Antimicrobial therapy approaches in the mastitis control driven by one health insights.

The use of antimicrobials in the dairy production environment for mastitis control must take etiology, clinical signs, economic impacts, and regulatory frameworks into consideration. The objective of the present review is to highlight important aspects of the dynamics of antimicrobial use in dairy production and the potential impacts on the main pathogens circulating in this environment, considering the parameters set by the World Health Organization (WHO) regarding the priority of monitoring as well as control strategies for these agents, such as the methicillin-resistant Staphylococcus and the beta-lactamase-producing Escherichia coli. Understanding the animal-environment-pathogen triad is crucial for establishing control measures and preventing the spread of bacterial resistance. Implementing mastitis prevention and control measures in dairy farms, considering process flow and personnel qualification, enables a reduction in antimicrobial usage and contributes to prevent the spread of resistant bacterial agents in the dairy production environment, minimizing the relapses and the chronicity of the infectious process.

A utilização de antimicrobianos no controle de mastite em ambiente de produção leiteira deve considerer alguns aspectos como a etiologia, os sinais clínicos, os impactos económicos e a legislação. O objetivo da presente revisão é destacar aspectos importantes na dinâmica do uso de antimicrobianos na produção leiteira e os potenciais impactos sobre os principais patógenos circulantes neste ambiente, considerando os parâmetros estabelecidos pela Organização Mundial da Saúde (OMS) quanto à prioridade de monitoramento, bem como estratégias de controle para esses agentes, como o Staphylococcus resistente à meticilina e a Escherichia coli produtora de beta-lactamase. Compreender a tríade animal-ambiente-patógeno é crucial para estabelecer medidas de controle e prevenir a propagação da resistência bacteriana. A implementação de medidas de prevenção e controle de mastites nas propriedades leiteiras, considerando o fluxo do processo e a qualificação do pessoal, permite a redução do uso de antimicrobianos e contribui para prevenir a propagação de agentes bacterianos resistentes no ambiente de produção leiteira, minimizando as recidivas e a cronicidade do processo infeccioso.

Poultry slaughterhouse wastewater as a source of bacterial antimicrobial resistance.

Slaughterhouses produce huge volumes of effluents throughout the production chain that, when discharged untreated into bodies of water, can become a source of environmental contamination. This is particularly worrisome if these effluents are used for irrigation since they increase contamination levels and spread pathogens and resistance determinants to humans and animals. Therefore, in this study, we assessed antimicrobial resistance in bacteria isolated from inlet water, equalization wastewater tanks, treatment plant wastewater, and treated wastewater in slaughterhouse facilities in Rio de Janeiro, Brazil. Four samples were collected at each of the collection points, between June 2021 and July 2022. Following bacterial isolation and identification, the samples were analyzed for antimicrobial resistance using the disk diffusion method to test aminoglycoside, beta-lactam, and fluoroquinolone antimicrobials. A total of 229 bacteria were isolated, with 74 isolates selected from the genera Citrobacter (12), Enterobacter (14), Klebsiella (35), Serratia (5), and Pseudomonas (8). Inlet water had the lowest number of isolates and was the only point with gentamicin-resistant isolates. Raw effluent from the equalization tank showed the highest number of isolated bacteria and resistance levels, followed by treated wastewater and the treatment plant. Across all samples, a high rate of cefoxitin-resistance was observed among the isolated bacteria. Klebsiella pneumoniae stood out as the species that demonstrated the greatest resistance to a variety of antimicrobials. These results highlight the importance of water quality monitoring in mitigating public health and environmental risks and high antimicrobial resistance levels.

Effect of long-term liquid dairy manure application on activity and structure of bacteria and archaea in no-till soils depends on plant in development.

