Antimicrobial efficiency of bromhexine hydrochloride against endometritis-causing Escherichia coli and Trueperella pyogenes in bovines.

In this study, the main agents associated with endometritis in cows in the state of Santa Catarina, Brazil, were identified and the resistance profile and virulence mechanisms of the bacterial isolates were evaluated. Isolates of Escherichia coli and Trueperella pyogenes were tested for their biofilm forming ability and the antimicrobial action of bromhexine hydrochloride in combination with other antimicrobials. A total of 37 uterine lavage samples were collected from cows with endometritis. Of the 55 bacteria isolated, 25.4% were identified as T. pyogenes and 16.3% as E. coli. The bacterial isolates showed greater resistance to sulfamethoxazole + trimethoprim (58.2%) and tetracycline (56.3%). Among the species, E. coli showed the highest resistance rates, with 100% of isolates showing resistance to amoxicillin, streptomycin, and gentamicin. The results of the minimum inhibitory concentration for the T. pyogenes isolates showed that 91.6% of the isolates were resistant to enrofloxacin and tetracycline, and 75% were resistant to ceftiofur and sulfamethoxazole + trimethoprim. All E. coli and T. pyogenes isolates showed biofilm forming ability. The plo, fimA, and nanH genes were identified in 100% of T. pyogenes isolates. In parallel, 100% of E. coli isolates had the fimH gene, and 11.1% had the csgD gene. Bromhexine hydrochloride showed antimicrobial activity against 100% of E. coli isolates and 66.6% of T. pyogenes isolates. Furthermore, when associated with antimicrobials, bromhexine hydrochloride has a synergistic and additive effect, proving to be an option in the treatment of endometritis in cows and an alternative for reducing the use of antimicrobials.

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.

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.

Resistance and virulence in Staphylococcus aureus by whole-genome sequencing: a comparative approach in blaZ-positive isolates.

Mastitis caused by Staphylococcus aureus is a worldwide problem in dairy farms, in part because of the pathogenicity of the bacteria, biofilm formation, and mechanisms of antimicrobial resistance that make the disease difficult to diagnose and treat, which is typically done with the use of beta-lactam antibiotics. The aim of the present study was to determine the virulence and resistance factors of S. aureus isolates from subclinical mastitis, blaZ + /mecA - /mecC - , resistant and sensitive to oxacillin. All isolates were classified as CC97 by MLST analysis, a clonal complex well adapted to the mammary gland and although STAU23 and STAU73 were resistant to oxacillin while STAU32 and STAU78 were sensitive, the genomic analysis identified only the blaZ operon corresponding to resistance to beta-lactams. However, the presence of the sdrC gene was revealed exclusively in resistant isolates, an important adhesin in the colonization process that potentiates pathogenicity in S. aureus. In addition, resistance islands (REIs) were identified in these isolates, suggesting more conserved REIs. In the analysis of SNPs throughout the genome, mutations were found in the trmB and smpB genes of the resistant isolates and in the murD and rimM genes of the sensitive isolates. This study highlights the potential benefit of genome-wide characterization tools to identify molecular mechanisms of S. aureus in bovine mastitis.

Synergy between polypyrrol and benzoic acid against antibiotic-resistant Salmonella spp.

AIMS: The purpose was to characterize Salmonella Heidelberg (SH) and Minnesota (SM) isolates in terms of their resistance and persistence profile and to assess the antimicrobial effect of benzoic acid (BA) and polypyrrole (PPy). METHODS AND RESULTS: The 20 isolates from broiler litter drag swabs were submitted to antibiogram and efflux pump expression. The minimum inhibitory/bactericidal concentration (MIC/MBC) of the compounds, synergistic activity, time kill, biofilm production, presence of related genes, and molecular docking between compounds and bacterial target sites were evaluated. All isolates showed multidrug resistance (MDR) and BA and PPy showed mean MIC (1750 and 342 µg ml-1) and MBC (3167 and 1000 µg ml-1), respectively. None of the isolates expressed an efflux pump. The compounds showed synergism against an SH isolate and reduced the count by 3 logs in the presence of the compounds after 4 h. Most isolates (16/20) produced weak to moderate biofilm and 17 showed genes related to biofilm. The compounds interacted with two essential proteins, 3,4-dihydroxy-2-butanone 4-phosphate synthase proteins and ferritin-like domain-containing protein, in bacterial metabolism at different target sites. CONCLUSIONS: It can be concluded that BA and PPy showed activity on SH and SM, MDR, and biofilm producers, with a potential synergistic effect.

