation of hospital clones. However, studies on the features of enterococci species co-colonizing healthy individuals are scarce. We investigated the prevalence, antibiotic resistance, and bacteriocin profiles of Enterococcus species in fecal samples from healthy adults in Portugal using culture-based methods, WGS, and bacteriocin inhibition assays. Results were compared with data from a 2001 study in the same region. Enterococcus spp. ( n = 315; 24% MDR) were recovered from all volunteers. Enterococcus lactis was the prevalent species (75%), followed by Enterococcus faecalis (65%) and Enterococcus faecium (47%). E. lactis prevalence increased 2.5-fold since 2001. Linezolid resistance genes ( optrA/poxtA ) were detected in E. faecium and Enterococcus thailandicus isolates, while a vancomycin-variable E. faecium was also identified. Virulence and plasmid profiles were diverse across species, with evidence of exchange of virulence markers and plasmid replicons between E. faecium and E. lactis . Bacteriocin gene repertoires were extensive and species-specific. Higher numbers of bacteriocin genes were associated with stronger inhibition profiles, and 25% of E. faecium and E. lactis isolates were capable of inhibiting relevant VRE clones. This study unveils the co-occurrence and ecological dynamics of Enterococcus species in the healthy human gut, reinforcing its role as a reservoir for key antibiotic resistance genes and potentially pathogenic strains. The shift toward E. lactis prevalence and the detection of linezolid resistance genes in healthy individuals underscore the need for ongoing surveillance of the gut microbiome to guide public health strategies and antibiotic stewardship efforts.IMPORTANCEThis study highlights the role of Enterococcus species in the healthy human gut, revealing important insights into their prevalence and antibiotic resistance. It emphasizes that the human gut serves as a significant reservoir for antibiotic-resistant strains and shows a notable increase and prevalence of Enterococcus lactis, which has been underappreciated due to identification challenges. The research also underscores the bacteriocins' role in microbial competition, where commensal strains inhibit clinical VRE, potentially aiding the restoration of the gut microbiota, after antibiotic treatment. The findings accentuate the need for ongoing surveillance to track changes in gut bacteria, especially with the emergence of resistance genes to last resort antibiotics. Such monitoring is crucial for shaping public health strategies and managing the growing threat of antibiotic-resistant infections. Profiling bacteriocins at the species and strain level can identify ecological adaptation factors and inform strategies to target high-risk clones.

Background/Objectives: Animals destined for human consumption play a key role in potentially transmitting bacteria carrying antibiotic resistance genes. However, there is limited knowledge about the carriage of antibiotic-resistant bacteria in native breeds. We aimed to characterize the phenotypic profiles and antibiotic resistance genes in Escherichia coli isolated from bovines, including three native Portuguese bovine breeds. Methods: Forty-nine E. coli isolates were selected from 640 fecal samples pooled by age group (eight adult or eight calf samples) from each farm, representing both dairy cattle raised in intensive systems and meat cattle raised in extensive systems in Northern Portugal. The presumptive E. coli colonies plated onto MacConkey agar were confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The antibiotic resistance profiles were screened by antimicrobial susceptibility testing (EUCAST/CLSI guidelines), and the antibiotic resistance genes by PCR. Results: Most isolates showed resistance to ampicillin (69%), tetracycline (57%), gentamicin (55%), and trimethoprim + sulfamethoxazole (53%), with no resistance to imipenem. Resistance to at least one antibiotic was found in 92% of isolates, while 59% exhibited multidrug resistance. Most calf isolates, including those from native breeds, showed a multidrug-resistant phenotype. Among the adults, this was only observed in Holstein-Friesian and Barrosã cattle. None of the Holstein-Friesian isolates were susceptible to all the tested antibiotics. ESBL-producing E. coli was identified in 39% of isolates, including those from Holstein-Friesian calves and adults, Cachena calves and Minhota adults. The sul 2 gene was detected in 69% of isolates, followed by bla CTX-M (45%), aac (3')-IV (41%), and aac (6')-Ib-cr (31%), with a higher prevalence in adults. Conclusions: This pioneering study highlights the concerning presence of multidrug-resistant E. coli in native Portuguese cattle breeds.

Multidrug-resistant Enterococcus faecium strains represent a major concern due to their ability to thrive in diverse environments and cause life-threatening infections. While antimicrobial resistance and virulence mechanisms have been extensively studied, the contribution of bacteriocins to E. faecium 's adaptability remains poorly explored. E. faecium , within the Bacillota phylum, is a prominent bacteriocin producer. Here, we developed a tailored database of 76 Bacillota bacteriocins (217 sequences, including 40 novel bacteriocins) and applied it to uncover bacteriocin distribution patterns in 997 quality-filtered E. faecium and Enterococcus lactis (former E. faecium clade B) genomes. Curated using computational pipelines and literature mining, our database demonstrates superior precision versus leading public tools in identifying diverse bacteriocins. Distinct bacteriocin profiles emerged between E. faecium and E. lactis , highlighting species-specific adaptations. E. faecium strains from hospitalized patients were significantly enriched in bacteriocins as enterocin A and bacteriocins 43 (or T8), AS5, and AS11. These bacteriocin genes were strongly associated with antibiotic resistance, particularly vancomycin and ampicillin, and Inc18 rep 2_pRE25-derivative plasmids, classically associated with vancomycin resistance transposons. Such bacteriocin arsenal likely enhances the adaptability and competitive fitness of E. faecium in the nosocomial environment. By combining a novel tailored database, whole-genome sequencing, and epidemiological data, our work elucidates meaningful connections between bacteriocin determinants, antimicrobial resistance, mobile genetic elements, and ecological origins in E. faecium and provides a framework for elucidating bacteriocin landscapes in other organisms. Characterizing species- and strain-level differences in bacteriocin profiles may reveal determinants of ecological adaptation, and translating these discoveries could further inform strategies to exploit bacteriocins against high-risk clones., Importance: This work significantly expands the knowledge on the understudied bacteriocin diversity in opportunistic enterococci, revealing their contribution in the adaptation to different environments. It underscores the importance of placing increased emphasis on genetic platforms carrying bacteriocins as well as on cryptic plasmids that often exclusively harbor bacteriocins since bacteriocin production can significantly contribute to plasmid maintenance, potentially facilitating their stable transmission across generations. Further characterization of strain-level bacteriocin landscapes could inform strategies to combat high-risk clones. Overall, these insights provide a framework for unraveling the therapeutic and biotechnological potential of bacteriocins., Competing Interests: The authors declare no conflict of interest.