This study aimed to evaluate the impact of long-term liquid dairy manure (LDM) application on the activity and structure of soil bacterial and archaea communities in two cropping seasons over 1 year of a no-till crop rotation system. The experiment was run in a sandy clay loam texture Oxisol, in Brazil, including LDM doses of 60, 120, and 180 m3 ha-1 year-1, installed in 2005. Soil sampling was conducted during spring 2018 and autumn 2019 at 0-10-cm depth. Microbial biomass carbon and nitrogen, 16S rRNA gene sequencing, microbial respiration and quotient were performed. Over the 14-year period, LDM application increased soil microbial community activity. Analysis of 16S rRNA gene sequencing revealed dominance by Proteobacteria, Acidobacteria, and Actinobacteria phyla (67% in spring and 70% in autumn). Genera Pirulla and Nitrososphaera showed enrichment at LDM doses of 120 and 180 m3 ha-1 year-1 doses, respectively. During spring, following black oat cropping, shifts in the relative abundance of Bacteroidetes, Proteobacteria, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Chloroflexi, Actinobacteria, and AD3 phyla were observed due to LDM application, correlating with soil chemical indicators such as pH, K, Ca, Mn, and Zn. Our findings indicate that plant development strongly influences microbial community composition, potentially outweighing the impact of LDM. Our findings indicate that the application of liquid dairy manure alters the soil bacterial activity and community; however, this effect depends on the developing plant.

Rhizosphere-associated microbiota of Canavalia ensiformis in sulfentrazone bioremediation.

The objective of this study was to determine the efficiency of the microbial rhizosphere (Canavalia ensiformis) in the phytoremediation of sulfentrazone using quantification methods (CO2 evolution, microbial biomass carbon, and metabolic quotient) and identification of bacteria (PCR-DGGE technique). The experiment was conducted in a completely randomized design, in a 2x4 factorial scheme, with four replications. The treatments were composed of rhizospheric soil (cultivated with C. ensiformis) and non-rhizosphere soil (uncultivated soil); and four levels of contamination by sulfentrazone (0, 200, 400, and 800 g ha-1 a.i.). The microbiota associated with the rhizosphere of C. ensiformis efficiently reduced sulfentrazone residues in the soil, with better performance at the dose of 200 g ha-1 a.i. Using the PCR-DGGE technique allowed the distinction of two profiles of bacteria in the rhizospheric activity of C. ensiformis. The second bacterial profile formed was more efficient in decontaminating soil contaminated with sulfentrazone residue. The microbiota associated with the rhizosphere of C. ensiformis has an efficient profile in decontaminating soils with residues equivalent to 200 g ha-1 a.i. the herbicide sulfentrazone.

Phytoremediation of soils contaminated with herbicide residues is a viable technique for decontamination of the environment.Canavalia ensiformis has an efficient profile in the decontamination of soils with residue equivalent to 200 g ha⁻¹ a.i. of the herbicide sulfentrazone.The PCR technique and microbial respiration used to analyze the diversity and estimate the bacterial population of a soil are viable tools to evaluate the phytoremediation potential of the microbiota associated with plant species.

Chemical attributes, bacterial community, and antibiotic resistance genes are affected by intensive use of soil in agro-ecosystems of the Atlantic Forest, Southeastern Brazil.

Soil is one of the largest reservoirs of microbial diversity in nature. Although soil management is vital for agricultural purposes, intensive practices can have a significant impact on fertility, microbial community, and resistome. Thus, the aim of this study was to evaluate the effects of an intensive soil management system on the chemical attributes, composition and structure of prevalent bacterial communities, and presence and abundance of antimicrobial resistance genes (ARGs). The chemical characterization, bacterial diversity and relative abundance of ARGs were evaluated in soils from areas of intensive vegetable cultivation and forests. Results indicate that levels of nutrients and heavy metals were higher in soil samples from cultivated areas. Similarly, greater enrichment and diversity of bacterial genera was detected in agricultural areas. Of the 18 target ARGs evaluated, seven were detected in studied soils. The oprD gene exhibited the highest abundance among the studied genes and was the only one that showed a significantly different prevalence between areas. The oprD gene was identified only from soil of the cultivated areas. The blaSFO, erm(36), oprD and van genes, in addition to the pH, showed greater correlation with in soil of cultivated areas, which in turn exhibited higher contents of nutrients. Thus, in addition to changes in chemical attributes and in the microbial community of the soil, intensive agricultural cultivation systems cause a modification of its resistome, reinforcing the importance of the study of antimicrobial resistance in a One Health approach.

Influence of nutrient, toxic metal and herbicide contents on the soil bacterial communities in tropical vegetable growing areas.

