Diversity and pathogenesis of Staphylococcus aureus from bovine mastitis: current understanding and future perspectives

Staphylococcus aureus is a leading cause of bovine mastitis worldwide. Despite some improved understanding of disease pathogenesis, progress towards new methods for the control of intramammary infections (IMI) has been limited, particularly in the field of vaccination. Although herd management programs have helped to reduce the number of clinical cases, S. aureus mastitis remains a major disease burden. This review summarizes the past 16 years of research on bovine S. aureus population genetics, and molecular pathogenesis that have been conducted worldwide. We describe the diversity of S. aureus associated with bovine mastitis and the geographical distribution of S. aureus clones in different continents. We also describe studies investigating the evolution of bovine S. aureus and the importance of host-adaptation in its emergence as a mastitis pathogen. The available information on the prevalence of virulence determinants and their functional relevance during the pathogenesis of bovine mastitis are also discussed. Although traits such as biofilm formation and innate immune evasion are critical for the persistence of bacteria, the current understanding of the key host-pathogen interactions that determine the outcome of S. aureus IMI is very limited. We suggest that greater investment in research into the genetic and molecular basis of bovine S. aureus pathogenesis is essential for the identification of novel therapeutic and vaccine targets

Bovine mastitis: evaluation of antigens for diagnosis of Staphylococcus aureus and role of a LysR regulator in host-pathogen interaction

Infecções intramamárias causadas por Staphylococcus aureus são em sua maioria de natureza subclínica e evoluem frequentemente para persistente. O diagnóstico preciso dos animais infectados é fundamental para evitar a dissiminação do patógeno e prevenir o surgimento de novas infecções. A cultura bacteriológica é utilizada como padrão-ouro na identificação de patógenos causadores de mastite, mas a demora na obtenção de resultados ainda é um grande entrave. Os imunoensaios são uma abordagem alternativa para o diagnóstico que podem ser rápidos, específicos e, em alguns casos, realizados em campo. Na busca por marcadores para o diagnóstico da mastite bovina causada por S. aureus, avaliou- se a capacidade de três proteínas na identificação da bactéria em amostras de leite mastítico. O potencial de IsaA e Nuc para o immunodiagnóstico de S. aureus de origem bovina foi mostrado, embora seja necessária a padronização do método para que a sensibilidade aumente. Sugere-se que apenas uma região da proteína seja usada no teste. Neste trabalho, o papel de um regulador transcricional putativo da família LysR (SAB2209) também foi avaliado para expandir nosso conhecimento sobre os mecanismos envolvidos na patogênese de S. aureus. A superexpressão de LysR afetou a formação de biofilme e a susceptibilidade a estresse oxidativo, no entanto sem afetar o crescimento bacteriano e hemólise. LysR também reduziu a colonização da bactéria em ensaios ex vivo e in vivo. Análise do transcriptoma mostrou que 34,8% dos genes de S. aureus RF122 foram alterados em resposta à superexpressão de SAB2209. O gene sortase A (srtA), que codifica uma transpeptidase que ancora adesinas na parede da célula, e os genes tagX e tagB, envolvidos na produção de ácido teicóico foram reduzidos 3,0; 7,3 e 11,2 vezes, respectivamente. Em contraste, a transcrição dos genes responsáveis pela biossíntese de purinas e adenosina sintase (adsA) foi estimulada. Os nossos resultados sugerem que SAB2209 influencia passos iniciais para a colonização de S. aureus e evasão do sistema imune do hospedeiro, através da alteração da expressão de genes associados com a função da parede celular e estratégias evasivas.Intramammary infections caused by Staphylococcus aureus are mostly subclinical and frequently evolves to a persistent form. The accurate detection of infected animals is vital to avoid pathogen spread and to prevent the occurrence of new infections. Bacteriological culture remains the gold standard for the identification of mastitis pathogens, but it is also time-demanding. Immunoassays are an alternative approach of diagnosis that can be fast, specific, and in some cases, suitable for field analysis. In a continuous pursuit for biomarkers that could diagnose bovine mastitis caused by S. aureus, we evaluated the ability of three proteins to identify the pathogen in milk samples collected from infected animals. IsaA and Nuc showed a potential for immunodiagnosis of S. aureus, but specificity still needs improvement perhaps by targeting a smaller region of the protein in the assay. In this work the role of a putative LysR type transcriptional regulator (SAB2209) was also evaluated to expand our knowledge of the mechanisms involved in S. aureus pathogenesis. LysR overexpression affected biofilm formation and oxidative stress susceptibility but no changes on bacterial growth and hemolysis were seen. LysR also reduced colonization in ex vivo and in vivo assays. Transcriptome analysis showed that 34.8% of the staphylococcal genes were altered in response to SAB2209 overexpression. The gene sortase A (srtA), that codes a transpeptidase that anchors adhesins to the cell wall, and the genes tagX and tagB, involved in teichoic acid production were reduced 3.0, 7.3, and 11.2-fold, respectively. In contrast, genes responsible for purine biosynthesis and adenosine synthase (adsA) were up-regulated. Our results suggest that SAB2209 influences initial steps to S. aureus colonization and immune evasion, by altering the genes associated with cell wall function and evasive strategies.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

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

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

The C-type lectin disrupts pre-formed staphylococcal biofilms.

<p><i>S</i>. <i>aureus</i> and <i>S</i>. <i>epidermidis</i> were cultivated in microplates for 22 h at 37°C. The bacteria were washed, and the biofilm cells attached to the microtiter plate wells were incubated with 100 µg/mL C-type lectin for an additional 2 or 3 h. After staining with crystal violet, the OD_560nm was measured. The percentage of biofilm disruption was calculated relative to the control (saline solution) which was set to 100%. The percentage of biofilm disruption was calculated relative to that observed with PBS, which served as a control. The values are the means ± SD from three independent experiments.</p

Scanning electron microscopy reveals disruption of staphylococcal biofilms by lectin.

<p><i>Staphylococcus aureus</i> NRS 155 (A–C) and <i>S</i>. <i>epidermidis</i> NRS 101 (B–D) were grown on a polystyrene surface for 22 h at 37°C in the presence (C–D) or absence (A–B) of 100 µg/mL lectin. Arrows indicate staphylococcal extracellular polymeric substance.</p

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

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