Cationic biocides (CBs), such as quaternary ammonium compounds and biguanides, are critical for controlling the spread of bacterial pathogens like Enterococcus spp., a leading cause of multidrug-resistant healthcare-associated infections. The widespread use of CBs in recent decades has prompted concerns about the potential emergence of Enterococcus spp. populations exhibiting resistance to both biocides and antibiotics. Such concerns arise from their frequent exposure to subinhibitory concentrations of CBs in clinical, food chain and diverse environmental settings. This comprehensive narrative review aimed to explore the complexity of the Enterococcus ' response to CBs and of their possible evolution toward resistance. To that end, CBs' activity against diverse Enterococcus spp. collections, the prevalence and roles of genes associated with decreased susceptibility to CBs, and the potential for co- and cross-resistance between CBs and antibiotics are reviewed. Significant methodological and knowledge gaps are identified, highlighting areas that future studies should address to enhance our comprehension of the impact of exposure to CBs on Enterococcus spp. populations' epidemiology. This knowledge is essential for developing effective One Health strategies that ensure the continued efficacy of these critical agents in safeguarding Public Health., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pereira, Antunes, Peixe, Freitas and Novais.)

BackgroundThe pet industry is expanding worldwide, particularly raw meat-based diets (RMBDs). There are concerns regarding the safety of RMBDs, especially their potential to spread clinically relevant antibiotic-resistant bacteria or zoonotic pathogens.AimWe aimed to investigate whether dog food, including RMBD, commercially available in Portugal can be a source of Salmonella and/or other Enterobacteriaceae strains resistant to last-line antibiotics such as colistin.MethodsFifty-five samples from 25 brands (21 international ones) of various dog food types from 12 suppliers were screened by standard cultural methods between September 2019 and January 2020. Isolates were characterised by phenotypic and genotypic methods, including whole genome sequencing and comparative genomics.ResultsOnly RMBD batches were contaminated, with 10 of 14 containing polyclonal multidrug-resistant (MDR) Escherichia coli and one MDR Salmonella . One turkey-based sample contained MDR Salmonella serotype 1,4,[5],12:i:- ST34/cgST142761 with similarity to human clinical isolates occurring worldwide. This Salmonella exhibited typical antibiotic resistance ( bla TEM  +  strA-strB  +  sul2  +  tet(B) ) and metal tolerance profiles ( pco  +  sil  +  ars ) associated with the European epidemic clone. Two samples (turkey/veal) carried globally dispersed MDR E. coli (ST3997-complexST10/cgST95899 and ST297/cgST138377) with colistin resistance (minimum inhibitory concentration: 4 mg/L) and mcr-1 gene on IncX4 plasmids, which were identical to other IncX4 circulating worldwide.ConclusionSome RMBDs from European brands available in Portugal can be a vehicle for clinically relevant MDR Salmonella and pathogenic E. coli clones carrying genes encoding resistance to the last-line antibiotic colistin. Proactive actions within the One Health context, spanning regulatory, pet-food industry and consumer levels, are needed to mitigate these public health risks.

Competing Interests: The author or authors declare that they have no conflict of interest.

Objectives: To investigate the global distribution of an optrA-harbouring linezolid-resistant Enterococcus faecalis ST476 clonal lineage., Methods: Comprehensive searches of the NCBI database were performed to identify published peer-reviewed articles and genomes of E. faecalis ST476. Each genome was analysed for resistome, virulome, OptrA variant and optrA genetic contexts. A phylogenetic comparison of ST476 genomes with publicly available genomes of other STs was also performed., Results: Sixty-six E. faecalis ST476 isolates from 15 countries (China, Japan, South Korea, Austria, Denmark, Spain, Czech Republic, Colombia, Tunisia, Italy, Malaysia, Belgium, Germany, United Arab Emirates and Switzerland) mainly of human and animal origin were identified. Thirty available ST476 genomes compared with genomes of 591 STs indicated a progressive radiation of E. faecalis STs starting from ST21. The closest ancestral node for ST476 was ST1238. Thirty E. faecalis ST476 genomes exhibited 3-916 SNP differences. Several antimicrobial resistance and virulence genes were conserved among the ST476 genomes. The optrA genetic context exhibited a high degree of or complete identity to the chromosomal transposon Tn6674. Only three isolates displayed an optrA-carrying plasmid with complete or partial Tn6674. The WT OptrA protein was most widespread in the ST476 lineage., Conclusions: Linezolid-resistant optrA-carrying E. faecalis of the clonal lineage ST476 is globally distributed in human, animal and environmental settings. The presence of such an emerging clone can be of great concern for public health. Thus, a One Health approach is needed to counteract the spread and the evolution of this enterococcal clonal lineage., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Enterococcus faecium ( Efm ) is a versatile pathogen, responsible for multidrug-resistant infections, especially in hospitalized immunocompromised patients. Its population structure has been characterized by diverse clades (A1, A2, and B (reclassified as E. lactis ( Ela )), adapted to different environments, and distinguished by their resistomes and virulomes. These features only partially explain the predominance of clade A1 strains in nosocomial infections. We investigated in vitro interaction of 50 clinical isolates (clade A1 Efm ) against 75 commensal faecal isolates from healthy humans (25 clade A2 Efm and 50 Ela ). Only 36% of the commensal isolates inhibited clinical isolates, while 76% of the clinical isolates inhibited commensal isolates. The most apparent overall differences in inhibition patterns were presented between clades. The inhibitory activity was mainly mediated by secreted, proteinaceous, heat-stable compounds, likely indicating an involvement of bacteriocins. A custom-made database targeting 76 Bacillota bacteriocins was used to reveal bacteriocins in the genomes. Our systematic screening of the interactions between nosocomial and commensal Efm and Ela on a large scale suggests that, in a clinical setting, nosocomial strains not only have an advantage over commensal strains due to their possession of AMR genes, virulence factors, and resilience but also inhibit the growth of commensal strains., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Enterococcus faecium ( Efm ) is a leading cause of hospital-associated (HA) infections, often enriched in putative virulence markers (PVMs). Recently, the Efm clade B was assigned as Enterococcus lactis ( Elts ), which usually lack HA- Efm infection markers. Available databases for extracting PVM are incomplete and/or present an intermix of genes from Efm and Enterococcus faecalis , with distinct virulence profiles. In this study, we constructed a new database containing 27 PVMs [ acm, scm, sgrA, ecbA, fnm, sagA, hylEfm, ptsD, orf1481, fms15, fms21-fms20 (pili gene cluster 1, PGC-1), fms14-fms17-fms13 (PGC-2), empA-empB-empC (PGC-3), fms11-fms19-fms16 (PGC-4), ccpA, bepA, gls20-glsB1, and gls33-glsB ] from nine reference genomes (seven Efm + two Elts ). The database was validated against these reference genomes and further evaluated using a collection of well-characterized Efm ( n = 43) and Elts ( n = 7) control strains, by assessing PVM presence/absence and its variants together with a genomic phylogeny constructed as single-nucleotide polymorphisms. We found a high concordance between the phylogeny and in silico findings of the PVM, with Elts clustering separately and mostly carrying Elts- specific PVM gene variants. Based on our validation results, we recommend using the database with raw reads instead of assemblies to avoid missing gene variants. This newly constructed database of 27 PVMs will enable a more comprehensive characterization of Efm and Elts based on WGS data. The developed database exhibits scalability and boasts a range of applications in public health, including diagnostics, outbreak investigations, and epidemiological studies. It can be further used in risk assessment for distinguishing between safe and unsafe enterococci.IMPORTANCEThe newly constructed database, consisting of 27 putative virulence markers, is highly scalable and serves as a valuable resource for the comprehensive characterization of these closely related species using WGS data. It holds significant potential for various public health applications, including hospital outbreak investigations, surveillance, and risk assessment for probiotics and feed additives., Competing Interests: The authors declare no conflict of interest.

