Antimicrobial and antibiofilm activity of highly soluble polypyrrole against methicillin-resistant Staphylococcus aureus.

AIMS: The purpose was to evaluate the antimicrobial activity of highly soluble polypyrrole (Hs-PPy), alone or combined with oxacillin, as well as its antibiofilm potential against methicillin-resistant Staphylococcus aureus strains. Furthermore, the in silico inhibitory mechanism in efflux pumps was also investigated. METHODS AND RESULTS: Ten clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and two reference strains were used. Antimicrobial activity was determined by broth microdilution, and the combination effect with oxacillin was evaluated by the checkerboard assay. The biofilm formation capacity of MRSA and the interference of Hs-PPy were evaluated. The inhibitory action of Hs-PPy on the efflux pump was evaluated in silico through molecular docking. Hs-PPy showed activity against the isolates, with inhibitory action between 62.5 and 125 µg ml-1 and bactericidal action at 62.5 µg ml-1, as well as synergism in association with oxacillin. The isolates ranged from moderate to strong biofilm producers, and Hs-PPy interfered with the formation of this structure, but not with mature biofilm. There was no in silico interaction with the efflux protein EmrD, the closest homolog to NorA. CONCLUSIONS: Hs-PPy interferes with biofilm formation by MRSA, has synergistic potential, and is an efflux pump inhibitor.

Chimeric proteins of Mycoplasma hyopneumoniae as vaccine and preclinical model for immunological evaluation.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a primary agent of porcine enzootic pneumonia, a disease that causes significant economic losses to pig farming worldwide. Commercial vaccines induce partial protection, evidencing the need for a new vaccine against M. hyopneumoniae. In our work, three chimeric proteins were constructed, composed of potentially immunogenic domains from M. hyopneumoniae proteins. We designed three chimeric proteins (Q1, Q2, and Q3) based on bioinformatics analysis that identified five potential proteins with immunogenic potential (MHP418, MHP372, MHP199, P97, and MHP0461). The chimeric proteins were inoculated in the murine model to evaluate the immune response. The mice vaccinated with the chimeras presented IgG and IgG1 against proteins of M. hyopneumoniae. There was induction of IgG in mice immunized with Q3 starting from 30 days post-vaccination, and groups Q1 and Q2 showed induction at 45 days. Mice of the group immunized with Q3 showed the production of IgA. In addition, the mice inoculated with chimeric proteins showed a proinflammatory cytokine response; Q1 demonstrated higher levels of TNF, IL-6, IL2, and IL-17. In contrast, animals immunized with Q2 showed an increase in the concentrations of TNF, IL-6, and IL-4, whereas those immunized with Q3 exhibited an increase in the concentrations of TNF, IL-6, IL-10, and IL-4. The results of the present study indicate that these three chimeric proteins can be used in future vaccine trials with swine because of the promising antigenicity.

Red light-emitting diode therapy minimizes the functional deleterious effects of the antiretroviral ritonavir on osteoblasts in vitro.

PURPOSE: Long-term human immunodeficiency virus (HIV)-infected patients are considered at higher risk for osteoporosis. Among the various causes that lead these patients to lower bone health, there is the use of antiretroviral drugs (ARVs), especially protease inhibitors (PI), such as ritonavir (RTV). In this context, emerge the potential benefits of LED therapy, whose effects on bone cells are currently being extensively studied, showing a modulation in cell differentiation. However, it remains unclear if photobiostimulation might interfere with RTV effects on osteoblast differentiation. METHODS: In the present study, we investigated the effects of red LED (625 nm) irradiation (15 mW/cm2, 0.2 J/cm2, and 8 mW/cm2, 0.12 J/cm2) on osteoblast cell line MC3T3-E1 treated with RTV (2.5, 5, and 10 µg/mL). RESULTS: Our results indicated that red LED irradiation was able to reverse, or at least minimize, the deleterious effects of RTV on the osteoblasts. Neither the ARV treatments 5 and 10 µg/mL (104.4% and 95.01%) nor the LED protocols (100.3% and 105.7%) statistically altered cell viability, assessed by the MTT assay. Also, the alkaline phosphatase activity and mineralization showed a decrease in osteoblast activity followed by ARV exposure (39.3-73%), which was attenuated by LED in more than 70% with statistical significance (p < 0.05). CONCLUSION: In conclusion, photobiostimulation with red LED at 625 nm was associated with improved beneficial biological effects as a potential inducer of osteogenic activity on RTV-affected cells. This is the first study that investigated the benefits of red LED irradiation over ARV-treated in vitro osteoblasts.

