Escherichia coli isolated from pyometra and cystitis in the same animal exhibit a wide phenotypic similarity.

AIMS: Pyometra and cystitis caused by Escherichia coli are common diseases identified in canine or feline females. The origin of pyometra infection remains uncertain, and effective prevention strategies for this disease are still unknown. This study aimed to provide a phenotypic characterization, including antimicrobial resistance and virulence profiles, of endometrial pathogenic (EnPEC) and uropathogenic (UPEC) E. coli strains isolated simultaneously from the same animal. METHODS AND RESULTS: Sixteen E. coli strains, from eight different animals, were analyzed in this study. The antimicrobial susceptibility profile of EnPEC and UPEC strains was determined using the disc diffusion method, which showed a similar susceptibility profile among strains (EnPEC and UPEC) from the same animal. The virulence profile of the strains was assessed through biofilm formation, as well as serum resistance abilities. EnPEC and UPEC strains from the same animal exhibited slight variations in their virulence and antimicrobial resistance capabilities. Overall, most of the strain pairs showed a high similarity in their ability to establish biofilms and survive in serum complement activity. CONCLUSIONS: Overall, strains of E. coli isolated from both pyometra and cystitis in the same animal, despite presenting distinct clinical diseases, exhibit a wide phenotypic similarity, suggesting a common origin for the strains.

Comparative genetic and pathogenic approaches of Escherichia coli isolated simultaneously from pyometra and urine of bitches.

Escherichia coli (E. coli) are widely related to pyometra and cystitis in dogs, and these infections can occur simultaneously. The goal of this study was to determine genetic and pathogenic insights of 14 E. coli isolated simultaneously from pyometra content and bladder urine of seven bitches. To achieve this, in silico and in vitro comparative analyses were conducted. Whole-genome comparisons demonstrated that E. coli isolated from pyometra and urine of the same animal were predominantly genetic extraintestinal E. coli clones belonging to the same Sequence Type and phylogroup. The E. coli clones identified in this study included ST372, ST457, ST12, ST127, ST646, and ST961. Five isolates (35.7%) belonged to the ST12 complex. Except for two E. coli, all other isolates belonged to the B2 Clermont phylogroup. Interestingly, some genomes of E. coli from urine carried more virulence genes than those E. coli from pyometra. Both pyometra and urine E. coli isolates demonstrated a strong affinity for adhering to HeLa and T24 cells, with a low affinity for invading them. However, certain isolates from urine exhibited a greater tendency to adhere to T24 cells in qualitative and quantitative assays compared to isolates from pyometra. In conclusion, this study revealed the high genomic similarity between pyometra and urine E. coli isolates, as well as the virulent capacity of both to colonize endometrial and urothelial cells. The findings of this study underscore the importance of concurrently managing both infections clinically and could potentially contribute to future resources for the prevention of cystitis and pyometra.

Alternative amplicon-PCR protocol for maximizing bacterial and fungal sequencing in low-biomass samples.

Determining bacterial and fungal communities from low-biomass samples remains a challenge for high-throughput sequencing. Due to the low microbial load and host contamination, some sites, including the female upper reproductive tract and the lower respiratory tract, were even considered sterile until recent years. Despite efforts to improve sampling and DNA isolation protocols, some samples provide insufficient microbial DNA input for library preparation and sequencing. Herein, we propose an alternative amplicon-PCR protocol to be used in bacterial and fungal sequencing in low-biomass samples, targeting 16S-rDNA and the internal transcribed spacer region (ITS), respectively. Similar to a nested-PCR, we performed two sequential PCR reactions to maximise the target amplicon. We compared metagenomic results from the original Illumina protocol (Protocol 1 - P1) and the alternative one (Protocol 2 - P2), using a mock community and clinical samples with different microbial loads. Our findings showed no significant differences in data generated by P1 and P2, indicating that the second amplification round does not bias the microbiota diversity rates. Thus, the alternative protocol can be applied for low-biomass samples when the original protocol results in spurious output, preventing library preparation and sequencing.

A cross-sectional study on Campylobacter fetus subsp. venerealis prevalence and associated factors in Brazilian southern cattle farms.