The reliability of Fourier-transform infrared (FT-IR) spectroscopy for Klebsiella pneumoniae typing and outbreak control has been previously assessed, but issues remain in standardization and reproducibility. We developed and validated a reproducible FT-IR with attenuated total reflectance (ATR) workflow for the identification of K. pneumoniae lineages. We used 293 isolates representing multidrug-resistant K. pneumoniae lineages causing outbreaks worldwide (2002-2021) to train a random forest classification (RF) model based on capsular (KL)-type discrimination. This model was validated with 280 contemporaneous isolates (2021-2022), using wzi sequencing and whole-genome sequencing as references. Repeatability and reproducibility were tested in different culture media and instruments throughout time. Our RF model allowed the classification of 33 capsular (KL)-types and up to 36 clinically relevant K. pneumoniae lineages based on the discrimination of specific KL- and O-type combinations. We obtained high rates of accuracy (89%), sensitivity (88%), and specificity (92%), including from cultures obtained directly from the clinical sample, allowing to obtain typing information the same day bacteria are identified. The workflow was reproducible in different instruments throughout time (>98% correct predictions). Direct colony application, spectral acquisition, and automated KL prediction through Clover MS Data analysis software allow a short time-to-result (5 min/isolate). We demonstrated that FT-IR ATR spectroscopy provides meaningful, reproducible, and accurate information at a very early stage (as soon as bacterial identification) to support infection control and public health surveillance. The high robustness together with automated and flexible workflows for data analysis provide opportunities to consolidate real-time applications at a global level. IMPORTANCE We created and validated an automated and simple workflow for the identification of clinically relevant Klebsiella pneumoniae lineages by FT-IR spectroscopy and machine-learning, a method that can be extremely useful to provide quick and reliable typing information to support real-time decisions of outbreak management and infection control. This method and workflow is of interest to support clinical microbiology diagnostics and to aid public health surveillance., Competing Interests: The authors declare no conflict of interest.

Background: Although Brucella abortus , Brucella suis, and Brucella canis may infect humans and dogs worldwide, no study to date has assessed and compared owners and their dogs between island and mainland seashore areas. Materials and Methods: Accordingly, the study herein has applied serological tests, including Microplate Agglutination Test with 2-Mercaptoethanol, immunochromatographic assay, and Rose Bengal Test, and a Brucella genus-specific PCR assay to 195 owners and their 148 dogs living on 1 mainland seashore area and three nearby oceanic islands of southern Brazil. Results: No seropositivity to B. abortus and B. suis was detected in owner or dog sera. Anti- B. canis seropositivity was observed in 3/148 (2.0%) dogs, but no owner sample was seropositive to B. canis . In addition, all blood samples from both owners and dogs were negative on Brucella genus-specific PCR assay. Conclusions: The seropositive dogs were not related and lived on the seashore mainland area of Guaraqueçaba city. The absence of seropositivity on the islands and the low seropositivity on the seashore mainland could be attributed to geographic isolation, and suggest the low impact of the disease in the region. Despite being a zoonotic disease, brucellosis by B. canis is not included in the National Program for Control and Eradication of Brucellosis, and its diagnosis and notification are not mandatory. The presence of seropositive dogs highlights the risk to human health and the importance of epidemiological surveillance actions in the region, as well as the need for the implantation of preventive measures to avoid the transmission of the pathogen.

Introduction The standard treatment of cancer has dramatically improved with immune checkpoint inhibitors (ICIs). Despite their proven advantage, many patients fail to exhibit a meaningful and lasting response. The beta-adrenergic signalling pathway may hold significant promise due to its role in promoting an immunosuppressive milieu within the tumour microenvironment. Inhibiting β-adrenergic signalling could enhance ICI activity; however, blocking this pathway for this purpose has yielded conflicting results. The primary objective of this study was to evaluate the effect of beta-blocker use on overall survival and progression-free survival during ICI therapy. Methods A multicentric, retrospective, observational study was conducted in four Portuguese institutions. Patients with advanced non-small cell lung cancer treated with ICIs between January 2018 and December 2019 were included. Those using beta blockers for non-oncological reasons were compared with non-users. Results Among the 171 patients included, 36 concomitantly received beta blockers and ICIs. No significant increase was found in progression-free survival among patients who took β-blockers (HR 0.74, 95% confidence interval (CI) 0.48-1.12, p = 0.151), and no statistically significant difference was found in overall survival. An apparent trend was observed towards better outcomes in the beta-blocker group, with a median overall survival of 9.93 months in the group not taking β-blockers versus 14.90 months in the β-blocker group (p = 0.291) and a median progression-free survival of 5.37 in the group not taking β-blockers versus 10.87 months in the β-blocker group (p = 0.151). Nine (25%) patients in the beta-blocker group and 16 (12%) in the non-beta-blocker group were progressive disease-free at the end of follow-up. This difference between the two groups is statistically significant (p = 0.047). Conclusion Our study found no statistically significant evidence that beta blockers enhance the effectiveness of immunotherapy. Using adrenergic blockade to modulate the immune system shows promise, warranting the need to develop prospective clinical studies., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Duarte Mendes et al.)

Whipple's disease (WD) is a chronic multisystemic infection caused by Tropheryma whipplei . It is a rare condition with a wide spectrum of clinical presentations, necessitating a high clinical suspicion to arrive at the diagnosis. We present the case of a 65-year-old woman who experienced chronic, intermittent, and migratory polyarthralgia, weight loss, anorexia, and pyrosis. She was admitted due to bilateral deep vein thrombosis (DVT). She exhibited lymphadenopathy without hepatosplenomegaly, and lymph node biopsy revealed reactive lymphadenitis with intrahistiocytic bacilli that reacted positively to periodic acid-Schiff staining. This led to the suspicion of WD, which was subsequently confirmed through small bowel biopsies. She initiated treatment with ceftriaxone and experienced rapid clinical improvement. WD poses a diagnostic challenge. The signs and symptoms are often nonspecific and can result in misdiagnosis as a rheumatic or neoplastic disease. The presentation with DVT, while unusual, has been reported as a manifestation of WD., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Porteiro et al.)