Molecular Characterization of Salmonella Phage Wara Isolated from River Water in Brazil.

Antimicrobial resistance is increasing despite new treatments being employed, so novel strategies are required to ensure that bacterial infections remain treatable. Bacteriophages (phages; bacteria viruses) have the potential to be used as natural antimicrobial methods to control bacterial pathogens such as Salmonella spp. A Salmonella phage, Wara, was isolated from environmental water samples at the Subaé River Basin, Salvador de Bahia, Brazil. The basin has environmental impacts in its main watercourses arising from the dumping of domestic and industrial effluents and agricultural and anthropological activities. The phage genome sequence was determined by Oxford Nanopore Technologies (ONT) MinION and Illumina HiSeq sequencing, and assembly was carried out by Racon (MinION) and Unicycler (Illumina, Illumina + MinION). The genome was annotated and compared to other Salmonella phages using various bioinformatics approaches. MinION DNA sequencing combined with Racon assembly gave the best complete genome sequence. Phylogenetic analysis revealed that Wara is a member of the Tequintavirus genus. A lack of lysogeny genes, antimicrobial resistance, and virulence genes indicated that Wara has therapeutic and biocontrol potential against Salmonella species in healthcare and agriculture.

Synergistic interaction of clove, cinnamon, and eucalyptus essential oils impregnated in cellulose acetate electrospun fibers as antibacterial agents against Staphylococcus aureus.

The development of antibiotic-free antibacterial strategies applied in the control of bacterial and biofilm proliferation on surfaces is an important topic in discussion in the literature. Essential oils have been explored as isolated and combined components to act as an antibacterial material that inhibits bacterial proliferation, avoiding the contamination of surfaces. Herein, cellulose acetate electrospun fibers impregnated with essential oils of clove, cinnamon and eucalyptus and their combination (clove + cinnamon, cinnamon + eucalyptus and clove + eucalyptus) were explored against the standard strain of Staphylococcus aureus (ATCC 25923). As isolated components, the best performance follows the order clove>cinnamon>eucalyptus essential oil. The association of clove and cinnamon into cellulose acetate electrospun fibers returned a promising and fast antibacterial and antibiofilm activity (improvement in 65%), as a piece of evidence that synergism is observed for the association of essential oils incorporated into electrospun fibers that preserves the antibacterial activity by encapsulation of components.

Virulent potential of methicillin-resistant and methicillin-susceptible Staphylococcus pseudintermedius in dogs.

Staphylococcus pseudintermedius is a zoonotic pathogen responsible for several infectious diseases in pet animals, yet its pathogenic potential is not fully understood. Thus, this study aims to unravel the virulence profile of S. pseudintermedius from canine origin. Methicillin-resistant (MRSP) and methicillin-susceptible (MSSP) strains were isolated from different infection sites and their genotypic and phenotypic features were compared to determine the clinical implications of MRSP and MSSP strains. Bacterial identification was performed using MALDI-TOF and 16S-rDNA sequencing. In addition, we used multilocus sequence typing (MLST) for strains' sequence type (ST) determination and phylogenetic relationship. The strains were screened for toxin genes, including cytotoxins (lukS, lukF), exfoliative toxin (siet), enterotoxins (sea, seb, sec, secCanine, sel, sem, and seq) and toxic shock syndrome toxin (tst-1). In vitro phenotypic analyses assessing antimicrobial susceptibility profile, biofilm formation ability, and expression of extracellular matrix components were performed. The investigated S. pseudintermedius strains belong to 17 unique ST, most of which were classified as ST71. MSSP and MRSP strains shared siet, lukS, and lukF virulence markers. Our findings showed that some MSSP strains also harbored sel, seq, and sem enterotoxin genes, suggesting a more diverse virulence profile. All MRSP strains and 77% of MSSP strains were classified as multidrug resistant (MDR). Moreover, all investigated S. pseudintermedius strains showed strong biofilm formation ability. In summary, our findings highlight the wide spread of highly virulent and drug-resistant zoonotic S. pseudintermedius strains, being a potential concern for One Health issues.