Synergism between molecules derived from Garcinia brasiliensis and antimicrobial drugs on field isolates of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the smallest free-living bacteria found in nature; it has an extremely small genome and lacks a cell wall. It is the main etiological agent of porcine enzootic pneumonia (EP), a chronic respiratory disease with worldwide distribution that causes significant losses in swine production. Due to the great economic impact caused by EP, new strategies for treating and controlling this agent are researched. The objective of this study was to verify the anti-M. hyopneumoniae activity of compounds derived from Garcinia brasiliensis and the synergism with the main antimicrobials used in the treatment of EP; this is the first study assessing the synergism between bioactive molecules and antimicrobial compounds in vitro against isolates of M. hyopneumoniae. The minimum inhibitory concentrations (MICs) of the antimicrobials tiamulin, valnemulin, and enrofloxacin, as well as the bioactive compounds guttiferone-A (Gut-A), 7-epiculsone (7-Epic), copper 7-epiculsone (7-Epic-Cu), and benzophenone, were determined. Subsequently, the interactions of antibiotics with the compounds were evaluated using the checkerboard method. Three field M. hyopneumoniae isolates were used, and the J strain was used as a control. The MIC values of the antimicrobials compared to the field isolates were equal to and lower than those of the reference strain J. Among the compounds used, 7-Epic-Cu showed the lowest MIC value. Synergistic association was observed for Gut-A with tiamulin and valnemulin, whereas 7-Epic and 7-Epic-Cu showed synergistic action with enrofloxacin. No synergistic effect was observed for benzophenone. Despite being an initial study, the results suggest that these combinations hold promise for the treatment of infections caused by M. hyopneumoniae.

Proteomic profiling of membrane vesicles from Mycobacterium avium subsp. paratuberculosis: Navigating towards an insilico design of a multi-epitope vaccine targeting membrane vesicle proteins.

Bacteria typically produce membrane vesicles (MVs) at varying levels depending on the surrounding environments. Gram-negative bacterial outer membrane vesicles (OMVs) have been extensively studied for over 30 years, but MVs from Gram-positive bacteria only recently have been a focus of research. In the present study, we isolated MVs from Mycobacterium avium subsp. paratuberculosis (MAP) and analyzed their protein composition using LC-MS/MS. A total of 316 overlapping proteins from two independent preparations were identified in our study, and topology prediction showed these cargo proteins have different subcellular localization patterns. When MVs were administered to bovine-derived macrophages, significant up-regulation of pro-inflammatory cytokines was observed via qRT-PCR. Proteome functional annotation revealed that many of these proteins are involved in the cellular protein metabolic process, tRNA aminoacylation, and ATP synthesis. Secretory proteins with high antigenicity and adhesion capability were mapped for B-cell and T-cell epitopes. Antigenic, Immunogenic and IFN-γ inducing B-cell, MHC-I, and MHC-II epitopes were stitched together through linkers to form multi-epitope vaccine (MEV) construct against MAP. Strong binding energy was observed during the docking of the 3D structure of the MEV with the bovine TLR2, suggesting that the putative MEV may be a promising vaccine candidate against MAP. However, in vitro and in vivo analysis is required to prove the immunogenic concept of the MEV which we will follow in our future studies. SIGNIFICANCE: Johne's disease is a chronic infection caused by Mycobacterium avium subsp. paratuberculosis that has a potential link to Crohn's disease in humans. The disease is characterized by persistent diarrhea and enteritis, resulting in significant economic losses due to reduced milk yield and premature culling of infected animals. The dairy industry in the United States alone experiences losses of approximately USD 250 million due to Johne's disease. The current vaccine against Johne's disease is limited by several factors, including variable efficacy, limited duration of protection, interference with diagnostic tests, inability to prevent infection, and logistical and cost-related challenges. Nevertheless, a multiepitope vaccine design approach targeting M. avium subsp. paratuberculosis has the potential to overcome these challenges and offer improved protection against Johne's disease.