Cattle farming is a major livestock activity with economic relevance in Rio Grande do Sul (RS), Brazil. However, this activity is still considered of intermediate to low technological level, and in this region, there are few epidemiologic reports of Campylobacter fetus subsp. venerealis (Cfv), the causative agent of bovine genital campylobacteriosis (BGC). Thus, we designed a cross-sectional study to assess the prevalence and Cfv-associated factors in cattle farms in RS, Brazil. In total, 99 farms were randomly selected to participate in the survey. Preputial mucus samples from selected bulls were collected twice (within a 15-day interval) and subjected to Cfv molecular detection. A farm was considered positive when at least one sample was positive for Cfv. Our findings indicate that the farm-level Cfv prevalence in RS is 67.67%. On average, the chance of a farm using natural service to be Cfv-positive increased approximately twice compared to farms that do not use natural service. We also determined that Cfv routine tests reduce the chance of a farm being positive by 92%. Therefore, both Cfv detection tests and the reduction of natural services decrease the chance of a farm being positive for Cfv. Finally, we conclude that Cfv is widely spread in Southern Brazil cattle farms and it is urgent the implementation of control measures to reduce Cfv prevalence in the target population.

Evaluating the potential of a new reactor configuration to enhance simultaneous organic matter and nitrogen removal in dairy wastewater treatment.

The dairy industry is a very productive sector worldwide and known for producing great volumes of wastewater that is rich in organic matter and nutrients. Apart from fat, the organic matter in such effluents is easily degradable, demanding an external carbon source for conventional denitrification. In this manner, new configurations of reactors promoting a suitable environment for more sustainable nitrogen removal are beyond required-they are paramount. Therefore, the performance of a structured-bed hybrid baffled reactor (SBHBR) with anaerobic and oxic/anoxic chambers was designed and assessed for treating different dairy wastewaters. A combination of baffled and biofilm-structured systems under intermittent aeration was the solution proposed to obtain a new method for nitrogen removal under low COD/TN ratios. The COD/TN ratios tested were 2.1 ± 0.6, 0.84 ± 0.5, and 0.35 ± 0.1 in the inlet of the O/A chambers for operational stages I, II, and III, respectively. The SBHBR provided COD removal efficiencies above 90% in all experimental stages. During stage III, the process had nitrification and denitrification efficiencies of 85.9 ± 17% and 85.2 ± 9%, respectively, resulting in a TN removal efficiency of 74.6 ± 14.7%. Stoichiometric calculations were used to corroborate the activity of bacteria that could perform the anammox pathways as their main mechanisms.

Enhanced biological nitrogen and phosphorus removal from sewage driven by fermented glycerol: comparative assessment between sequencing batch- and continuously fed-structured fixed bed reactor.

The nutrient biological removal from sewage, especially from anaerobic reactor effluents, still represents a major challenge in conventional sewage treatment plants. In this work, the nitrogen and phosphorus removal from anaerobic pre-treated domestic sewage in an up-flow anaerobic sludge blanket (UASB) reactor was assessed in a structured fixed bed reactor (SFBR) operated in a continuous and in a batch mode using polyurethane foam as material support for biomass and fermented glycerol as the exogenous carbon source. The SFBR was operated as a sequencing batch reactor with cycles of 90, 120, and 150 min under anaerobic, oxic, and anoxic conditions, respectively, reaching average efficiencies for total nitrogen and phosphorus removal of 88% and 56%, respectively. Fermented glycerol was added during the non-aerated periods. Under continuous feeding, the SFBR was operated with aeration/non-aeration periods of 2/1 (h) and 3/1 (h), hydraulic retention time of 12 h, and a recirculation ratio of 3. Without fermented glycerol addition, the maximum removal of total nitrogen (TN) reached 42%, while adding glycerol in the non-aerated period improved TN removal to 64.9% (2/1 h) and 69.5% (3/1 h). During continuous operation, no phosphorus removal was observed, which was released during the non-aerated period, remaining in the effluent. Optical microscopy analyses confirmed the presence of polyphosphate granules and of the phosphorus accumulating organisms in the reactor biofilm. It was concluded that the batch feeding method was determinant for phosphorus removal. The structured fixed bed reactor with polyurethane foam proved to be feasible in the removal of organic matter and nutrients remaining in the UASB reactor effluent.

Epitope identification of SARS-CoV-2 structural proteins using in silico approaches to obtain a conserved rational immunogenic peptide.

