Molecular characterization of Staphylococcus aureus from bovine mastitis and antibiofilm activity of a C-type lectin

Staphylococcus aureus é um dos principais patógenos responsáveis à mastite bovina subclínica que, frequentemente, evolui para a forma crônica. A caracterização de cepas circulantes em rebanhos leiteiros é importante para o desenvolvimento de métodos de diagnóstico precoces e identificação de marcadores de prognóstico. Esse trabalho foi dividido em três capítulos. No capítulo I, isolados bovinos coletados em diversas fazendas dos Estados de Minas Gerais e Rio de Janeiro foram caracterizados com base em características moleculares e fisiológicas. Observou-se a predominância do spa t605 e do grupo agr tipo II sobre os demais. A maioria dos isolados apresentou moderada produção de biofilme e uma alta sensibilidade aos antimicrobianos testados. Esses resultados aumentam o conhecimento sobre as cepas disseminadas em rebanhos brasileiros e contribuem para definição de estratégias de combate à mastite no país. No capítulo II, realizou-se uma busca por marcadores de prognóstico em 285 isolados de S. aureus originários do Canadá. Os isolados foram coletados de animais com manifestação de mastite clínica ou subclínica, ao longo do ciclo lactacional da vaca, sendo a persistência validada por método molecular. Os isolados que persistiam do período seco até a próxima lactação produziram mais biofilme que os não persistentes. Os isolados clínicos foram menores produtores de biofilme quando comparados aos isolados subclínicos. A produção de biofilme foi inversamente porporcional à expressão do gene hld. Dezoito tipos de spa foram encontrados, sendo o t529 o mais predominante. Para os isolados em lactação, o gene seg foi associado com a redução da chance da bactéria ser persistente. No capítulo III, uma busca por substâncias com atividade antipatogênica em veneno de Bothrops jararacussu foi realizada. Frações obtidas por cromatografia de exclusão molecular mostraram atividade anti-biofilme quando testadas sobre S. aureus e S. epidermidis. Espectrometria de massas identificou uma lectina que foi purificada por cromatografia de afinidade, cuja atividade anti-biofilme foi comprovada sobre diferentes espécies de bactérias causadoras de mastite bovina. Esse trabalho revelou uma nova atividade biológica para lectinas do tipo C, que pode ser testada como estratégia complementar no combate de infecções causadas por S. aureus.Staphylococcus aureus is a major pathogen responsible for subclinical bovine mastitis that often progresses to a chronic form. Molecular characterization of strains circulating in dairy herds is important to develop methods for early diagnosis and to identify prognostic markers which may help in the definition of appropriate mastitis control. This work was divided into three chapters. In Chapter I, bovine isolates collected from several farms in the states of Minas Gerais and Rio de Janeiro were characterized based on molecular and physiological characteristics. The spa type 605 and the agr type II predominated over other groups. Most isolates showed a moderate biofilm production and a high sensitivity to the tested antibiotics. These results increase our knowledge of the strains disseminated in Brazilian herds and improve herd management and disease control in the country. In Chapter II, we performed a search for prognostic markers in 285 isolates of S. aureus originated from Canada. The isolates were collected from animals showing clinical or subclinical mastitis during the lactational cycle of the cow, and persistence was validated by a molecular approach. The isolates that persisted during dry period through freshening produced more biofilm than non-persistent ones. Clinical isolates produced less biofilm than subclinical isolates. Biofilm production inversely correlated to hld expression level. Eighteen spa types were found, but t529 was more prevalent. For subclinical lactational isolates, the gene seg was associated with a reduction in the odds of the bacteria being persistent. In Chapter III, a search for substances found with anti-pathogenic activity was performed in the venom of Bothrops jararacussu. Fractions obtained by size exclusion chromatography showed anti-biofilm activity when tested on S. aureus and S. epidermidis. Mass spectrometry identified a lectin that was purified by affinity chromatography, whose anti-biofilm activity was demonstrated on different species of bovine mastitis bacteria. This work revealed a new biological activity for C-type lectins that can be tested as a complementary strategy to combat infections caused by S. aureus.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Gene expression of Staphylococcus aureus isolated from bovine mastitis in response to subinhibitory concentrations of antibiotics

