NaOCl effect on biofilm produced by Staphylococcus aureus isolated from the milking environment and mastitis infected cows / Efeito do hipoclorito de sódio em biofilmes produzidos por Staphylococcus aureus isolados do ambiente de ordenha e de vacas com mastite

Biofilms constitute a physical barrier, protecting the encased bacteria from detergents and sanitizers. The objective of this work was to analyze the effectiveness of sodium hypochlorite (NaOCl) against strains of Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis and Staphylococcus aureus isolated from the milking environment (blowers and milk conducting tubes). The results revealed that, in the presence of NaOCl (150ppm), the number of adhered cells of the twelve S. aureus strains was significantly reduced. When the same strains were evaluated in biofilm condition, different results were obtained. It was found that, after a contact period of five minutes with NaOCl (150ppm), four strains (two strains from milk , one from the blowers and one from a conductive rubber) were still able to grow. Although with the increasing contact time between the bacteria and the NaOCl (150ppm), no growth was detected for any of the strains. Concerning the efficiency of NaOCl on total biofilm biomass formation by each S. aureus strain, a decrease was observed when these strains were in contact with 150 ppm NaOCl for a total period of 10 minutes. This study highlights the importance of a correct sanitation protocol of all the milk processing units which can indeed significantly reduce the presence of microorganisms, leading to a decrease of cow´s mastitis and milk contamination.

Biofilmes são constituídos de bactérias aderidas a uma superfície e aderidas entre si envolvidas por um polissacarídeo de constituição proteica, lipídica e glicídica que conferem uma barreira física às bactérias dentro deste microambiente. O objetivo deste trabalho foi analisar a eficácia do hipoclorito de sódio (NaOCl) contra estirpes de Staphylococcus aureus isoladas de leite cru de vacas com mastite subclínica e Staphylococcus aureus isolados do ambiente de ordenha (borrachas de ordenhadeiras e mangueiras condutoras de leite). Os resultados revelaram que, na presença de hipoclorito de sódio (150ppm), o número de células aderidas das 12 estirpes de S. aureus analisadas foi significativamente reduzido. Quando as mesmas estirpes foram avaliadas em condições de biofilme, diferentes resultados foram obtidos. Verificou-se que, após um período de contato de cinco minutos com NaOCl (150ppm), quatro estirpes (duas estirpes de leite, uma estirpe das borrachas das ordenhadeiras e uma estirpe de uma mangueira condutora de leite) ainda eram capazes de crescer. Com o aumento do tempo de contato do hipoclorito e as bactérias, cada vez maior, na concentração de 150ppm, não foi detectado o crescimento das estirpes. Em relação à eficácia do NaOCl na formação total da biomassa do biofilme por cada uma das estirpes de S. aureus, observou-se decréscimo da biomassa dos biofilmes quando estas estirpes estavam em contato com o NaOCl na concentração de 150ppm durante um tempo total de 10 minutos. O estudo demonstra a importância de um protocolo de saneamento correto de todas as unidades de processamento de leite, que pode, efetivamente, reduzir a presença de microrganismos de forma significativa, conduzindo a uma diminuição da mastite e da contaminação do leite.

N-acetylcysteine and vancomycin alone and in combination against staphylococci biofilm

INTRODUCTION: The ability of staphylococci to produce biofilm is an important virulence mechanism that allows bacteria both to adhere and to live on artificial surfaces and to resist to the host immune factors and antibiotics. Staphylococcal infections have become increasingly difficult to treat due their antibiotic resistance. Therefore, there is a continuous need for new and effective treatment alternatives against staphylococcal infections. The main goal of this study was to test N-acetylcysteine (NAC) and vancomycin alone and in combination against S. epidermidis and S. aureus biofilms. METHODS: Biofilms were treated with NAC at minimum inhibitory concentration (MIC) and 10 × MIC concentrations and vancomycin at MIC and peak serum concentrations. RESULTS: The use of NAC 10 × MIC alone showed a significant antibactericidal effect, promoting a 4-5 log10 CFU/ mL reduction in biofilm cells. The combination of NAC 10 × MIC with vancomycin (independently of the concentration used) reduced significantly the number of biofilm cells for all strains evaluated (5-6 log10). CONCLUSION: N-acetylcysteine associated to vancomycin can be a potential therapeutic strategy in the treatment of infections associated to biofilms of S. epidermidis or S. aureus.

Evaluation of antimicrobial activity of certain combinations of antibiotics against in vitro Staphylococcus epidermidis biofilms.