Importance: Enterococcus faecalis causes life-threatening invasive hospital- and community-associated infections that are usually associated with multidrug resistance globally. Although E. faecalis infections cause opportunistic infections typically associated with antibiotic use, immunocompromised immune status, and other factors, they also possess an arsenal of virulence factors crucial for their pathogenicity. Despite this, the relative contribution of these virulence factors and other genetic changes to the pathogenicity of E. faecalis strains remain poorly understood. Here, we investigated whether specific genomic changes in the genome of E. faecalis isolates influence its pathogenicity-infection of hospitalized and nonhospitalized individuals and the propensity to cause extraintestinal infection and intestinal colonization. Our findings indicate that E. faecalis genetics partially influence the infection of hospitalized and nonhospitalized individuals and the propensity to cause extraintestinal infection, possibly due to gut-to-bloodstream translocation, highlighting the potential substantial role of host and environmental factors, including gut microbiota, on the opportunistic pathogenic lifestyle of this bacterium., Competing Interests: The authors declare no conflict of interest.

Ovarian cancer is one of the most common gynecological malignancies and has low survival rates. One of the main determinants of this unfavorable prognosis is the high rate of peritoneal metastasis at diagnosis, closely related to its morbidity and mortality. The mechanism underlying peritoneal carcinomatosis is not clearly defined, but a clear preference for omental spread has been described. Growing evidence suggests that adipose tissue plays a role in promoting cancer onset and progression. Moreover, obesity can lead to changes in the original functions of adipocytes, resulting in metabolic and inflammatory changes in the adipose tissue microenvironment, potentially increasing the risk of tumor growth. However, the specific roles of adipocytes in ovarian cancer have not yet been fully elucidated. Due to the undeniable link between obesity and cancer, the adipose tissue microenvironment could also present a promising therapeutic target that warrants further research. This review discusses the complex relationship between ovarian cancer and the adipose tissue microenvironment.

The use of antibiotics in animal production is linked to the emergence and spread of antibiotic-resistant bacteria, a threat to animal, environmental and human health. Copper (Cu) is an essential element in poultry diets and an alternative to antibiotics, supplementing inorganic or organic trace mineral feeds (ITMF/OTMF). However, its contribution to select multidrug-resistant (MDR) and Cu tolerant Enterococcus, a bacteria with a human-animal-environment-food interface, remains uncertain. We evaluated whether feeding chickens with Cu-ITMF or Cu-OTMF contributes to the selection of Cu tolerant and MDR Enterococcus from rearing to slaughter. Animal faeces [2-3-days-old (n = 18); pre-slaughter (n = 16)] and their meat (n = 18), drinking-water (n = 14) and feed (n = 18) from seven intensive farms with ITMF and OTMF flocks (10.000-64.000 animals each; 2019-2020; Portugal) were sampled. Enterococcus were studied by cultural, molecular and whole-genome sequencing methods and Cu concentrations by ICP-MS. Enterococcus (n = 477; 60 % MDR) were identified in 80 % of the samples, with >50 % carrying isolates resistant to tetracycline, quinupristin-dalfopristin, erythromycin, streptomycin, ampicillin or ciprofloxacin. Enterococcus with Cu tolerance genes, especially tcrB ± cueO, were mainly found in faeces (85 %; E. faecium/E. lactis) of ITMF/OTMF flocks. Similar occurrence and load of tcrB ± cueO Enterococcus in the faeces was detected throughout the chickens' lifespan in the ITMF/OTMF flocks, decreasing in meat. Most of the polyclonal MDR Enterococcus population carrying tcrB ± cueO or only cueO (67 %) showed a wild-type phenotype (MIC CuSO4  ≤ 12 mM) linked to absence of tcrYAZB or truncated variants, also detected in 85 % of Enterococcus public genomes from poultry. Finally, < 65 μg/g Cu was found in all faecal and meat samples. In conclusion, Cu present in ITMF/OTMF is not selecting Cu tolerant and MDR Enterococcus during chickens' lifespan. However, more studies are needed to assess the minimum concentration of Cu required for MDR bacterial selection and horizontal transfer of antibiotic resistance genes, which would support sustainable practices mitigating antibiotic resistance spread in animal production and the environment beyond., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The first medical oncology appointment serves as a platform for patients to comprehend their diagnosis and prognostic implications of cancer. This study aimed to determine patients' communication preferences during their first medical oncology appointment and to assess the disparities between patients' preferences and perceptions., Methods: A total of 169 cancer patients participated by completing the Communication in First Medical Oncology Appointment Questionnaire (C-FAQ), a two-section questionnaire designed to assess patients' preferences and perceptions regarding Content (information provided and its extent), Facilitation (timing and location of information delivery), and Support (emotional support) during their first medical oncology appointment. A comparative analysis was conducted to assess the variations between preferences and perceptions., Results: Content emerged as the most significant dimension compared to Facilitation and Support. The physician's knowledge, honesty, and ability to provide clear information were considered the most important attributes. Patients evaluated most of their preferences as "very important". Patients' perception of the communication dimensions present during their appointment was below preferences for 11 items, indicating significant discrepancies in clinical practice., Conclusions: Patients highly valued their preferences concerning Content, Facilitation, and Support dimensions of communication. However, patient preferences were more prominently oriented towards the Content dimension. The discrepancies between preferences and perceptions should be viewed as an opportunity for enhancing communication skills through training., (© 2023 The Authors. Psycho-Oncology published by John Wiley & Sons Ltd.)

The relevance of postmortem microbiological examinations has been controversial for decades, but the boom in advanced sequencing techniques over the last decade is increasingly demonstrating their usefulness, namely for the estimation of the postmortem interval. This comprehensive review aims to present the current knowledge about the human postmortem microbiome (the necrobiome), highlighting the main factors influencing this complex process and discussing the principal applications in the field of forensic sciences. Several limitations still hindering the implementation of forensic microbiology, such as small-scale studies, the lack of a universal/harmonized workflow for DNA extraction and sequencing technology, variability in the human microbiome, and limited access to human cadavers, are discussed. Future research in the field should focus on identifying stable biomarkers within the dominant Bacillota and Pseudomonadota phyla, which are prevalent during postmortem periods and for which standardization, method consolidation, and establishment of a forensic microbial bank are crucial for consistency and comparability. Given the complexity of identifying unique postmortem microbial signatures for robust databases, a promising future approach may involve deepening our understanding of specific bacterial species/strains that can serve as reliable postmortem interval indicators during the process of body decomposition. Microorganisms might have the potential to complement routine forensic tests in judicial processes, requiring robust investigations and machine-learning models to bridge knowledge gaps and adhere to Locard's principle of trace evidence.