Unveiling deception: Characterizing false amber necklace messages on Facebook.

BACKGROUND: Messages promoting the benefits of amber necklaces for children are common on social media, despite their health risks. AIM: This study characterized Facebook posts with false content about the efficacy of amber necklaces in teething. DESIGN: A sample of 500 English-language Facebook posts was analyzed by two investigators to determine the motivations, author's profile, and sentiments of posts. Latent Dirichlet Allocation topic modeling was used to identify salient terms and topics. An intertopic distance map was created to calculate the topic similarity. These data were analyzed using descriptive analysis, the Mann-Whitney U test, Cramer's V test, and multiple logistic regression models, regarding the time since initial posting and interaction metrics. RESULTS: Most posts were made by business profiles and expressed positive sentiments, with social, psychological, and financial motivations. The posts were categorized into the topics "giveaway," "healing features," and "sales." Overperforming scores and total interaction increased with time since the initial posting. Posts with links had higher overperforming scores. CONCLUSION: The findings suggest that Facebook posts about the efficacy of amber necklaces in teething are motivated by financial interests, using psychological and social mechanisms to achieve greater interaction with their target audience.

A genetic and virulence characterization of Brazilian strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is considered the primary causative agent of porcine enzootic pneumonia (EP), a chronic contagious respiratory disease that causes economic losses. Obtaining new pathogenic isolates and studying the genome and virulence factors are necessary. This study performed a complete sequencing analysis of two Brazilian strains, UFV01 and UFV02, aiming to characterize the isolates in terms of the virulence factors and sequence type. The complete genome analysis revealed the main virulence genes (mhp385, mhp271, MHP_RS03455, p102, p97, p216, MHP_RS00555, mhp107) and ST-123, the presence of three toxin-related genes (tlyC, PLDc_2 and hcnC), and some genetic groups specific to these two isolates. Subsequently, the pathogenicity of the isolates was evaluated via an experimental infection conducted in a swine model. The study was divided into three groups, namely a negative control group (n = 4) and two test groups (n = 8), totaling 20 animals. They were challenged at 35 days of age with 107 CCU (Color Changing Units) M. hyopneumoniae via the intratracheal route. The UFV01 group showed earlier and higher seroconversion (IgG) (100%), while only 50% of the UFV02 group seroconverted. The same trend was observed when analyzing the presence of IgA in the bronchoalveolar lavage fluid (BALF) at 35 days post-infection (dpi). The UFV01 group had a mean macroscopic lesion score of 11.75% at 35 dpi, while UFV02 had 3.125%. Microscopic lesions were more severe in the UFV01 group. Based on laryngeal swab samples evaluated by qPCR, and the detection began at 14 days. The UFV01 group showed 75% positivity at 14 dpi. The UFV02 group also started excreting at 14 dpi, with a positivity rate of 37.5%. The results indicate that the UFV01 isolate exhibits higher virulence than UFV02. These findings may aid in developing new vaccines and diagnostic kits and establishing experimental models for testing.

Antimicrobial Resistance Profiles of Multidrug-Resistant Enterobacteria Isolated from Feces of Weaned Piglets.