Background & objectives: Staphylococcus epidermidis is the most common pathogen associated with infections of surgical implants and other prosthetic devices owing to its adhesion and biofilm-forming ability on biomaterials surfaces. The objective of this study was to compare susceptibilities of biofilm-grown cells to single antibiotic and in combination with others to identify those that were effective against S. epidermidis biofilms. Methods: Biofilms were grown in the MBEC™ assay system. The use of this methodology allowed a rapid testing of an array of antibiotics alone (eight) and in combination (25 double combinations). The antibacterial effect of all treatments tested was determined by colony forming units (cfu) enumeration method. Results: The MBEC™ assay system produced multiple and reproducible biofilms of S. epidermidis. Although none of the antibiotics tested have demonstrated an antimicrobial effect (log reduction >3) against all S. epidermidis isolates biofilms, but combinations containing rifampicin showed in general a broader spectrum namely rifampicin-gentamicin and rifampicin-clindamycin. Levofloxacin in combination with rifampicin showed a killing effect against three isolates but failed to attain a bactericidal action against the other two. Interpretation & conclusions: Our findings showed that rifampicin should be a part of any antibiotic therapy directed against S. epidermidis biofilms. However, the efficient antibiotics combination might be dependent on S. epidermidis isolate being tested.

Farnesol in combination with N-acetylcysteine against Staphylococcus epidermidis planktonic and biofilm cells

Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections, in which biofilm formation is considered to be the main virulence mechanism. In biofilm environment, microbes exhibit enhanced resistance to antimicrobial agents. This fact boosted the search of possible alternatives to antibiotics. Farnesol and N-acetylcysteine (NAC) are non-antibiotic drugs that have demonstrated antibacterial properties. In this study, the effect of farnesol and NAC isolated or in combination (farnesol+NAC) was evaluated. NAC at 10 × MIC caused a total cell death in planktonic cells. On the other hand, S. epidermidis biofilms exhibited 4 log reduction in viable cell number after a 24h treatment with NAC at the former concentration. Our results demonstrated that there was a higher CFU log reduction of S. epidermidis planktonic cells when farnesol was combined with NAC at 1 × MIC relatively to each agent alone. However, these results were not relevant because NAC alone at 10 × MIC was always the condition which gave the best results, having a very high killing effect on planktonic cells and a significant bactericidal effect on biofilm cells. This study demonstrated that no synergy was observed between farnesol and NAC. However, the pronounced antibacterial effect of NAC against S. epidermidis, on both lifestyles, indicates the use of NAC as a potential therapeutic agent in alternative to antibiotics.

Adhesion of Salmonella Enteritidis to stainless steel surfaces

Adhesion of microorganisms to food processing surfaces and the problems it causes are a matter of strong concern to the food industry. Contaminated food processing surfaces may act as potential sources of transmission of pathogens in food industry, catering and in the domestic environments. Several studies have shown that adhesion of bacteria to surfaces partly depends upon the nature of the inert surfaces and partly upon the bacterial surface properties. The aim of this study was to compare the adhesion of four different strains of Salmonella Enteritidis to stainless steel 304 (SS 304). The effect of surface hydrophobicity and surface elemental composition on the adhesion process was also analysed. Hydrophobicity was evaluated through contact angle measurements using the sessile drop method. All the strains studied showed positive values of the degree of hydrophobicity (deltaGlwl) and so can be considered hydrophilic while stainless steel revealed a hydrophobic character. Bacterial cell surface composition was measured using X-ray photoelectron spectroscopy (XPS). The XPS results corroborated the similarity of the values of the degree of hydrophobicity obtained by contact angles. The different Salmonella strains showed similar elemental composition and cell surface physico-chemical properties. Nevertheless, S. Enteritidis MUSC presented higher adhesion ability to SS 304 (p<0.05). It can be concluded that the physico-chemical properties of the strain does not explain the ability of adhesion to stainless steel. Other factors like the production of polysaccharides must be considered.

A adesão de microrganismos a superfícies de processamento de alimentos e os problemas que daí resultam são matéria de grande preocupação para a indústria alimentar. Superfícies de processamento de alimentos contaminadas podem actuar como uma potencial fonte de transmissão de patogénicos na indústria alimentar, restauração e em ambientes domésticos. Diversos estudos têm demonstrado que a adesão de bactérias a superfícies depende, por um lado, da natureza das superfícies inertes e, por outro, das propriedades superficiais das bactérias. O objectivo deste trabalho consistiu na comparação da capacidade de adesão de 4 cepas diferentes de Salmonella Enteritidis ao aço inoxidável 304 (SS 304). Analisou-se também o efeito da hidrofobicidade e da composição elementar no processo de adesão. A hidrofobicidade foi determinada através da medição de ângulos de contacto usando o método da gota séssil. Todas as cepas apresentaram valores positivos do grau de hidrofobicidade (deltaGlwl) podendo, assim, ser consideradas hidrofílicas enquanto o aço inoxidável revelou um carácter hidrofóbico. A composição elementar da superfície das células bacterianas foi medida através de espectroscopia de fotoelectrões X (XPS). Os resultados do XPS corroboraram a similaridade de valores do grau de hidrofobicidade obtidos por ângulos de contacto. As diferentes cepas de Salmonella apresentaram uma composição elementar e propriedades físico-químicas semelhantes. No entanto, a Salmonella MUSC apresentou uma capacidade de adesão ao aço inoxidável mais elevada (p<0.05). Pode então concluir-se que as propriedades físico-químicas das cepas não explicam a capacidade de adesão ao aço inoxidável, devendo ser considerados outros factores tais como a produção de exopolissacáridos.