Action of crude ethanol extract of Hymenaea martiana leaf, gallic acid, and polypyrrole (PPy) against Aeromonas hydrophila.

Pisciculture represents one of the industries with the fastest growth rates worldwide. However, it presents obstacles to its development, such as bacteriosis, which is conventionally treated with antibiotics. The indiscriminate and inappropriate use of antibiotics can lead to bacterial resistance, thus alternatives to the use of antibiotics have been researched. The study aimed to analyze the potential of crude ethanol extract (CEE) from Hymenaea martiana leaf, gallic acid (GA), and polypyrrole (PPy) against Aeromonas hydrophila. Tests were performed to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compounds individually and in synergy (checkerboard) against A. hydrophila and in silico tests between the compounds evaluated. The CEE of H. martiana leaf and PPy were effective against A. hydrophila with MBC results of 3125 µg/mL for the CEE of H. martiana and 125 µg/mL for PPy. Evaluating the GA, a MIC and MBC of 125 µg/mL was obtained. In the interaction tests (checkerboard, using PPy/CEE and PPy/GA), there was a significant reduction in individual introductions. Thus, for the PPy/CEE tests, we had a reduction of MIC/MBC to 1.95 and 781.25 µg/mL, and for the synergy tests between PPy/GA to 7.8125 and 31.125 µg/mL, respectively. The synergy tests are encouraging, and it is possible to verify a decrease of up to 98% in the introduction of PPy, 75% in CEE for H. martiana and 75.1% for GA, when compared to their individual tests. The tests with GA are encouraging due to GA's effectiveness as an antimicrobial agent and high synergy with polypyrrole, both in vitro results and molecular docking experiments showed the actions at the same activation site in A. hydrophila. In vivo tests evaluating isolated components of CEE from H. martiana in synergy with PPy should be performed, to verify the quality of the interactions and the improvement of the immune responses of the animals. It was evidenced that gallic acid, a substance isolated from the extract, tends to have more promising results. This is relevant since the industry has been developing these compounds for different uses, thus providing easier access to the product. Thus, the present study indicates an efficient alternative in the use of bioactive compounds as substitutes for conventional antimicrobials.

Oomicidal activity of polypyrrole nanoparticles against Pythium insidiosum.

This study evaluated in-vitro action of a new molecule, the polypyrrole nanoparticles (Ppy-NP), against Pythium insidiosum isolates using M38-A2/CLSI; the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Additionally, changes in the hyphae wall of P. insidiosum CBS 575.85 treated with Ppy-NP were evaluated by scanning electron microscopy (SEM). The MIC100 and MOC for all isolates ranged from 8 to 32 µg mL-1, and the MIC90 and MIC50 were 16 µg mL-1. The SEM showed structural damage to the hyphae of P. insidisoum treated with Ppy-NP, as hyphae surfaces with less turgidity were found, thereby showing scaling and ruptures compared to the control (untreated hyphae). Our findings highlighted the anti-P. insidiosum properties of Ppy-NP proved to be a promising candidate for research using pythiosis experimental models.

Grape skin flour obtained from wine processing as an antioxidant in beef burgers.