The relationship between bacterial diversity and the bioavailability of nutrients, toxic metals and the herbicide oxyfluorfen in a tropical vegetable growing area was evaluated. The study was conducted in a vegetable growing area located in the mountainous region of Rio de Janeiro (Brazil), and samples were collected in areas of vegetable cultivation and areas of environmental reserve. Fertility analyses and determination of the pseudototal levels of toxic metals in the soil samples were performed. The profile of the soil bacterial community was determined by amplification of the 16S rRNA gene and separation by DGGE. The results showed that the levels of toxic metals and elements associated with soil fertility were higher in vegetable production areas. These differences in the physical and chemical characteristics of the soil favored the presence of a greater number of OTUs in the cultivation areas (17.3-27 OTUs) than in the areas of environmental reserve (13-22 OTUs). Therefore, this study demonstrates that the presence of toxic metals and the herbicide oxyfluorfen and the increase in fertility in soils in areas with intensive vegetable cultivation resulting from the intensive management adopted in these areas promotes a differentiation of the bacterial profiles in soils in tropical vegetable growing areas.

Crotalaria juncea L. enhances the bioremediation of sulfentrazone-contaminated soil and promotes changes in the soil bacterial community.

Sulfentrazone (STZ) is an efficient tool for the pre- and post-emergence control of monocotyledonous and dicotyledonous weeds in fields of crops such as pineapple, coffee, sugarcane, citrus, eucalyptus, tobacco, and soybean. However, this herbicide persists in the soil, causing phytotoxicity in the subsequent crop. Therefore, it is important to use efficient strategies for the remediation of STZ-contaminated areas. The aim of this study was to evaluate the effects of Crotalaria juncea L. on the remediation of STZ-contaminated soil and on the microbial activity and bacterial community structure therein. The study was conducted in three stages: (i) cultivation of C. juncea in soil contaminated with 200, 400, and 800 g ha-1 STZ; (ii) determination of the soil microbial activity (basal respiration, microbial biomass carbon, and bacterial community structure); and (iii) cultivation of a bioindicator species and determination of the residual fraction of STZ. The soil microbial activity was impacted by the soil type and STZ dose. Soil previously cultivated with C. juncea (rhizospheric soil) displayed higher CO2 and lower qCO2 values than non-rhizospheric soil (no previous C. juncea cultivation). Increasing doses of STZ reduced the activity and lowered the diversity indices of the soil microorganisms. The bacterial community structure was segregated between the rhizospheric and non-rhizospheric soils. Regardless of soil type, the bioindicator of remediation (Pennisetum glaucum R.Br.) grew only at the STZ dose of 200 g ha-1, and the plant intoxication level was also lower in rhizospheric soil treated with this herbicide dose. All P. glaucum plants died in the soils treated with 400 and 800 g ha-1 STZ. Previous cultivation of C. juncea in soils contaminated with 200, 400, and 800 g ha-1 STZ reduced the residual fraction of the herbicide by 4.8%, 12.5%, and 17.4%, respectively, compared with that in the non-rhizospheric soils. In conclusion, previous cultivation with C. juncea promoted increases in the soil bacterial activity and diversity indices, mitigated the deleterious effects of STZ on the bioindicator crop, and reduced the residual fraction of the herbicide in the soil.

Entomopathogenic fungus treatment changes the gut bacterial diversity of Rhipicephalus microplus ticks.

BACKGROUND: Ticks are obligate bloodsucking parasites responsible for significant economic losses and concerns with human and animal health, mainly due to the transmission of pathogens. Entomopathogenic fungi have been intensively studied as an alternative strategy for tick control that can be used in combination with synthetic acaricides in the integrated management of ticks. Here, we investigated how the gut bacterial community of Rhipicephalus microplus is shaped after Metarhizium anisopliae treatment and how the tick susceptibility to the fungus is affected after disrupting gut bacterial microbiota. METHODS: Partially engorged tick females were artificially fed with pure bovine blood or blood plus tetracycline. Two other groups received the same diet and were topically treated with M. anisopliae. The guts were dissected, and the genomic DNA was extracted 3 days after the treatment; the V3-V4 variable region of the bacterial 16S rRNA gene was amplified. RESULTS: The gut of ticks that received no antibiotic but were treated with M. anisopliae exhibited lower bacterial diversity and a higher occurrence of Coxiella species. The Simpson diversity index and Pielou equability coefficient were higher in the gut bacterial community when R. microplus were fed with tetracycline and fungus-treated. Ticks from fungus-treated groups (with or without tetracycline) exhibited lower survival than untreated females. Previous feeding of ticks with the antibiotic did not change their susceptibility to the fungus. Ehrlichia spp. were not detected in the gueated groups. CONCLUSIONS: These findings suggest that myco-acaricidal action would not be impacted if the calf hosting these ticks is under antibiotic therapy. Moreover, the hypothesis that entomopathogenic fungi can affect the bacterial community in the gut of R. microplus engorged females is endorsed by the fact that ticks exposed to M. anisopliae exhibited a dramatic reduction in bacterial diversity. This is the first report of an entomopathogenic fungus affecting the tick gut microbiota.