The short time between the first cases of COVID-19 and the declaration of a pandemic initiated the search for ways to stop the spread of SARS-CoV-2. There are great expectations regarding the development of effective vaccines that protect against all variants, and in the search for it, we hypothesized the obtention of a predicted rational immunogenic peptide from structural components of SARS-CoV-2 might help the vaccine research direction. In the search for a candidate of an immunogenic peptide of the SARS-CoV-2 envelope (E), membrane (M), nucleocapsid (N), or spike (S) proteins, we access the predicted sequences of each protein after the genome sequenced worldwide. We obtained the consensus amino acid sequences of about 14,441 sequences of each protein of each continent and the worldwide consensus sequence. For epitope identification and characterization from each consensus structural protein related to MHC-I or MHC-II interaction and B-cell receptor recognition, we used the IEDB reaching 68 epitopes to E, 174 to M, 245 to N, and 833 to S proteins. To select an epitope with the highest probability of binding to the MHC or BCR, all epitopes of each consensus sequence were aligned. The curation indicated 1, 4, 8, and 21 selected epitopes for E, M, N, and S proteins, respectively. Those epitopes were tested in silico for antigenicity obtaining 16 antigenic epitopes. Physicochemical properties and allergenicity evaluation of the obtained epitopes were done. Ranking the results, we obtained one epitope of each protein except for the S protein that presented two epitopes after the selection. To check the 3D position of each selected epitope in the protein structure, we used molecular homology modeling. Afterward, each selected epitope was evaluated by molecular docking to reference MHC-I or MHC-II allelic protein sequences. Taken together, the results obtained in this study showed a rational search for a putative immunogenic peptide of SARS-CoV-2 structural proteins that can improve vaccine development using in silico approaches. The epitopes selected represent the most conserved sequence of new coronavirus and may be used in a variety of vaccine development strategies since they are also presented in the described variants of SARS-CoV-2.

Co-digestion of biofuel by-products: Enhanced biofilm formation maintains high organic matter removal when methanogenesis fails.

Ethanol and biodiesel industries generate large volumes of by-products, such as vinasse and glycerol, which are suitable for biogas exploitation. This paper assessed the applicability and process performance of the anaerobic structured-bed reactor (AnSTBR) for the mesophilic (30 °C) continuous (105 days) anaerobic co-digestion of sugarcane vinasse and distilled glycerol under increasing organic loading rates (OLR) (0.5-5.0 kgCOD m-3d-1). The highest methane yield (211 NmL g-1CODrem.) and volumetric production (668 NmL L-1d-1) occurred at an OLR of 3.5 kgCOD m-3d-1. The performance of the AnSTBR showed high removal efficiencies of total COD (77.1%), carbohydrates (81.9%), and glycerol content (99.7%). Biofilm growth enhancement within the reactor offset the impairment of methanogenesis activity at high organic loads. The prompt biodegradability of glycerol reinforced the importance of gradually increase the organic load to prevent the buildup of volatile acids and maintain a stable long-term co-digestion system.

Cows' reproductive performances and parity order influences the cervicovaginal fungal community.

Fungi have already been described as etiological agents of reproductive diseases such as endometritis and infertility in cows. However, few studies have been developed to elucidate the entire cervicovaginal fungal communities in cows. Therefore, our study aimed to characterize the fungal community present in the cervix of cows with different reproductive performances. Cervicovaginal mucus was collected from 36 Angus breed cows (1.5-12 years old) on a commercial beef cattle ranch. Twenty-one cows had a history of infertility in the year prior to the collection, showing early return to estrus. Ten cows were sampled at 60-70 days postpartum being considered fertile cows. Additionally, five non-sexually active heifers were employed as control group. Ascomycota and Basidiomycota were the predominant fungal phyla in the analyzed animals. Diversity metrics of the cervicovaginal fungal community revealed statistical differences in the composition of the fungal community among infertile cows, fertile cows and non-sexually active heifers. In addition, the cervicovaginal fungal microbiota had significative increased richness and evenness in nulliparous cows and non-sexually active heifers, while in multiparous cows a decreased richness and evenness of the fungal microbiota were identified. These results provide an unprecedented understanding of the cervicovaginal fungal structure associated with infertility and parity order.