Staphylococcus aureus é um dos principais micro-organismos causadores da mastite bovina, doença que provoca as maiores perdas na pecuária leiteira mundial. Este patógeno possui diversos fatores de virulência que contribuem para a grande diversidade genética observada entre isolados e auxiliam no estabelecimento das infecções. Nos últimos anos, vários trabalhos têm demonstrado que o uso de antibióticos em concentrações subinibitórias modula a expressão gênica influenciando a virulência de patógenos bacterianos. Este trabalho teve por objetivo investigar o efeito de concentrações subinibitórias de ampicilina, gentamicina, oxacilina e tilosina, antibióticos usados em formulações veterinárias para o tratamento da mastite, na expressão de genes de duas cepas de S. aureus de origem bovina. Inicialmente, foi realizada uma investigação da presença de genes que codificam alguns fatores de virulência em 85 bactérias isoladas de animais com manifestação de mastite bovina. Os genes clfB e sdr foram os mais prevalentes, sendo detectados em 83,5% e 75,3% dos isolados, respectivamente. A diversidade genética dos isolados, avaliada por PCR multiplex, também foi alta e permitiu a discriminação de mais de 60 grupos. Esses resultados nortearam a escolha de S. aureus 4006 e 4125, com dissimilaridade genética de 80%, para os ensaios posteriores. RNA total das duas culturas crescidas em valores equivalentes a 0,5X, 0,25X e 0,125X da concentração inibitória mínima definida para cada antibióticos foi extraído e usado em análises de RT-PCR em tempo real. A expressão dos genes spa, clfB, sdrC, fnBP, icaD, icaR, murF e sarA foi normalizada para o gene gyrB. Todos os antibióticos testados causaram alteração na expressão dos genes avaliados. Um mesmo antibiótico usado em diferentes doses, assim como diferentes antibióticos em dose similar, modulou diferencialmente a expressão dos genes. Para o isolado 4006, a proteína regulatória SarA, que regula a transcrição de vários fatores de virulência, foi muita expressa em vários tratamentos. Já para o isolado 4125, o regulador icaD foi positivamente influenciado, quando diferentes condições foram testadas. Em suma, os dados confirmam que genes de S. aureus se expressam de forma diferenciada em resposta a concentrações de antibióticos abaixo das consideradas inibitórias e que a variação existente entre cepas de S. aureus dificulta que um padrão de expressão seja estendido a toda espécie.Staphylococcus aureus is one of the main micro-organisms causing bovine mastitis, a disease that causes the greatest losses in dairy farming worldwide. This pathogen has several virulence factors that contribute to the great genetic diversity observed among isolates and assist in the establishment of infections. In recent years, several studies have shown that the use of antibiotics in subinhibitory concentrations modulates gene expression, influencing the virulence of bacterial pathogens. This study aimed to investigate the effect of subinhibitory concentrations of ampicillin, gentamicin, oxacillin and tylosin, antibiotics used in veterinary formulations for the treatment of mastitis, in the expression of genes from two strains of S. aureus of bovine origin. Initially, we conducted a preliminary investigation of the presence of genes encoding some virulence factors in 85 bacteria isolated from animals with bovine mastitis outbreak. Genes clfB and sdrCDE were the most prevalent, detected in 83.5% and 75.3% of the isolates, respectively. The genetic diversity of isolates, assessed by multiplex PCR, was also high and allowed the discrimination of more than 60 groups. These results guided the choice of S. aureus 4006 and 4125, with genetic similarity of 80% for the later trials. Total RNA from two cultures grown in values equivalent to 0.5X, 0.25X, 0.125X and the minimum inhibitory concentration defined for the four antibiotics was extracted and used in the analysis of real time RT-PCR. The expression of genes spa, clfB, sdrC, fnBP, icaD, icaR, murf and sarA was normalized to the gyrB gene. All antibiotics tested caused alterations in the expression of genes evaluated. The same antibiotics used in different doses, as well as different antibiotics at the same dose, differentially modulate the expression of genes. Only for 4006, the SarA regulatory protein, which regulates the transcription of several virulence factors, was expressed in many different treatments. As for 4125 isolate, the icaD regulator gene was positively influenced when different conditions were tested. In short, the data confirm that genes of S. aureus are expressed differently in response to concentrations of antibiotics below the considered inhibitory concentration and that the variance between strains of S. aureus makes difficult a pattern of expression is extended to all species.Conselho Nacional de Desenvolvimento Científico e Tecnológic

A C-Type Lectin from <i>Bothrops jararacussu</i> Venom Disrupts Staphylococcal Biofilms

<div><p>Bovine mastitis is a major threat to animal health and the dairy industry. <i>Staphylococcus aureus</i> is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of <i>Bothrops jararacussu</i> for antibiofilm activity against <i>S</i>. <i>aureus</i> NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against <i>S</i>. <i>aureus</i>. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against <i>S</i>. <i>epidermidis</i> NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of <i>S</i>. <i>aureus</i> and <i>S</i>. <i>epidermidis</i> biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed <i>S</i>. <i>epidermidis</i> biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of <i>S</i>. <i>aureus</i>, <i>S</i>. <i>hyicus</i>, <i>S</i>. <i>chromogenes</i>, <i>Streptococcus agalactiae</i>, and <i>Escherichia coli</i>. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model.</p></div

The C-type lectin prevents biofilm production in different bacterial species.