The objective of this study was to determine the best level of wine making by-product meal (WBM) as a natural antioxidant to replace butylhydroxytoluene (BHT) in beef burger stored at -20 °C for up to 120 days. The treatments consisted of control (basic formulation - BF, without antioxidant); BF with BHT; and BF with WBM0.5, WBM1.0, WBM1.5, and WBM2.0, with 0.5, 1.0, 1.5 and 2.0 g WBM/100 g BF, respectively. Up to 60 days of storage, the lipid oxidation value between BHT and WBM0.5 treatments did not differ and were lower than the values presented by the other treatments. On day 90 and 120, the lipid oxidation values of treatments BHT, WBM0.5, and WBM1.0 did not differ and were lower than the values presented by WBM1.5 and WBM2.0 treatments. Burgers from all treatments with WBM inclusion had crude fiber values above 3 g/100 g. WBM1.5 and WBM2.0 treatments had the worst scores for appearance, aroma, juiciness and tenderness, in addition to the highest cooking losses. WBM can be used at up to 1 g/100 g to replace BHT in frozen beef burgers. Higher levels of WBM inclusion increased lipid oxidation and negatively affected the sensory quality of burgers.

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.

Bovine brucellosis seroprevalence and flow network analysis in slaughterhouses in the state of Ceará.

The present study aimed to assess the prevalence and risk factors associated with bovine brucellosis in slaughterhouses in the state of Ceará using spatial distribution and flow network analysis. Four slaughterhouses were sampled in Ceará: two under municipal inspection and two under state inspection. Blood samples were randomly collected from bovine animals, resulting in a total of 964 samples. The collected sera were subjected to the Acidified Buffered Antigen (AAT) test, and the complement fixation test (FC) was performed for positive cases. An epidemiological questionnaire was applied to 38 producers who slaughter animals at the sampled facilities to assess the risk factors for brucellosis. An apparent prevalence of 1.55% (15) was found in the AAT test and 0.2% (n=2) in the FC test. A higher percentage of reactive animals was observed (66.6%) in properties where cattle farming is not the main activity, with OR = 4.75. The absence of contact with neighboring animals is a factor associated with protection, with a lower prevalence of seroreactive animals (23.5%) when animals were raised without contact with others (OR = 0.30). Therefore, bovine brucellosis in herds and animals can be considered low in the studied region and under all production systems. Nevertheless, despite the importance of this disease to the economic and public health aspects and the advances of the PNCEBT Program, brucellosis is still circulating in Ceará.

Objetivou-se conhecer a prevalência e fatores de risco associados a brucelose em bovinos em abatedouros no estado do Ceará, determinando sua distribuição espacial e análise da rede de fluxo. Foram amostrados quatro abatedouros no Ceará referente a três mesorregiões do estado (Centro Sul, Sertões e Sul cearense), sendo dois abatedouros sob inspeção municipal e dois sob inspeção estadual para colheita de amostras de sangue em animais bovinos de forma aleatória, gerando um total de 964 amostras. Os soros colhidos foram submetidos ao teste do Antígeno Acidificado Tamponado (AAT) e para os casos positivos foi realizado o teste da Fixação de Complemento (FC). Foi aplicado um questionário epidemiológico para avaliação dos fatores de risco para brucelose, sendo entrevistados um total de 38 produtores que abatiam seus animais nos estabelecimentos participantes da pesquisa. Foi encontrada uma prevalência aparente de 1,5% (n=15) na prova do AAT e 0,2% (n=2) na FC. Duas variáveis apresentaram-se associadas à presença de bovinos positivos na AAT, a existência de atividades econômicas paralelas à bovinocultura (OR = 4,75). A ausência de contato com animais da vizinhança (OR = 0,30), apresentando-se como fator protetor. Pode-se afirmar que a prevalência da brucelose bovina na área estudada é baixa, no entanto, ainda é observada a circulação do agente no estado do Ceará, sendo importante a manutenção das medidas de controle previstas no Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose (PNCEBT) e inclusão de medidas adicionais, tendo em vista a relevância da enfermidade na Saúde Pública e no cenário agropecuário do país.

General biosafety measures for laboratory environments, outpatient clinics, medical centers, and veterinary hospitals during the SARS-CoV-2 pandemic.