Bacteria and antimicrobial resistance profile during the composting process of wastes from animal production.

Livestock waste is widely used in agriculture. Although they provide benefits to the soil, and consequently to plants, they have the potential to contaminate the environment, as they contain pathogenic microorganisms and determinants of antimicrobial resistance, if not properly managed. Therefore, this study aims to evaluate the effect of composting horse bedding and poultry litter in organic and conventional production systems on the occurrence of bacteria in the Enterobacteriales order and to identify their antimicrobial resistance profiles. Bacterial strains were isolated from Salmonella-Shigella and eosin methylene blue solid media from animal waste during the composting process that was conducted for 125 days. After isolation, the strains were identified by the MALDI-TOF technique; the disk diffusion test was then performed for phenotypic detection of antimicrobial resistance. A total of 158 bacterial strains were isolated during composting of three wastes. The Enterobacteriaceae family was the most abundant, whereas Proteus mirabilis and Escherichia coli were the species with the highest percentage in the wastes, which also exhibited a multi-resistance profile. Poultry litter showed a greater abundance of resistant bacteria than horse bedding did. Similarly, a greater number of resistant bacteria was detected in conventional poultry litter than in organic poultry litter. The results obtained reinforce that animal wastes are reservoirs of pathogenic bacteria that are resistant to antimicrobials and highlight the importance of developing management strategies that aim to reduce and/or eliminate these contaminants to guarantee their safe use in agriculture.

Bacterial diversity and detection of resistance genes to broad-spectrum betalactams in dairy family farm soils / Diversidade bacteriana e detecção de genes de resistência a betalactâmicos de amplo espectro em solos de propriedade familiar leiteira

Bovine mastitis is a complex disease that brings great losses to the dairy producer. The microbial diversity of the soils, as well as the presence of resistance genes in the environment directly influence the maintenance of mastitis in the farm. The objective of this work was to analyze the bacterial diversity in pasture soils of a dairy family farm, detecting enterobacteria that may be involved in the etiology of bovine mastitis, and to detect genes that encode broad-spectrum betalactamases in these soils. Twelve soil samples, representative of different areas of the farm located in the municipality of Barra do Piraí, Rio de Janeiro, were collected at different times of the year. Total DNA was extracted from the samples, gene amplified by Nested-PCR and then the amplification products were separated by DGGE (Denaturing Gradient Gel Electrophoresis). With the DGGE it was possible to construct dendograms that effectively represented the bacterial diversity of these soils. Eight of the soil samples were used to amplify the genes encoding the betalactamase enzymes TEM (blaTEM gene), SHV (blaSHV gene) and CTX (blaCTXM gene). In three of the eight soil samples, the blaSHV gene was found to be present. The blaTEM and blaCTX-M genes were not detected in any of the samples. The detection of genes encoding broad-spectrum betalactamases in dairy cattle pasture soils is of concern, because the transfer of gene material between pathogenic and non-pathogenic bacteria in this environment is a reality