Quaternary ammonium compounds (QACs) have been extensively used in the community, healthcare facilities, and food chain, in concentrations between 20 and 30,000 mg/L. Enterococcus faecalis and Enterococcus faecium are ubiquitous in these settings and are recognized as nosocomial pathogens worldwide, but QACs' activity against strains from diverse epidemiological and genomic backgrounds remained largely unexplored. We evaluated the role of Enterococcus isolates from different sources, years, and clonal lineages as hosts of QACs tolerance genes and their susceptibility to QACs in optimal, single-stress and cross-stress growth conditions. Only 1% of the Enterococcus isolates included in this study and 0.5% of publicly available Enterococcus genomes carried qacA/B, qacC, qacG, qacJ, qacZ, qrg, bcrABC or oqxAB genes, shared with >60 species of Bacillota, Pseudomonadota, Actinomycetota, or Spirochaetota. These genes were generally found within close proximity of antibiotics and/or metals resistance genes. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of benzalkonium chloride (BC) and didecyldimethylammonium chloride ranged between 0.5 and 4 mg/L (microdilution: 37°C/20 h/pH = 7/aerobiosis) for 210 E. faecalis and E. faecium isolates (two isolates carrying qacZ ). Modified growth conditions (e.g., 22°C/pH = 5) increased MIC BC /MBC BC (maximum of eightfold and MBC BC = 16 mg/L) and changed bacterial growth kinetics under BC toward later stationary phases in both species, including in isolates without QACs tolerance genes. In conclusion, Enterococcus are susceptible to in-use QACs concentrations and rarely carry QACs tolerance genes. However, their potential gene exchange with different microbiota, the decreased susceptibility to QACs under specific environmental conditions, and the presence of subinhibitory QACs concentrations in various settings may contribute to the selection of particular strains and, thus, require a One Health strategy to maintain QACs effectiveness. IMPORTANCE Despite the increasing use of quaternary ammonium compounds (QACs), the susceptibility of pathogens to these antimicrobials remains largely unknown. Enterococcus faecium and Enterococcus faecalis are susceptible to in-use QACs concentrations and are not main hosts of QACs tolerance genes but participate in gene transfer pathways with diverse bacterial taxa exposed to these biocides. Moreover, QACs tolerance genes often share the same genetic contexts with antibiotics and/or metals resistance genes, raising concerns about potential co-selection events. E. faecium and E. faecalis showed increased tolerance to benzalkonium chloride under specific environmental conditions (22°C, pH = 5), suggesting that strains might be selected in settings where they occur along with subinhibitory QACs concentrations. Transcriptomic studies investigating the cellular mechanisms of Enterococcus adaptation to QACs tolerance, along with longitudinal metadata analysis of tolerant populations dynamics under the influence of diverse environmental factors, are essential and should be prioritized within a One Health strategy.

Acid stress poses a common challenge for bacteria in diverse environments by the presence of inorganic (e.g., mammals' stomach) or organic acids (e.g., feed additives; acid-based disinfectants). Limited knowledge exists regarding acid-tolerant strains of specific serotypes, clonal lineages, or sources in human/animal pathogens: namely, non-typhoidal Salmonella enterica (NTS) and Enterococcus faecium (Efm). This study evaluated the acidic pH (Mueller-Hinton acidified with HCl) and peracetic acid (PAA) susceptibility of Efm ( n = 72) and NTS ( n = 60) from diverse epidemiological/genetic backgrounds and with multiple antibiotic resistance profiles. Efm minimum growth/survival pH was 4.5-5.0/3.0-4.0, and for NTS it was 4.0-4.5/3.5-4.0. Efm distribution among acidic pH values showed that only isolates of clade-non-A1 (non-hospital associated) or the food chain were more tolerant to acidic pH compared to clade-A1 (hospital-associated clones) or clinical isolates ( p < 0.05). In the case of NTS, multidrug-resistant (MDR) isolates survived better in acidic pH ( p < 0.05). The PAA MIC/MBC for Efm was 70-120/80-150 mg/L, and for NTS, it was 50-70/60-100 mg/L. The distribution of Efm among PAA concentrations showed that clade-A1 or MDR strains exhibited higher tolerance than clade-non-A1 or non-MDR ones ( p < 0.05). NTS distribution also showed higher tolerance to PAA among non-MDR and clinical isolates than food chain ones ( p < 0.05) but there were no differences among different serogroups. This unique study identifies specific NTS or Efm populations more tolerant to acidic pH or PAA, emphasizing the need for further research to tailor controlled measures of public health and food safety within a One Health framework.

The physical and chemical characteristics of the soil can influence plant growth. When sewage sludge (SS) is applied as a soil fertilizer, the accumulation of non-essential elements contained in it can be toxic for plants. The aim of this study was to understand the effect of SS dosage on the cell cycle of Lactuca sativa L. meristematic cells and on the initial growth of L. sativa and Passiflora alata Curtis. Nine concentrations of SS + distilled water (mg dm -3 ) corresponding to 0, 20, 40, 60, 80, 120, 160, 320, and 520 t ha -1 were tested in four replicates of 25 seeds. Chemical analysis showed an increase in pH of the sludge from 0 to 80 t ha -1 SS followed by its stabilization thereafter. The highest electrical conductivity was observed at 520 t ha -1 SS. SS negatively affected the germination and initial growth of seedlings from P. alata and L. sativa. Cytogenetic analysis on 6000 L. sativa meristematic cells for each treatment revealed that SS could adversely affect the genetic stability of this species. SS concentrations above 120 t ha -1 adversely affected the germination and early seedling growth of L. sativa and P. alata. At high concentrations (120 t ha -1 ), SS induced genetic lesions in L. sativa, along with chromosomal and nuclear alterations., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Leptospirosis has been described as a disease neglected worldwide. Affecting humans and animals, the disease is often related to poor environmental conditions such as lack of sanitation and presence of synanthropic rodents. Despite being considered as a One Health issue, no study has focused on comparing owner-dog seroprevalence between islands and seashore mainland. Accordingly, the present study assessed anti- Leptospira spp. antibodies by applying microscopic agglutination test (MAT) methods to Leptospira and assessing associated risk factors via univariate and multivariate logistic regression analysis of owners and their dogs in islands and seashore mainland of southern Brazil. No anti- Leptospira spp. Seropositivity was found in 330 owner serum samples, while dogs presented an overall seroprevalence of 5.9%. All seropositive dogs reacted to serogroups of Leptospira interrogans , including 66.7% of Pyrogenes, 44.4% Canicola, 22.2% Icterohaemorrhagiae, 16.7% Australis; six reacted to more than one serogroup. No association was found among seropositivity and epidemiological variables, except that neighborhood dogs were less likely to be seropositive. Although no seropositivity was observed in owners, seropositivity in dogs had the potential to indicate such species as being sentinels for environmental exposure and potential human risk of infection.