Estratégia de saúde da família e adesão ao tratamento do diabetes: fatores facilitadores / Family health strategy and diabetes treatment adherence: facilitating factors / Estrategia de salud familiar y adherencia al tratamiento de diabetes: factores facilitadores

O diabetes mellitus é um importante e crescente problema de saúde, estando entre as doenças crônicas não transmissíveis mais prevalentes no mundo. Este estudo teve como objetivo abordar a percepção dos profissionais de saúde em relação aos aspectos facilitadores da adesão ao tratamento do diabetes mellitus. Realizou-se um estudo clínico-qualitativo em formato de grupo focal, que teve como público-alvo os profissionais da Estratégia Saúde da Família (ESF) de um município de pequeno porte do Centro-Oeste de Minas Gerais. Os profissionais apontaram as seguintes práticas como favorecedoras da adesão: construção de vínculo, participação da família, utilização de linguagem acessível, estímulo à participação ativa do paciente no tratamento, realização de acolhimento, práticas que incluem a intersetorialidade, a multidisciplinaridade e a interdisciplinaridade, ações de apoio matricial, interconsultas, atendimentos compartilhados e trabalhos com grupos. Percebe-se que a adesão depende de uma série de fatores interligados, que incluem a proposta de política pública, participação da gestão municipal, organização dos serviços, formação dos profissionais e suas concepções a respeito do processo saúde-doença, concepção de clínica, dinâmica de trabalho das equipes e capacidade dos profissionais de interagirem com a população. A experiência descrita neste artigo pode servir de inspiração para melhorias nas intervenções dos profissionais de saúde na Atenção Primária à Saúde (APS).

Diabetes Mellitus is a major health issue, consisting of one the most prevalent chronic noncommunicable diseases worldwide. This study addresses the perception of health personnel regarding the facilitating aspects of diabetes mellitus treatment adherence. A clinical-qualitative study based on a focus group was carried out with professionals from the Family Health Strategy (FHS) of a small city in Minas Gerais, Brazil. Respondents pointed to the following practices as favoring adherence: bonding, family participation, use of accessible language, stimulus to the patient's active participation in the treatment, user embracement, intersectoral, multidisciplinary and interdisciplinary practices, matrix support actions, inter-consultations, shared care, and group work. Results show that adherence depends on a series of interconnected factors, which include public policy proposals, participation of municipal management, organization of services, training of professionals and their conceptions about the health-disease process, clinical concept, team dynamics, and the professionals' ability to interact with patients. The experience described here can help improve the interventions of Primary Health Care (PHC) professionals.

La diabetes mellitus es un problema de salud importante y creciente, y se encuentra entre las enfermedades crónicas no transmisibles más prevalentes en el mundo. Este estudio tuvo como objetivo abordar la percepción de los profesionales de la salud en relación a los aspectos que facilitan la adherencia al tratamiento de la diabetes mellitus. Se realizó un estudio clínico-cualitativo en formato de grupo focal, con un público objetivo de profesionales de la Estrategia de Salud Familiar (ESF) de una pequeña ciudad del centro-oeste de Minas Gerais (Brasil). Los profesionales señalaron las siguientes prácticas como favorecedoras de la adherencia: construcción del vínculo, participación familiar, uso de lenguaje accesible, fomento de la participación activa del paciente en el tratamiento, acogida, prácticas que incluyen la intersectorialidad, multidisciplinariedad e interdisciplinariedad, matriz de acciones de apoyo, interconsulta, servicios compartidos y trabajo con grupos. Se observó que la adherencia depende de una serie de factores interconectados que incluyen la propuesta de política pública, la participación de la gestión municipal, la organización de los servicios, la formación de los profesionales y sus concepciones sobre el proceso salud-enfermedad, concepción de la clínica, dinámica de trabajo de equipos y la capacidad de los profesionales para interactuar con la población. La experiencia descrita en este artículo puede servir de inspiración para mejorar las intervenciones de los profesionales sanitarios en Atención Primaria.