<p>The bacteria <i>E</i>. <i>coli</i> (<i>Escherichia coli</i>), <i>S</i>. <i>agal</i>. (<i>Streptococcus agalactiae</i>), <i>S</i>. <i>chro</i>. (<i>Staphylococcus chromogenes</i>), <i>S</i>. <i>hyic</i>. (<i>Staphylococcus hyicus</i>), <i>Sa</i> 2878 (<i>Staphylocccus aureus</i> 2878), <i>Sa</i> 4082 (<i>S</i>. <i>aureus</i> 4082), <i>Sa</i> 4130 (<i>S</i>. <i>aureus</i> 4130), <i>Sa</i> 4157 (<i>S</i>. <i>aureus</i> 4157) and <i>Sa</i> 4651 (<i>S</i>. <i>aureus</i> 4651) were grown in BHIg containing 50 µg/mL lectin for 22 h at 37°C. Biofilm production was monitored by reading the OD_560nm after staining with crystal violet. The percentage of biofilm disruption is shown relative to that observed with PBS, which was used as a control. The values are the means (± SD) of three independent experiments.</p

The C-type lectin prevented biofilm formation in a dose-dependent manner.

<p>The C-type lectin was incubated at 37°C for 22 h in serial dilutions from 100 µg/mL to 0.19 µg/mL. The bacterial growth (OD_600nm) and biofilm production (OD_560nm) of <i>S</i>. <i>aureus</i> NRS155 (A) and <i>S</i>. <i>epidermidis</i> NRS101 (B) were monitored. The percentage of bacterial growth and biofilm production was calculated relative to that observed with PBS and expressed in percentage. The results are the average of three independent experiments ± SD.</p

A C-type lectin was identified by TripleTOF 5600 MS.

<p>The mass spectrometer was fitted with a nanospray III ion source and coupled to an Agilent 1200 HPLC. A typical spectrum of a single peptide [(K)IFNELKAWK(D), m/z, 574.83] is shown (A). The mass and the amino acid sequence of the C-type lectin are shown and the sequence obtained from spectrometry is underlined (B). This protein was identified as Q7T228 in Uniprot database with coverage of 94%.</p

Draft genome sequences of Staphylococcus aureus strains isolated from subclinical bovine mastitis in Brazil

Here, we present the draft genome sequences of four Staphylococcus aureus strains isolated from mastitic milk collected from animals with subclinical manifestations. Three of them were typed as sequence type 126 (ST126), a genotype with no genome sequence available. ST126 is found in several herds of southern Brazil and is described as a bovine pathogen strongly associated with milk around the world

Effect of fractions 15 and 16 on bacterial growth and biofilm production.

<p><i>Staphylococcus aureus</i> NRS155 (A) and <i>S</i>. <i>epidermidis</i> NRS101 (B) were grown at 37ºC in BHIg containing fraction 15 or 16 (or saline for the control). The bacterial growth (OD_600nm) and biofilm biomass (OD_560nm) were measured using a multidetection microplate reader. Inlets Biofilm production measured for <i>S</i>. <i>aureus</i> (A) and <i>S</i>. <i>epidermidis</i> (B) grown in for 22 h at 37°C in BHIg containing 20 µg/mL of fraction 15 or 16. The percentage of biofilm production was calculated relative to the control (BHIg containing saline instead of fraction 15 or 16) which was set to 100%. Results represent the average of three independent experiments ± SD.</p

Effect of fractions purified from <i>Bothrops jararacussu</i> venom on <i>Staphylococcus aureus</i> growth and biofilm biomass.

<p><i>Staphylococcus aureus</i> NRS155 was grown in BHI with 0.25% glucose at 37°C for 22 h in contact with the fractions. Bacterial growth was determined by measuring OD_600nm (A), and the biofilm biomass was determined by measuring OD_560nm (B). The results are the average of three independent experiments ± SD.</p

Affinity chromatography of the C-type lectin.

<p>The lectin from <i>Bothrops jararacussu</i> venom was purified using a D-galactose column (3 cm x 1 cm, I.D.). The adsorbed proteins (lectin) were eluted with PBS (pH 7.4) containing 300 mM galactose (dashed line) (A). The protein concentration was monitored by reading the OD_280nm (solid line). SDS-PAGE analysis of the purified lectin from <i>B</i>. <i>jararacussu</i> venom (B). The gel was loaded with 25 µL of the following samples: Molecular Mass Marker (MM, kDa), crude venom diluted 1:100 in PBS (venom), proteins not bound to D-galactose column (waste), purified protein eluted with 300 mM galactose (lectin).</p