A mastite bovina é uma doença complexa que traz grandes prejuízos ao produtor de leite. A diversidade micro-biana dos solos, bem como a presença de genes de resistência no ambiente, influencia diretamente a manutenção da mastite na fazenda. O objetivo deste trabalho foi analisar a diversidade bacteriana em solos de pastagem de uma propriedade familiar leiteira, detectando enterobactérias que possam estar envolvidas na etiologia da mastite bovina, e detectar genes que codificam betalactamases de amplo espectro nesses solos. Doze amostras de solo, representativas de diferentes áreas da fazenda localizada no município de Barra do Piraí, Rio de Janeiro, foram coletadas em diferentes épocas do ano. O DNA total foi extraído das amostras, o gene foi amplificado por Nested-PCR e, em seguida, os produtos de amplificação foram separados por DGGE (Denaturing Gradient Gel Electrophoresis). Com a DGGE, foi possível construir dendogramas que representavam efetivamente a diversidade bacteriana desses solos. Oito das amostras de solo foram usadas para amplificar os genes que codificam as enzimas betalactamase TEM (gene blaTEM), SHV (gene blaSHV) e CTX (gene blaCTXM). Em três das oito amostras de solo, foi constatada a presença do gene blaSHV. Os genes blaTEM e blaCTX-M não foram detectados em nenhuma das amostras. A detecção de genes que codificam betalactamases de amplo espectro em solos de pastagens de gado leiteiro é preocupante, pois a transferência de material genético entre bactérias patogênicas e não patogênicas nesse ambiente é uma realidade

Production of extended-spectrum beta-lactamases in Escherichia coli isolated from poultry in Rio de Janeiro, Brazil.

The overuse of antimicrobials in poultry has led to the development and dissemination of multidrug-resistant bacteria in the poultry industry. One of the most effective mechanisms of resistance found in Escherichia coli is the production of extended-spectrum ß-lactamases (ESBL); there are several ESBLs, including the TEM, SHV, and CTX-M families. This resistance mechanism and the risks associated with transmitting these resistant microorganisms between animals, the environment, and humans can occur through direct contact and consumption of infected animals. This study aimed to determine the prevalence of E. coli in samples isolated from three broiler farms in Rio de Janeiro, Brazil, and screen the isolates for ESBL genes. The findings of this study demonstrated the presence of ESBL-producing E. coli in all farms studied. The findings of this study highlight the urgency for a program to monitor the poultry industry value chains at the regional level to control the spread of antimicrobial resistance. Therefore, we recommend that the enzyme subtypes produced by bacterial isolates should be determined to effectively characterize the distribution of genes related to antimicrobial resistance.

O uso excessivo de antimicrobianos em frangos de corte tem contribuído para o desenvolvimento e disseminação de bactérias multirresistentes, e um dos mais relevantes mecanismos de resistência encontrados em Escherichia coli é a produção de enzimas denominadas ß-lactamases de espectro estendido (ESBL). CTX-M, SHV e TEM são as ß-lactamases mais comumente encontradas nesta espécie e as ESBL mais prevalentes globalmente. Esse mecanismo de resistência e o risco associado à transmissão desses microrganismos resistentes entre animais, meio ambiente e seres humanos se devem principalmente ao contato direto e ao consumo de origem animal. Este trabalho buscou elucidar a prevalência de E. coli em amostras de três granjas de frangos de corte localizadas no Rio de Janeiro, Brasil, e caracterizá-las de acordo com seu genótipo. O estudo demonstrou uma presença consistente de E. coli produtora de ESBL com presença abundante do gene bla SHV nos isolados de todas as fazendas estudadas. Deste modo, este estudo teve como objetivo contribuir com dados epidemiológicos relativos à distribuição de genes relacionados às ß-lactamases na produção animal, conscientizando sobre a transmissão desses microrganismos resistentes entre animais, meio ambiente e seres humanos contribuindo com dados epidemiológicos e de sua importância em uma perspectiva de saúde única.

Fecal Microbiome Responses to Sudden Diet Change in Mangalarga Marchador horses.

Sudden changes in horses' diet have been previously associated with gastrointestinal disease. This study evaluated the effects of a sudden change of diet composed exclusively of Coastcross hay (CHD) to a complete extruded diet (CED) on the fecal microbiome of horses. A completely randomized design with repeated measurements was used. The study started with eight adult horses randomly split into group A, fed with CHD, and group B, fed with CED. After 34 days of diet adaptation, the diets were abruptly changed between the groups. Fecal samples were collected at 0, 24, and 96 hours after the diet change, and the pH and microbiome analyses of the feces were subsequently evaluated. Changing from CHD to CED reduced the alpha diversity 24 hours after the alteration, with a decrease in the relative abundance of Firmicutes and an increase of Bacteroidetes. Fecal pH decreased and the relative abundance of Verrucomicrobia increased 96 hours after changing the diets. The community structure was also different after 96 hours of diet change. In contrast, 24 hours after changing from CED to CHD reduced fecal pH and abundance of Synergistetes. After 96 hours, there was an increase in the alpha diversity, and the abundance of the phylum Lentisphaerae. Group B showed no changes in the community structure when its diet was changed. Concluding, diet composition influenced the response of the equine fecal microbiome to sudden dietary changes.