The aim of the present work was to modify the exuded gum of Sterculia striata tree by an amination reaction. The viscosity and zero potential of the chicha gum varied as a function of pH. The modification was confirmed by X-ray diffraction (XRD), infrared spectroscopy (FTIR), size exclusion chromatography (SEC), zeta potential, thermogravimetric analysis (TG), and differential scanning calorimetry (DSC). Furthermore, the chemical modification changed the molar mass and surface charge of the chicha gum. In addition, the gums were used in tests for ex vivo mucoadhesion strength, antibacterial activity against the standard strain of Staphylococcus aureus (ATCC 25923), inhibitory activity of α-glucosidase, antioxidant capacity, and viability of Caco-2 cells. Through these tests, it was found that amination caused an increase in the mucoadhesive and inhibitory activity of chicha gum against the bacterium Staphylococcus aureus. In addition, the gums (pure and modified) showed antioxidant capacity and an inhibitory effect against the α-glucosidase enzyme and did not show cytotoxic potential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Poultry meat has been a vehicle of antibiotic resistant bacteria and genes. Yet, the diversity of selective pressures associated with their maintenance in the poultry-production chain remains poorly explored. We evaluated the susceptibility of Enterococcus spp. from chicken meat collected 20 years apart to antibiotics, metals, acidic pH and peracetic acid-PAA. Contemporary chicken-meat samples (n = 53 batches, each including a pool of neck skin from 10 single carcasses) were collected in a slaughterhouse facility using PAA as disinfectant (March-August 2018, North of Portugal). Broilers were raised in intensive farms (n = 29) using CuSO 4 and organic acids as feed additives. Data were compared with that of 67 samples recovered in the same region during 1999-2001. All 2018 samples had multidrug resistant-MDR isolates, with >45 % carrying Enterococcus faecalis, Enterococcus faecium or Enterococcus gallinarum resistant to tetracycline, erythromycin, ampicillin, quinupristin-dalfopristin, ciprofloxacin, chloramphenicol or aminoglycosides. Resistance rates were similar (P > 0.05) to those of 1999-2001 samples for all but five antibiotics. The decrease of samples carrying vancomycin-resistant isolates from 46 % to 0 % between 1999-2001 and 2018 was the most striking difference. Isolates from both periods were similarly susceptible to acid pH [minimum-growth pH (4.5-5.0), minimum-survival pH (3.0-4.0)] and to PAA (MIC 90  = 100-120 mg/L/MBC 90  = 140-160 mg/L; below concentrations used in slaughterhouse). Copper tolerance genes (tcrB and/or cueO) were respectively detected in 21 % and 4 % of 2018 and 1999-2001 samples. The tcrB gene was only detected in E. faecalis (MIC CuSO4  > 12 mM), and their genomes were compared with other international ones of chicken origin (PATRIC database), revealing a polyclonal population and a plasmid or chromosomal location for tcrB. The tcrB plasmids shared diverse genetic modules, including multiple antimicrobial resistance genes (e.g. to tetracyclines, chloramphenicol, macrolide-lincosamide-streptogramin B-MLS B , aminoglycosides, bacitracin, coccidiostats). When in chromosome, the tcrB gene was co-located closely to merA (mercury) genes. Chicken meat remains an important vehicle of MDR Enterococcus spp. able to survive under diverse stresses (e.g. copper, acid) potentially contributing to these bacteria maintenance and flux among animal-environment-humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

It was recently proposed that Enterococcus faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis. Our goals were to develop a PCR assay to rapidly differentiate these species and to discuss the main phenotypic and genotypic differences from a clinical perspective. The pan-genome of 512 genomes of E. faecium and E. lactis strains was analyzed to assess diversity in genes between the two species. Sequences were aligned to find the best candidate gene for designing species-specific primers, and their accuracy was tested with a collection of 382 enterococci. E. lactis isolates from clinical origins were further characterized by whole-genome sequencing (Illumina). Pan-genome analysis resulted in 12 gene variants, with gene gluP (rhomboid protease) being selected as the candidate for species differentiation. The nucleotide sequence of gluP diverged by 90 to 92% between sets, which allowed species identification through PCR with 100% specificity and no cross-reactivity. E. lactis strains were greatly pan-susceptible and not host specific. Hospital E. lactis isolates were susceptible to clinically relevant antibiotics, lacked infection-associated virulence markers, and were associated with patients presenting risk factors for enhanced bacterial translocation. Here, we propose a PCR-based assay using gluP for easy routine differentiation between E. faecium and E. lactis that could be implemented in different public health contexts. We further suggest that E. lactis, a dominant human gut species, can cross the gut barrier in severely ill, immunodeficient, and surgical patients. Knowing that bacterial translocation may be a sepsis promoter, the relevance of infections caused by E. lactis strains, even if they are pan-susceptible, should be explored. IMPORTANCE Enterococcus faecium is a WHO priority pathogen that causes severe and hard-to-treat human infections. It was recently proposed that E. faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis; therefore, some of the human infections occurring globally are being misidentified. In this work, we developed a PCR-based rapid identification method for the differentiation of E. faecium and E. lactis and discussed the main phenotypic and genotypic differences of these species from a clinical perspective. We identified the gluP gene as the best candidate, based on the phylogenomic analysis of 512 published pan-genomes, and validated the PCR assay with a comprehensive collection of 382 enterococci obtained from different sources. Further detailed analysis of clinical E. lactis strains showed that they are highly susceptible to antibiotics and lack the typical virulence markers of E. faecium but are able to cause severe human infections in immunosuppressed patients, possibly in part due to gut barrier translocation.

The tumor microenvironment is crucial in tumourigenesis, response to therapy, and elimination of tumor cells. Tumor-infiltrating lymphocytes (TILs) promote the host immune response and are associated with a better prognosis in colorectal cancer (CRC). This multicentric retrospective study evaluated the relationship between the presence and intensity of TILs and survival outcomes. A total of 651 patients from four Portuguese oncological centers who underwent surgical resection for stages II or III colorectal adenocarcinoma between 2016 and 2019 were included in this study. The mean age of the study population was 70 years; 58.2% were males. The median overall survival was 58.03 ± 1.29 months (95% confidence interval (CI) 55.50 - 60.56), and the median disease-free survival (DFS) was 53.02 ± 1.39 months (95% CI 50.29 - 55.74). Patients with high infiltrate (including those with moderate, abundant, or Crohn-like infiltrate) had significantly longer DFS i.e., 58.48 ± 1.84 months (95% CI 54.87 - 62.09 months) vs 49.22 ± 1.75 months (95% CI 45.79 - 52.64 months) in the group with absent or minimal infiltrate; p = 0.003. Assessing the side of the tumor, high infiltrate was associated with higher DFS (59.86 ± 2.36 months (95% CI 55.23 - 64.50 months) vs 49.60 ± 2.40 months (95% CI 44.90 - 54.29 months), p = 0.011). This work reinforces the importance of research into possible prognostic and predictive factors in patients with CRC., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2022, Vitorino et al.)