Nosocomial infections caused by multidrug-resistant enterobacteria have become a major challenge in global public health. Previous studies have indicated that use of antibiotics in livestock production chains is linked to the rising threat of antibiotic resistance in humans. In this study, we aimed to evaluate the distribution of genes encoding resistance to tetracycline, ß-lactams, and colistin in multidrug-resistant enterobacteria isolated from feces of weaned pigs. Ninety-four enterobacteria isolates were submitted to antibiotic susceptibility test by minimum inhibitory concentration (MIC). In addition, we performed conjugation experiments to verify if plasmid-bearing isolates containing the mcr-1 gene could transfer their resistance determinant to a colistin-sensitive recipient strain. Our results demonstrated a positive association between the detection of antibiotic resistance genes in enterobacteria and the phenotypic resistance profiles of the bacterial isolates. At least one of the extended-spectrum ß-lactamases (ESBL) genes (blaCTX-M, blaTEM, or bla SHV) and tetA was found among most bacterial genera analyzed. In addition, results revealed that the mcr-1 gene can be transferred from E. coli UFV-627 isolate to an F- recipient (Escherichia coli K12) by conjugation. Our findings support the hypothesis that swine represents an important reservoir of antibiotic resistance genes and suggest that horizontal transfer mechanisms (e.g., conjugation) may mediate the spread of these genes in the swine gastrointestinal tract.

Multidrug Efflux System-mediated resistance in Staphylococcus aureus under a One Health approach.

The aim of the study was to track the spread of antimicrobial resistance among the different sectors of One Health through the detection of Multidrug-Efflux-System in multidrug-resistant Staphylococcus aureus isolates. Multidrug-resistant (MDR) and methicillin-resistant (MRSA) S. aureus isolates were selected: 25 of human, one of animal and eight of food origin. The efflux system genes norA, norB, norC, LmrS, tet38 and msrA were screened by PCR. The activity of the efflux systems was determined by the minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin in the presence and absence of CCCP and in the quantification of ethidium bromide efflux. Furthermore, biofilm formation was determined in the presence and absence of the CCCP. The molecular epidemiology of the isolates was traced with the aid of PFGE. The gene norC was the most prevalent, detected in all isolates and msrA was the least prevalent, detected in only two isolates from humans. There was no difference in the MICs of tetracycline and ciprofloxacin in the presence of CCCP, but 55.9% of isolates showed ethidium bromide efflux. The presence of CCCP decreased the biofilm formation. Regarding the molecular epidemiology, in three clusters was a mixture of the isolates from different origins. Therefore, S. aureus MDR with active multidrug efflux systems are circulating between One Health domains and it is necessary to consider strategies to decrease this circulation in order to prevent the dissemination of resistance mediated by MES.

Review of methods for detection and characterization of non-tuberculous mycobacteria in aquatic organisms.

Mycobacteriosis is an emerging and often lethal disease of aquatic organisms caused by several non-tuberculous mycobacteria (NTM) species. Early diagnosis of mycobacteriosis in aquaculture and aquatic settings is critical; however, clinical diagnoses and laboratory detection are challenging, and the available literature is scarce. In an attempt to fill the gap, here we review the most relevant approaches to detect and characterize mycobacteria in clinical specimens of aquatic organisms. Emphasis is given to recent advances in molecular methods used to differentiate NTM species spanning from targeted gene sequencing to next-generation sequencing. Further, given that there are major gaps in our understanding of the prevalence of the different NTM species, partially because of their distinct requirements for in vitro growth, we also reviewed the most relevant NTM species reported to cause disease in aquatic organisms and their specific in vitro growth conditions. We also highlight that traditional bacterial culture continues to be relevant for NTM identification, particularly in non-automated laboratories. However, for NTM species discrimination, a high level of accuracy can be achieved with MALDI-TOF MS and molecular approaches, especially targeted gene sequencing applied from clinical specimens or from pure NTM isolates.

High-Risk Regions of African Swine Fever Infection in Mozambique.