Acinetobacter calcoaceticus-Acinetobacter baumannii complex in animals: identification and antimicrobial resistance profile / Complexo Acinetobacter calcoaceticus-Acinetobacter baumannii em animais: identificação e perfil de resistência antimicrobiana

Acinetobacter spp. is emerging as an important human and veterinary pathogen, mostly due to intrinsic and acquired resistance to antimicrobials. Despite its public health relevance, little is known about the prevalence, role of different Acinetobacter species and antimicrobial resistance profile of animal-origin isolates. Traditional phenotypic tests may fail to discriminate Acinetobacter species, therefore molecular analyses are often required as a complementary approach. The objectives of this study were to evaluate the occurrence of strains of the Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex isolated from animal infections including urinary tract infections, otitis, piodermitis and pododermatitis, and its resistance profile against different antimicrobial classes, including carbapenems. All Gram-negative coccobacilli isolates were characterized by MALDI-TOF and multiplex PCR, and the disk diffusion test was used to investigate multi-drug resistance (MDR) and carbapenem resistance genes by PCR as preconized by the standard guidelines. MALDI-TOF technique identified 21 strains belonging to the Acb complex (10 A. pittii, 8 A. baumannii, 3 A. nosocomialis, 1 A. ursingii, and 1 A. venetianus). Multiplex PCR confirmed the results of MALDI-TOF for 20 strains. Eight strains (34.78%) were classified as MDR, being 50% (4/8) A. baumannii, 37.5% (3/8) A. pittii, and 12.5% (1/8) A. nosocomialis. None of the isolates presented phenotypic carbapenemase production. Considering the carbapenem resistance genes, 26.09% (6/23) of the isolates presented one or more carbapenemase genes. From these, 50% (3/6) presented only bla VIM, 33.33% (2/6) presented only blaIMP, and 16.67% (1/6) presented blaIMP e blaVIM, simultaneously. These genes were detected among A. pittii isolates mostly (66.67%, 4/6). This study provides further insights into the occurrence and resistance profile of Acinetobacter of animal origin.

Acinetobacter spp. está emergindo como um importante patógeno humano e veterinário, principalmente devido à resistência intrínseca e adquirida aos antimicrobianos. Apesar de sua relevância para a saúde pública, pouco se sabe sobre a prevalência, o papel das diferentes espécies de Acinetobacter e o perfil de resistência antimicrobiana de isolados de origem animal. Testes fenotípicos tradicionais podem falhar em discriminar espécies de Acinetobacter, portanto, análises moleculares são frequentemente necessárias como uma abordagem complementar. Os objetivos deste estudo foram avaliar a ocorrência de cepas do complexo Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) isolados de infecções de animais, incluindo infecções do trato urinário, otite, piodermite e pododermatite, e seu perfil de resistência a diferentes classes de antimicrobianos, incluindo os carbapenêmicos. Todas as cepas cocobacilos Gram-negativas foram caracterizados por MALDI-TOF e PCR multiplex, e o teste de difusão em disco foi usado para investigar genes de resistência a múltiplas drogas (MDR) e resistência a carbapenêmicos por PCR conforme preconizado pelas diretrizes padrão. A técnica MALDI-TOF identificou 21 cepas pertencentes ao complexo Acb (10 A. pittii, 8 A. baumannii, 3 A. nosocomialis, 1 A. ursingii e 1 A. venetianus). Multiplex PCR confirmou os resultados de MALDI-TOF para 20 cepas. Oito cepas (34.78%) foram classificadas como MDR, sendo 50% (4/8) A. baumannii, 37.5% (3/8) A. pittii e 12.5% (1/8) A. nosocomialis. Nenhum dos isolados apresentou produção fenotípica de carbapenemases. Considerando os genes de resistência a carbapenemas, 26.09% (6/23) dos isolados apresentaram um ou mais genes de carbapenemases. Destes, 50% (3/6) apresentaram apenas bla VIM, 33.33% (2/6) apresentaram apenas bla IMP e 16.67% (1/6) apresentaram bla IMP e bla VIM, simultaneamente. Esses genes foram detectados principalmente entre os isolados de A. pittii (66.67%, 4/6). Este estudo fornece mais informações sobre a ocorrência e perfil de resistência de Acinetobacter de origem animal.