This study aimed to guide professionals working in veterinary laboratories, outpatient clinics, medical centers, and hospitals regarding the biosafety measures that should be adopted during the novel coronavirus (SARS-CoV-2) pandemic. While the population is not yet fully immunized by vaccines, the adoption of biosafety measures is essential to control the spread of circulating strains of the new coronavirus. Thus, the importance of professionals and collaborators following biosafety guidelines in different veterinary work environments is highlighted. The main protocols on biosafety to be adopted include frequent handwashing with water and soap or using 70% alcohol-based hand sanitizers, using personal protective equipment (PPE) (including gloves, lab coat, face mask), avoiding the contact of the hands with mucous membranes (eyes, nose and mouth), not sharing personal objects, keeping environments clean and well ventilated, social distancing of 1.5 m between individuals, and maintaining objects and surfaces regularly clean throughout the work environment. The transformation of work processes, such as various biosafety practices, is necessary within the context of the COVID-19 pandemic and improves the safety of professionals in their work environment and other people and animals, decreasing contamination risks in order to reduce the spread of this viral agent.

Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

AIMS: Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. METHODS AND RESULTS: The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. CONCLUSION: Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

Analysis of Gtpases Rab 5 and Rab 7 expression from macrophages infected with biofilm-producing and non biofilm-producing strains of Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a facultative intracellular pathogen that uses various mechanisms to survive within macrophages. In phagocytosis, this survival can be attributed to the ability to inhibit phagosome-lysosome fusion. In this fusion, some proteins, including Rabs GTPases, are involved in the maturation process and are responsible for regulating membrane vesicle trafficking. Thus, to better understand these mechanisms, the capacity of biofilm-producing and non biofilm-producing strains of Corynebacterium pseudotuberculosis for modulating the expression of endosomal proteins GTPases Rab 5 and Rab 7 was evaluated in an in vitro study of infection of goat macrophages. Blood was collected from ten Canindé goats, infected with biofilm-producing and non biofilm-producing strains of C. pseudotuberculosis. Blood cells were separated in colloidal silica-polyvinylpyrrolidone gradients (GE Healthcare®). These cells were maintained at 37 °C, with 5% of CO2. After differentiation, macrophages were infected with the mentioned strains. The bacterial pellets were marked with Rab 5 and Rab 7 antibodies, and their expression was observed by flow cytometry. Both strains of C. pseudotuberculosis (biofilm-producing and non biofilm-producing) were observed to be capable of altering the expression of Rab proteins in macrophages cultivated in vitro. Macrophages from the animals infected with the biofilm-producing strain had an increase in the expression of Rab 5 protein, mainly when these macrophages were treated with the non biofilm-producing strain. The same mechanism was shown to function with Rab 7 protein, however at a lower intensity of expression when compared with Rab 5.

Comparative Proteomic Analyses Between Biofilm-Forming and Non-biofilm-Forming Strains of Corynebacterium pseudotuberculosis Isolated From Goats.

Caseous lymphadenitis (CLA) is a chronic disease that affects small ruminants and causes economic losses in the associated breeding system. The causative agent of CLA is Corynebacterium pseudotuberculosis, a Gram-positive bacterium that exhibits tropism for external and internal lymph nodes and induces abscess formation in the host. Bacterial communities often produce a biofilm matrix that serves various functions, including protection against hostile environmental conditions, antibiotics, and the host immune response. Although biofilm formation has been reported for C. pseudotuberculosis, not all strains demonstrate this property in culture. In this work, we report the first comparative proteomic analysis of one biofilm-forming (CAPJ4) and one biofilm-non-forming strain (CAP3W) of C. pseudotuberculosis isolated from goats. Bacterial whole cell protein extracts were obtained for mass spectrometry analyses. Using LC-MS/MS, our studies reveal three and four proteins exclusively found in the CAPJ4 and CAP3W proteome, respectively. In addition, label-free quantitative analysis identified 40 proteins showing at-least 2-fold higher values in CAPJ4 compared CAP3W proteome Notably, CAPJ4 differentially synthesized the penicillin-binding protein, which participates in the formation of peptidoglycans. CAPJ4 also exhibited upregulation of N-acetylmuramoyl-L-alanine amidase and galactose-1-phosphate uridylyltransferase, which are involved in biofilm formation and exopolysaccharide biosynthesis. Here, we demonstrate that biofilm formation in C. pseudotuberculosis is likely associated with specific proteins, some of which were previously shown to be associated with virulence and biofilm formation in other organisms. Our findings may drive studies related to the bacterial mechanisms involved in the biofilm formation, in addition to providing targets for the treatment of CLA.