African swine fever (ASF) is a transboundary infectious disease that can infect wild and domestic swine and requires enhanced surveillance between countries. In Mozambique, ASF has been reported across the country, spreading between provinces, mainly through the movement of pigs and their by-products. Subsequently, pigs from bordering countries were at risk of exposure. This study evaluated the spatiotemporal distribution and temporal trends of ASF in swine in Mozambique between 2000 and 2020. During this period, 28,624 cases of ASF were reported across three regions of the country. In total, the northern, central, and southern regions presented 64.9, 17.8, and 17.3% of the total cases, respectively. When analyzing the incidence risk (IR) of ASF per 100,000 pigs, the Cabo Delgado province had the highest IR (17,301.1), followed by the Maputo province (8868.6). In the space-time analysis, three clusters were formed in each region: (i) Cluster A involved the provinces of Cabo Delgado and Nampula (north), (ii) Cluster B involved the province of Maputo and the city of Maputo (south), and (iii) Cluster C consisted of the provinces of Manica and Sofala (central) in 2006. However, when analyzing the temporal trend in the provinces, most were found to be decreasing, except for Sofala, Inhambane, and Maputo, which had a stationary trend. To the best of our knowledge, this is the first study to evaluate the spatial distribution of ASF in Mozambique. These findings will contribute to increasing official ASF control programs by identifying high-risk areas and raising awareness of the importance of controlling the borders between provinces and countries to prevent their spread to other regions of the world.

Distinguishing the milk microbiota of healthy goats and goats diagnosed with subclinical mastitis, clinical mastitis, and gangrenous mastitis.

Mastitis, mainly caused by bacterial intramammary infections, is the main problem in the breeding of dairy animals. The inflammations of the mammary gland is separated by types of mastitis, being subclinical, clinical, and the most severe, gangrenous mastitis. Here, we used 16S rRNA amplicon sequencing to characterize the bacterial microbiota of goat milk in the different types of goat mastitis caused by bacteria. We used 72 goat milk samples from a region of the state of Minas Gerais in Brazil, of which 12 were from clinically healthy animals, 42 from animals diagnosed with subclinical mastitis, 16 from animals with clinical mastitis, and 2 from animals with gangrenous mastitis. The group related to gangrenous mastitis was the most divergent in terms of alpha and beta diversity. The most abundant genus among samples of the groups was Staphylococcus spp., and we found a high abundance of Mycoplasma sp. in the milk of animals diagnosed with clinical mastitis. The most statistically relevant microorganisms among the groups were Prevotella sp., Ruminococcaceae, Prevotella ruminicola sp., and Providencia sp. We highlight a new association of bacterial agents in gangrenous mastitis among Escherichia sp./Shigella sp. and Enterococcus sp. and provide the second report of the genus Alkalibacterium sp., in milk samples. Only the taxa Staphylococcus sp., Bacteroides sp., Enterococcus, and Brevidabacterium sp., were present in all groups. The superpathway of L-tryptophan biosynthesis metabolites and the sucrose degradation III (sucrose invertase) pathway were the most prominent ones among the groups. In this study, we demonstrate how a rich microbiota of goat milk from healthy animals can be altered during the aggravation of different types of mastitis, in addition to demonstrating new bacterial genera in milk not previously detected in other studies as well as new associations between agents.

Identification of extracellular vesicles from J strain and wild isolate of Mycoplasma hyopneumoniae.

Respiratory diseases constitute a major health challenge for the worldwide pork industry. Porcine enzootic pneumonia (PES) is caused by Mycoplasma hyopneumoniae (Mhyo). Mycoplasmas have the ability to produce extracellular vesicles (EVs), which can be useful for pathogenicity studies and as delivery systems for vaccines. The aim of this study was to demonstrate and compare, under laboratory conditions, EVs produced by Mhyo strain J and wild isolate in stressed and non-stressed in vitro conditions. Using differential centrifugation, density gradient ultracentrifugation, and transmission electron microscopy, the ability of Mhyo strains to produce EVs was demonstrated under favorable and unfavorable conditions.

Multilocus sequence analysis reveals genetic diversity in Staphylococcus aureus isolate of goat with mastitis persistent after treatment with enrofloxacin.