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis / Expressão dos genes icaA e icaD na formação de biofilme em isolados de Staphylococcus aureus de mastite subclínica bovina

Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.(AU)

Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.(AU)

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis

ABSTRACT: Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.

RESUMO: Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.

Sulfonamide resistance genes in soils treated with waste from animal production in an organic production system / Genes de resistência a sulfonamida em solos tratados com resíduos da produção animal em sistema orgânico de produção

Animal waste is widely used in organic production systems. However, these residues can increase antimicrobial determinants in the soil. In this perspective, this study was developed to evaluate the presence of sulfonamide resistance genes in soils from an organic production system that received animal waste as organic fertilizer. Soil samples were collected from four properties with different management practices to increase soil fertility. Three properties use the animal waste from the conventional system and the other use plant residues as soil cover and a legal reserve. The extraction of total DNA from soil was carried out followed by the amplification of genes encoding sulfonamide resistance (sul1 and sul2) by the PCR (polymerase chain reaction) technique. The sul1 and sul2 genes were detected only in soils treated with animal waste. The genes were not detected in soils from the legal reserve and the property that used plant residues as soil cover. These results indicate that the use of animal waste as agricultural fertilizer can increase genes for resistance to antimicrobials in the soil and the composting process may not be enough to eliminate them. This information reiterates the need to implement standards that establish quality parameters for animal waste, considering resistance to antimicrobials, as well as the development of management strategies that reduce the risk of spreading resistance to antimicrobials when these residues are applied to soils.(AU)

Resíduos animais são amplamente utilizados em sistemas de produção orgânicos. No entanto, esses resíduos podem incrementar determinantes antimicrobianos no solo. Nesta perspectiva, este estudo foi desenvolvido para avaliar a presença de genes de resistência à sulfonamida em solos do sistema de produção orgânico que receberam resíduos de animais como fertilizante orgânico. Amostras de solo foram coletadas de quatro propriedades com diferentes formas de manejo para aumentar a fertilidade do solo. Três utilizaram resíduos animais do sistema convencional; uma utilizou resíduos vegetais como cobertura do solo, além de uma reserva legal. Foi realizada a extração do DNA total do solo, seguida da amplificação dos genes que codificam resistência sulfonamida (sul1 e sul2) pela técnica de PCR (Polymerase Chain Reaction). Os genes sul1 e sul2 foram detectados apenas nos solos tratados com dejetos de animais. Não foram detectados nos solos da reserva legal e das propriedades que utilizavam resíduos vegetais como cobertura do solo. Esses resultados indicam que o uso de resíduos animais como fertilizante agrícola pode incrementar genes de resistência aos antimicrobianos no solo e que o processo de compostagem pode não ser suficiente para eliminá-los. Essas informações reiteram a necessidade de implementar padrões que estabeleçam parâmetros de qualidade para os resíduos animais, levando em conta a resistência aos antimicrobianos, bem como o desenvolvimento de estratégias de manejo que reduzam o risco de disseminação da resistência aos antimicrobianos quando esses resíduos são aplicados no solo.(AU)

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis / Expressão dos genes icaA e icaD na formação de biofilme em isolados de Staphylococcus aureus de mastite subclínica bovina

Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.(AU)

Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.(AU)

Sulfonamide resistance genes in soils treated with waste from animal production in an organic production system / Genes de resistência a sulfonamida em solos tratados com resíduos da produção animal em sistema orgânico de produção