Profiles of Staphyloccocus aureus isolated from goat persistent mastitis before and after treatment with enrofloxacin.

BACKGROUND: Staphylococcus aureus is one of the main causative agents of mastitis in small ruminants. Antimicrobial use is the major treatment, but there are many flaws linked to resistance, tolerance or persistence. This study aimed to verify changes in resistance, virulence and clonal profiles of S. aureus isolated from persistent mastitis goat milk before and after enrofloxacin treatment. RESULTS: MIC increased to at least one antimicrobial in S. aureus isolates after enrofloxacin treatment compared to before. The most detected resistance genes before and after treatment were tetK, tetM, and blaZ, with more resistance genes detected after enrofloxacin treatment (p < 0.05). Occasional variations in efflux system gene detection were observed before and after treatment. Nine virulence genes (hla, fnbA, fnbB, eta, etb, sea, sec, seh, and sej) were detected at both times, and between these, the hla and eta genes were detected more in isolates after treatment. All isolates of S. aureus belonged to the same sequence type (ST) 133, except for two S. aureus isolates prior to enrofloxacin treatment which were classified as ST5 and the other as a new one, ST4966. Isolates of S. aureus 4, 8, and 100 from before and after treatment had identical pulse types, while others obtained from other animals before and after treatment were classified into distinct pulse types. CONCLUSION: There were occasional changes in the studied profiles of S. aureus isolated before and after treatment of animals with enrofloxacin, which may have contributed to the permanence of bacteria in the mammary gland, even when using traditional treatment, resulting in persistent mastitis.

Anti-biofilm Effect of ß-Lapachone and Lapachol Oxime Against Isolates of Staphylococcus aureus.

Antimicrobial resistance in bacteria, such as Staphylococcus aureus, has been the subject of many assistance studies of alternatives for the treatment of infections. These studies aim to solve this problem for bacteria, such as biofilm formation. Aiming to control the emergence of the problem or enhance antibiotic activity, the data sources are inserted into new therapeutic alternatives for the treatment of infections. ß-Lapachone and Lapachol Oxime are semi-synthetic derivatives of Lapachol with antimicrobial potential. Clinical isolates from human blood cultures were used in this study. Scanning electron microscopy (SEM) was performed following the glutaraldehyde fixation protocol. The presence of ß-Lapachone and Lapachol Oxima interfered in the biofilm formation state. In the MEV, the effect was observed in the reduction of the population of biofilm-forming cells. Therefore, it was possible to conclude the promising potential of the anti-biofilm of substances, justifying the nature of the natural products as agents of inspiration for the detection of new compounds with the biological function.

Nutritional interference for phenotypic biofilm quantification in Aeromonas spp. isolates containing the fla gene.

The Aeromonas genus has several virulence factors associated with the development of diseases in aquatic organisms, leading to losses in aquaculture. One of these factors is the flagella's formation which allows the biofilm's formation that provides the microorganisms a greater pathogenicity, greater protection to certain substances such as antibiotics. The aim of the study was to verify the presence of the fla gene, related to biofilm production in isolates of Aeromonas spp. from fishes and also to determine the best quantification condition of phenotypic biofilm production in vitro. Polymerase Chain Reactions were performed to obtain the amplification of the region comprising the fla gene. To determine the best condition for the production biofilm, the microplate adhesion test was carried out under different concentrations of TSB broth and it combined with glucose. Of the 43 isolates of Aeromonas spp. analyzed, 28 were positive for the fla gene and, in the quantification of the biofilm, all these were able to form biofilm in the TSB broth without dilution and without addition of glucose, being this the best condition tested. It was observed that the isolates of Aeromonas spp. analyzed have potential for biofilm formation, and hence potential for virulence.