Staphylococcus aureus is one of the main bacterial agents responsible for cases of mastitis in ruminants, playing an important role in the persistence and chronicity of diseases treated with antimicrobials. Using the multilocus sequence typing technique, network approaches and study of the population diversity of microorganisms, we performed analyzes of S. aureus (ES-GPM) isolated from goats with persistent mastitis (GPM). The most strains of ES-GPM were categorically different phylogenetically from the others and could be divided into two lineages: one with a majority belonging to ES-GPM and the other to varied strains. These two lineages were separated by 27 nuclear polymorphisms. The 43 strains comprised 22 clonal complexes (CCs), of which the ES-GPM strains were present in CC133, CC5 and a new complex formed by the sequence type 4966. The genetic diversity of some alleles showed be greater diversity and polymorphism than others, such as of the aroE and yqiL genes less than glpF gene. In addition, the sequences ES-GPM to the arc gene and glpF alleles showed the greatest number of mutations for ES-GPM in relation to non-ES-GPM. Therefore, this study identified genetic polymorphisms characteristic of S. aureus isolated from milk of goats diagnosed with persistent mastitis after the failed treatment with the antibiotic enrofloxacin. This study may help in the future to identify and discriminate this agent in cases of mastitis, and with that, the most appropriate antibiotic treatment can be performed in advance of the appearance of persistent mastitis caused by the agent, reducing the chances of premature culling and animal suffering.

Spatiotemporal distribution and temporal trends of brucellosis and tuberculosis in water buffalo (Bubalus bubalis) in Brazil.

The buffalo herds in Brazil have been an alternative for increasing the economy in different biomes. For this reason, knowledge of the spatial distribution of diseases of mandatory notification in buffalo herds, such as brucellosis and tuberculosis, is essential to guarantee the quality of exported animal products, as well as assist in strategies of national control and eradication programs. In the present study, we aimed to evaluate the spatiotemporal distribution and temporal trends of brucellosis and tuberculosis in buffalo in Brazilian states between 2012-2019. During this period, 474 cases of brucellosis and 604 cases of tuberculosis were observed in buffalo in Brazil, with no significant differences between the total number of cases and incidence risk. The spatial distribution for the states was mostly heterogeneous, showing similarities of occurrences for both diseases in the south, north, and the states of Minas Gerais and Pernambuco. In the eight years evaluated, tuberculosis showed cyclical variation every 1-2 years; however, for brucellosis, there was a cyclical trend only between 2012-2015, with a significant decrease until 2018. Among Brazilian states, Pará had greater disease case numbers, with 34 % for brucellosis and 40.6 % for tuberculosis. Temporal trend analysis showed an increase for Pernambuco (annual percentage change [APC]: 21.0 [CI = 20.3; 21.8]), Paraná (APC: 27.1 [CI = 5.6; 53.0]), and Santa Catarina (APC: 10.4 [CI = 0.8; 21.0]) for brucellosis, and for tuberculosis, only for Santa Catarina state (APC: 24.1 [CI = 15.5; 33.3]). Spatiotemporally, there were four high-risk brucellosis clusters with a primary cluster, cluster A (relative risk [RRs] = 53.42, P < 0.001), involving the state of Amazonas between 2014-2015. For tuberculosis, there were three states of high risk, with a primary cluster, cluster E (RRs = 28.18, P < 0.001), involving the states of Pará, Roraima, Amazonas, Rondônia, and Acre in 2014. In conclusion, brucellosis and tuberculosis in buffalo in Brazil are heterogeneously distributed, with well-defined regions of high historical risk of infection. Among these regions, the states of Amazonas, Pará, Amapá, and Minas Gerais stand out due to the higher risk of infection for both brucellosis and tuberculosis, alerting Brazilian authorities to the need for disease control actions.

Public Policies and One Health in Brazil: The Challenge of the Disarticulation.