Campylobacter fetus in abomasal fluid from spontaneously aborted bovine and ovine fetuses

Pregnancy losses are a major concern in livestock industry due to their economic impact on producers. Campylobacter fetus subspecies fetus (Cff) and C. fetus subspecies venerealis (Cfv) are directly related to reproductive failures in ruminants. Cff colonizes the gastrointestinal tract of a wide range of hosts leading to abortion, while Cfv is restricted to genital tract being generally associated to infertility in bovine. Considering the great economic losses related to campylobacteriosis in cattle and ovine herds, this study aims to investigate the occurrence of C. fetus, considering Cff and Cfv subspecies, in bovine and ovine spontaneously aborted fetuses in state of Rio Grande do Sul, Brazil. In this study, samples of abomasal fluid collected from 30 spontaneously aborted bovine (n = 18) and ovine (n = 12) fetuses were investigated for the detection of Campylobacter fetus throughout conventional PCR. Positive fetuses for C. fetus presence were further analyzed by molecular assays for Cff and Cfv detection, in order to determine subspecies identification. When available, samples of the main organs of the thoracic and abdominal cavities, as well as the brain, skeletal muscle, eyelid, skin, and placenta were collected for further histopathological analyses and bacterial culture, aiming to assess the presence of infection lesions and pathogens in those sites, respectively. Additionally, RT-qPCR assays were also performed for the detection of ruminant pestivirus, in order to detect bovine viral diarrhea cases. Throughout the present methodology, C. fetus was detected in the abomasal fluid samples of 2 bovine fetuses, being both identified as Cfv subspecies by PCR. Histopathological analyses demonstrated that macroscopic and microscopic changes found in the Cfv-positive animals were not either specific or directly related to Campylobacter infections. Moreover, no significant bacterial growth was observed in microbiological culture from the collected tissues, and both fetuses were negative for ruminant pestivirus. Differently, there was no detection of C. fetus in any of the analyzed ovine fetuses. Considering that abortion diagnosis rates reported in cattle and sheep industry are highly variable among the published studies, and that abortion diagnoses are commonly inconclusive due to difficulties in sampling methodology and inadequate identification of the pathogen involved, it is important to investigate the etiological causes of abortion the herds for better understanding the causes of pregnancy issues and monitoring their occurrence. In addition, the absence of pathognomonic lesions in the tissues investigated in the histopathological analyses observed in this study strongly suggests that well-known etiological agents commonly associated to abortion, such as Leptospira spp., Toxoplasma spp., Chlamydia spp. and Neospora caninum, are unlikely to be the cause of infection of the analyzed fetuses. Taking this into account, the presence of C. fetus in the abomasal fluid samples from two bovine fetuses demonstrated in the present study suggests the possible association of Cfv not only with infertility, but also with cases of bovine abortion, highlighting the importance of investigating unusual causal agents of abortions in sheep and cattle. Overall, an adequate diagnosis is essential for establishing better prevention strategies to avoid the circulation of abortion-related infectious agents in the herds.(AU)

Frozen bovine preputial mucus as a suitable sample for the direct molecular diagnosis of Campylobacter fetus subsp. venerealis.

Long transportation times remain a challenge to the satisfactory diagnosis of Campylobacter fetus subsp. venerealis (Cfv). Here we demonstrated that samples of frozen bovine preputial mucus maintained at -20 °C for 10 days can be used as an alternative source for molecular diagnosis of Cfv. This approach will improve the analysis of this bacterium.

Quantificação de bactérias lácticas em produtos lácteos com potencial probiótico comercializados nos supermercados de Volta Redonda e Barra Mansa / Quantification of lactic bacteria with potential probiotic dairy products sold in supermarket of Volta Redonda and Barra Mansa

No setor lácteo, os alimentos funcionais que são benéficos à saúde atingiram um bom mercado consumidor. Um produto para ser considerado probiótico deve apresentar contagem de microrganismos maior ou igual a 1x107 células viáveis/g ou mL do produto. O objetivo do trabalho foi avaliar a contagem de bactérias lácticas viáveis em produtos comerciais que declaram a presença de microrganismos nos rótulos. Foram analisadas 24 amostras de 5 diferentes marcas de iogurtes e leites fermentados comercializadas em supermercados de Volta Redonda/RJ e de Barra Mansa/RJ. Foram realizadas a avaliação microbiológica de viabilidade das bactérias lácticas, a análise de pH e a acidez titulável. Todas as marcas avaliadas apresentaram contagens superiores a 107 UFC/mL de bactérias láticas. Os valores de pH e acidez titulável atendem os critérios legais.