Non-fatal cases of rickettsial infection with different clinical features than the classic BSF (Brazilian Spotted Fever) have been reported in seashore areas of Paraná state, southern Brazil. In addition, Amblyomma ovale tick infected by Rickettsia parkeri strain Atlantic rainforest has been also described in this area. Accordingly, the aim of this study was to investigate the occurrence of anti-Rickettsia spp. antibodies in human and dog populations, and Rickettsia spp. infection in ticks from oceanic islands and seashore mainland cities of southern Brazil. Serum samples were collected from 328 persons and their 282 dogs from three islands and two seashore mainland cities. A total of 211 ticks were collected from dogs, identified as A. ovale and R. sanguineus. In overall, 40 of 328 (12.2%) human samples were seropositive for Rickettsia spp., including 21 of 190 (11.1%) on islands and 19 of 138 (13.7%) on seashore mainland, and 62 of 282 (22.0%) dog samples, including 31 of 153 (20.3%) on islands and 31 of 129 (24.0%) in seashore mainland areas. In overall, nine of 82 (11.0%) ticks were positive to real-time PCR assay targeting a fragment of the rickettsial gltA gene, including two of 64 (3.1%) Rickettsia sanguineus and seven of 18 (38.9%) A. ovale, of which four were infected with the R. parkeri strain Atlantic rainforest. Despite no association between risk factors and Rickettsia spp. seropositivity was found in human beings, access to natural areas (p = .011) and tick infestation (p = .004) was significantly associated to dog seropositivity. The serological and molecular findings herein have confirmed previous tick and clinical case reports and enlarged the geographical occurrence of A. ovale infected by R. parkeri strain Atlantic rainforest in oceanic islands and seashore mainland cities of Paraná State, indicating a new likely transmission area of this new rickettsial infection in human beings and dogs of southern Brazil., (© 2022 Wiley-VCH GmbH.)

Although toxoplasmosis has been considered among the most neglected zoonoses worldwide, no study has focused on the frequency and associated risk factors of owners and their dogs living on an island and mainland seashore areas. Accordingly, anti- Toxoplasma gondii IgG antibodies were screened by indirect fluorescent antibody test (IFAT) in owners and dogs from three oceanic islands and two nearby mainland harbor areas, with associated risk factors for toxoplasmosis assessed by univariable and multivariable logistic regression models. Overall, anti- T. gondii seropositivity was observed in 59/328 (18.0%) owners and 66/283 (23.3%) dogs, with no statistical difference between owners ( p = 0.360) and dogs ( p = 0.655) from islands and mainland areas. Consumption of local water springs ( p = 0.016; OR = 2.11) was an associated risk factor for T. gondii seropositivity, and owners with the habit of spring water intake were twice more likely seropositive ( p = 0.014; OR = 2.14). Presence of anti- T. gondii antibodies in dogs was associated with seropositive owners ( p = 0.008; OR = 2.81), household consumption of beef meat ( p = 0.042; OR = 1.7) and chicken ( p = 0.026; OR = 2.9). Despite being lower than the worldwide prevalence, toxoplasmosis seropositivity in owners and their dogs in southern Brazil was influenced by the positive owner, water source, and meat consumption, and not by inhabiting islands or seashore mainland areas, presence of dogs, cats, or both. In addition, drinking water quality should always be considered a critical risk factor for toxoplasmosis on islands.

Chlorhexidine (CHX) is widely used to control the spread of pathogens (e.g., human/animal clinical settings, ambulatory care, food industry). Enterococcus faecalis, a major nosocomial pathogen, is broadly distributed in diverse hosts and environments facilitating its exposure to CHX over the years. Nevertheless, CHX activity against E. faecalis is understudied. Our goal was to assess CHX activity and the variability of ChlR-EfrEF proteins (associated with CHX tolerance) among 673 field isolates and 1,784 E. faecalis genomes from the PATRIC database from different sources, time spans, clonal lineages, and antibiotic-resistance profiles. The CHX MIC (MIC CHX ) and minimum bactericidal concentration (MBC CHX ) against E. faecalis presented normal distributions (0.5 to 64 mg/L). However, more CHX-tolerant isolates were detected in the food chain and recent human infections, suggesting an adaptability of E. faecalis populations in settings where CHX is heavily used. Heterogeneity in ChlR-EfrEF sequences was identified, with isolates harboring incomplete ChlR-EfrEF proteins, particularly the EfrE identified in the ST40 clonal lineage, showing low MIC CHX (≤1mg/L). Distinct ST40-E. faecalis subpopulations carrying truncated and nontruncated EfrE were detected, with the former being predominant in human isolates. This study provides a new insight about CHX susceptibility and ChlR-EfrEF variability within diverse E. faecalis populations. The MIC CHX /MBC CHX of more tolerant E. faecalis (MIC CHX = 8 mg/L; MBC CHX = 64 mg/L) remain lower than in-use concentrations of CHX (≥500 mg/L). However, increased CHX use, combined with concentration gradients occurring in diverse environments, potentially selecting multidrug-resistant strains with different CHX susceptibilities, signals the importance of monitoring the trends of E. faecalis CHX tolerance within a One Health approach. IMPORTANCE Chlorhexidine (CHX) is a disinfectant and antiseptic used since the 1950s and included in the World Health Organization's list of essential medicines. It has been widely applied in hospitals, the community, the food industry, animal husbandry and pets. CHX tolerance in Enterococcus faecalis, a ubiquitous bacterium and one of the leading causes of human hospital-acquired infections, remains underexplored. Our study provides novel and comprehensive insights about CHX susceptibility within the E. faecalis population structure context, revealing more CHX-tolerant subpopulations from the food chain and recent human infections. We further show a detailed analysis of the genetic diversity of the efrEF operon (previously associated with E. faecalis CHX tolerance) and its correlation with CHX phenotypes. The recent strains with a higher tolerance to CHX and the multiple sources where bacteria are exposed to this biocide alert us to the need for the continuous monitoring of E. faecalis adaptation toward CHX tolerance within a One Health approach.

Aim: To assess the bioequivalence of two sodium valproate formulations in healthy subjects of both sexes., Materials and Methods: The study was conducted using an open, randomized, two-period crossover design with a 2-week washout interval. Plasma samples were obtained over a 96-hour period. Plasma concentrations of valproate were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC/MS) with negative ion electrospray ionization. From the sodium valproate plasma concentration vs. time curves, the following pharmacokinetic parameters were obtained C max , AUC, t max , Ke, and T 1/2 ., Results: The geometric mean with corresponding 90% confidence interval for test/reference percent ratios were 104.43% (90% CI 100.42 - 108.61%) for C max , 98.11% (90% CI = 94.66 - 101.70%) for AUC last , and 96.71% (90% CI = 92.97 - 100.60%) for AUC 0-inf ., Conclusion: Since the 90% CI for C max and AUC last ratios were all inside the 80 - 125% interval proposed by the US Food and Drug Administration Agency (FDA), it was concluded that the new sodium valproate formulation (epilenil 500-mg coated tablet) without food elaborated by Biolab Sanus Farmaceutica Ltda is bioequivalent to depakene formulation for both the rate and the extent of absorption.