Working the One health strategy in developing countries is a challenge, due to structural weaknesses or deprivation of financial, human, and material resources. Brazil has policies and programs that would allow continuous and systematic monitoring of human, animal, and environmental health, recommending strategies for control and prevention. For animals, there are components of the Epidemiological Surveillance of zoonosis and Animal Health Programs. To guarantee food safety, there are Health Surveillance services and support of the Agropecuary Defense in the inspection of these products, productive environments, and their inputs. Environmental Surveillance Services monitor water and air quality, which may influence health. For human health, these and other services related to Health Surveillance, such as Worker Health and Epidemiological Surveillance, which has a training program responsible for forming professionals groups to respond effectively to emergencies in public health are available. Therefore, Brazil has instruments that may allow integrated planning and intervention based on the One Health initiative. However, the consolidation of this faces several challenges, such as insufficient resources, professional alienation, and lack of the recognition of the importance of animal and environmental health for the maintenance of human and planetary well-being. This culminates in disarticulation, lack of communication, and integration between organizations. Thus, efforts to share attributions and responsibilities must be consolidated, overcoming the verticality of the actions, promoting efficiency and effectiveness. Finally, this perspective aims to describe the government instruments that constitute potential national efforts and the challenges for the consolidation of the One Health initiative in Brazil.

Interaction of Mycobacterium avium subsp. paratuberculosis with bovine sperm.

Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for Paratuberculosis mainly affecting domestic ruminants. The interaction between MAP and sperm and/or germ cells has not yet been established, however the adherence between MAP and the host cell surface is associated to the 85 complex proteins that bind to the host cell's fibronectin. Therefore, this study aimed to evaluate the binding of MAP to bovine sperm and to verify changes in these cells by the presence of MAP before and after sperm cryopreservation. Polyclonal antibodies to MAP 85 complex proteins were produced and utilized in the analyzes. Two Nelore bulls were used for semen collection and MAP dilutions (103-108 CFU/mL) were inoculated in the samples; sperm motility and vigor were evaluated using light microscopy at different times before and after cryopreservation and in the presence and absence of the antibodies 85A and 85B. Interaction of MAP and sperm, interaction of MAP and sperm in the presence of Ab 85A and in the presence of Ab 85B were analyzed by scanning electron microscopy. The viability of MAP after sperm cryopreservation were evaluated by plating the samples after thawing. It was observed that sperm in the presence of MAP shows a decrease in motility and vigor, and that the higher the MAP concentration, the lower the sperm performance. It was possible to determine the viability of MAP after cryopreservation in samples of higher concentrations, which demonstrates the potential of transmission of this pathogen through artificial insemination. The interaction of MAP with bovine sperm occurs mainly in the midpiece and may be linked to the proteins 85A and 85B present in the MAP membrane.

Antimicrobial activity of polypyrrole nanoparticles and aqueous extract of Moringa oleifera against Staphylococcus spp. carriers of multi-drug efflux system genes isolated from dairy farms.

Our objectives were to identify genes of the multi-drug efflux system and to evaluate the antimicrobial activities of polypyrrole nanoparticles (PPy-NPs) and aqueous extract of Moringa oleifera against Staphylococcus spp. isolated from dairy farms in Northeast Brazil. Initially, 162 Staphylococcus spp. isolates were subjected to in vitro antimicrobial sensitivity tests. Of these, 35 presented antimicrobial multi-drug resistance phenotypes. These 35 isolates were then referred for the detection of norA, norB, norC, msrA, mgrA, tet-38, and lmrS genes, all of which feature in multi-drug efflux systems. In the isolates carrying the genes, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of PPy-NPs and Moringa oleifera aqueous extract were determined. In the molecular analysis of the 35 isolates norA, norC, tet-38, and msrA genes were detected and for the other genes norB, lmrS and mgrA there was no amplification. Antimicrobial activity was verified of PPy-NPs and aqueous extract of Moringa oleifera in Staphylococcus spp. carrying multi-drug efflux system genes. We concluded that there are multi-drug efflux system genes present in the Staphylococcus spp. from the agricultural environment in Northeast Brazil, and that aqueous extract of Moringa oleifera and PPy-NPs show bactericidal activity against these isolates.