Animal waste is widely used in organic production systems. However, these residues can increase antimicrobial determinants in the soil. In this perspective, this study was developed to evaluate the presence of sulfonamide resistance genes in soils from an organic production system that received animal waste as organic fertilizer. Soil samples were collected from four properties with different management practices to increase soil fertility. Three properties use the animal waste from the conventional system and the other use plant residues as soil cover and a legal reserve. The extraction of total DNA from soil was carried out followed by the amplification of genes encoding sulfonamide resistance (sul1 and sul2) by the PCR (polymerase chain reaction) technique. The sul1 and sul2 genes were detected only in soils treated with animal waste. The genes were not detected in soils from the legal reserve and the property that used plant residues as soil cover. These results indicate that the use of animal waste as agricultural fertilizer can increase genes for resistance to antimicrobials in the soil and the composting process may not be enough to eliminate them. This information reiterates the need to implement standards that establish quality parameters for animal waste, considering resistance to antimicrobials, as well as the development of management strategies that reduce the risk of spreading resistance to antimicrobials when these residues are applied to soils.

Resíduos animais são amplamente utilizados em sistemas de produção orgânicos. No entanto, esses resíduos podem incrementar determinantes antimicrobianos no solo. Nesta perspectiva, este estudo foi desenvolvido para avaliar a presença de genes de resistência à sulfonamida em solos do sistema de produção orgânico que receberam resíduos de animais como fertilizante orgânico. Amostras de solo foram coletadas de quatro propriedades com diferentes formas de manejo para aumentar a fertilidade do solo. Três utilizaram resíduos animais do sistema convencional; uma utilizou resíduos vegetais como cobertura do solo, além de uma reserva legal. Foi realizada a extração do DNA total do solo, seguida da amplificação dos genes que codificam resistência sulfonamida (sul1 e sul2) pela técnica de PCR (Polymerase Chain Reaction). Os genes sul1 e sul2 foram detectados apenas nos solos tratados com dejetos de animais. Não foram detectados nos solos da reserva legal e das propriedades que utilizavam resíduos vegetais como cobertura do solo. Esses resultados indicam que o uso de resíduos animais como fertilizante agrícola pode incrementar genes de resistência aos antimicrobianos no solo e que o processo de compostagem pode não ser suficiente para eliminá-los. Essas informações reiteram a necessidade de implementar padrões que estabeleçam parâmetros de qualidade para os resíduos animais, levando em conta a resistência aos antimicrobianos, bem como o desenvolvimento de estratégias de manejo que reduzam o risco de disseminação da resistência aos antimicrobianos quando esses resíduos são aplicados no solo.

Conjugative plasmidic AmpC detected in Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae human clinical isolates from Portugal.

AmpC is a type of ß-lactamase enzyme produced by bacteria; these enzymes are classified in Class C and Group 1, and these confer resistance to cephamycin. Enterobacterales producing AmpC are reported worldwide and have great clinical importance due to therapeutic restriction and epidemiological importance once the easy dissemination by plasmidic genes to other bacteria is a real threat. These genes are naturally found in some enterobacteria as Enterobacter cloacae, Morganella morganii, and Citrobacter freundii, but other species have demonstrated similar resistance phenotype of AmpC production. Genes carried in plasmids have been described in these species conferring resistance to cefoxitin and causing therapeutic failure in some bacterial infections. This work detected and described five clinical strains of Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae that presented plasmid ampC (pAmpC) isolated from the north of Portugal collected in 2009. AmpC production was confirmed by inhibition of the enzyme by cloxacillin and boronic acid in agar diffusion tests. Also, PCR (polymerase chain reaction) was performed for the detection of gene universal to AmpC, blaampC, and others to AmpC group: blaACC, blaCIT, blaCMY, blaDHA, and blaEBC. The conjugation in liquid medium for 24 h was realized to determine if gene is localized in chromosome or plasmid. The isolates and their conjugants showed phenotypic characteristics and blaCMY and blaCIT were detected by PCR corroborating the AmpC characteristics observed in these bacteria. Confirmation of transfer of plasmid containing genes encoding AmpC is of high epidemiological relevance to the hospital studied and demonstrated the importance of AmpC surveillance and studies in hospital and community environments in order to choose the appropriate therapy for bacterial infections.

Accuracy of PCR universal primer for methicillin-resistant Staphylococcus and comparison of different phenotypic screening assays.

Characterization of methicillin-resistant Staphylococcus (MRS) is a continuous challenge at diagnostic laboratories. The phenotypic methods present heterogeneous results and the occurrence of variants of mecA gene turned this goal even more challenging to achieve. The present study provided an accurate and highly discriminatory screening tool for MRS, improving its detection.