Multidrug-resistant (MDR) Enterococcus faecium ( Efm ) infections continue to increase worldwide, although epidemiological studies remain scarce in lower middle-income countries. We aimed to explore which strains circulate in E. faecium causing human infections in Tunisian healthcare institutions in order to compare them with strains from non-human sources of the same country and finally to position them within the global E. faecium epidemiology by genomic analysis. Antibiotic susceptibility testing was performed and transfer of vancomycin- vanA and ampicillin- pbp5 resistance was performed by conjugation. WGS-Illumina was performed on Tunisian strains, and these genomes were compared with Efm genomes from other regions present in the GenBank/NCBI database ( n = 10,701 Efm genomes available May 2021). A comparison of phenotypes with those predicted by the recent ResFinder 4.1-CGE webtool unveiled a concordance of 88%, with discordant cases being discussed. cgMLST revealed three clusters [ST18/CT222 ( n = 13), ST17/CT948 strains ( n = 6), and ST203/CT184 ( n = 3)], including isolates from clinical, healthy-human, retail meat, and/or environmental sources in different countries over large time spans (10-12 years). Isolates within each cluster showed similar antibiotic resistance, bacteriocin, and virulence genetic patterns. pbp5 -AmpR was transferred by VanA-AmpR-ST80 (clinical) and AmpR-ST17- Efm (bovine meat). Identical chromosomal pbp5 -platforms carrying metabolic/virulence genes were identified between ST17/ST18 strains of clinical, farm animal, and retail meat sources. The overall results emphasize the role of high-resolution genotyping as provided by WGS in depicting the dispersal of MDR- Efm strains carrying relevant adaptive traits across different hosts/regions and the need of a One Health task force to curtail their spread.

Toxocariasis, caused by Toxocara spp. nematodes, is among the top 5 neglected parasitic diseases worldwide; however, no comprehensive study to date has serologically compared infections in people and their dogs and environmentally contaminated soil or sand of mainland and island locations. Accordingly, this study aimed to assess the seroprevalence of anti- Toxocara antibodies in traditional human seashore populations, the presence of eggs in dogs' feces and hair, and the presence of eggs in environmental samples from islands compared to the adjacent mainland of southern Brazil. Overall, 212/328 (64.6%) people were positive for Toxocara spp. antibodies, including 125/190 (65.8%) island and 87/138 (63.0%) mainland residents. For dog samples, 12/115 (10.43%) were positive for the presence of Toxocara spp. eggs, all from dogs living in islands, and 22/104 (21.15%) dog hair samples contained eggs of Toxocara spp. Environmental contamination with Toxocara spp. eggs was observed in 50/130 (38.46%) samples from all sampled sites. No significant association was found between risk factors (age, sex, educational level, monthly income, owning dogs or cats, ingestion of treated water, and consumption of raw or uncooked meat) and Toxocara spp. seropositivity. The present study is the first concurrent report on people, their dogs, and environmental contamination of Toxocara spp. The high prevalence we observed in the seashore populations of both in island and mainland areas may be caused by exposure to contaminated sand and climatic factors favoring frequent exposure to Toxocara spp. In conclusion, seashore lifestyle and living conditions of both island and mainland areas may have predisposed higher contact with infected pets and contaminated soil, favoring the high prevalence of toxocariasis., Competing Interests: We declare that we do not have any conflict of interest associated with manuscript., (© 2021 Published by Elsevier B.V.)

The increase in the number of pets in recent years has been followed by an exponential growth of the industrial pet food sector, which has been accompanied by new food safety risks, namely antibiotic resistance. The aim of this study was to investigate whether dog food commercially available in Portugal is a reservoir of clinically-relevant antibiotic resistant Enterococcus. Fifty-five samples (25 brands; 22 wet, 14 raw frozen, 8 dry, 7 treats and 4 semi-wet) were collected on 9 commercial surfaces in the Porto region (September 2019 to January 2020). Most samples were obtained from brands that are commercialized worldwide (n = 21/25). Sample (25 g) processing included pre-enrichment and enrichment steps in culture media without/with 3 antibiotics, and then plating into selective media without/with the same antibiotics. Susceptibility was studied for 13 antibiotics (disk diffusion; Etest; microdilution) according to EUCAST/CLSI. Clinically-relevant species (E. faecium and E. faecalis), antibiotic resistance (vanA, vanB, optrA, poxtA) and virulence (e.g. ptsD, esp, sgrA) genes were identified by PCR. Other species of Enterococcus were identified by MALDI-TOF MS. Clonality was established by MLST in selected isolates. Enterococcus (n = 184; 7 species; >85% E. faecium and E. faecalis) were detected in 30 samples (54%) of different types (14 raw, 16 heat treated-7 dry, 6 wet, 3 treats). E. faecium and E. faecalis were more frequent in dry and wet samples, respectively. More than 40% of enterococci recovered were resistant to erythromycin, tetracycline, quinupristin-dalfopristin, streptomycin, gentamicin, chloramphenicol, ampicillin or ciprofloxacin, and to a lesser extent to linezolid (23%; optrA, poxtA) or vancomycin and teicoplanin (2% each; vanA). Multidrug-resistant isolates (31%), including to vancomycin and linezolid, were obtained mostly from raw foods, although also detected in wet samples or treats, and mainly from culture media supplemented with antibiotics. Samples subjected to thermal treatment mostly carried non-MDR isolates. The variety of clones observed included strains previously identified in hospitalized patients (E. faecium ST17/ST80; E. faecalis ST40), farm animals, pets and environmental strains. This study shows that dog food from international brands is a vehicle of clinically-relevant enterococci carrying resistance to last resort antibiotics and relevant virulence genes, thus positioning pet food as an important source of antibiotic resistance spread within the One Health context. The high incidence of Enterococcus in a variety of dog food samples indicates the need to review selection of raw materials, manufacturing and hygiene practices in an emerging food sector growing worldwide., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The sediment contamination by trace metals in coastal aquatic ecosystems is a worldwide environmental problem, since metals can be toxic, persistent, and bioaccumulated. In case of natural events, such as storms, or anthropogenic activities, like dredging, the sediment resuspension to the water column occurs and can solubilize metals, probably increasing their bioavailability and consequently the risk to aquatic life. This study evaluated the bioavailability on reactive trace metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) in estuarine sediments from Iguaçu and Meriti Rivers, both in the drainage basin of Guanabara Bay (Rio de Janeiro, Brazil). Additionally, a discussion about the anthropogenic interference throughout time of six short sediments cores, calculating three different indexes (contamination factors, CF; potential ecological risk index for a single heavy metal, E i f for short; potential ecological risk, PERI) was performed. It was considered as reactive phase, the metal concentrations obtained using a weak acid extraction (in HCl 1 mol L -1 solution). Zn presented high concentrations after resuspension, being above effect range medium (ERM) (52.81 to 1337.4 mg kg -1 ). The CF indicated very high contamination degree for Cu (14.62 to 17.96) and Zn (27.80 to 35.85) for both rivers. The E i f for short presented higher risk to Cu and Zn for Iguaçu and Meriti rivers. PERI index classified Meriti River samples as severely contaminated (238.10 to 351.62)., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)