scholarly journals Influence of biofilm growth age, media and antibiotics exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro

2020 ◽  
Author(s):  
Xiaofeng Chen ◽  
Yijuan Xu ◽  
Heinz Winkler ◽  
Trine Rolighed Thomsen
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Treatment Duration ◽  
Time Dependent ◽  
Growth Media ◽  
Biofilm Growth ◽  
Biofilm Removal ◽  
Mueller Hinton ◽  
Experimental Parameters

Abstract Biofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported Minimum Biofilm Eradication Concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic treatment duration and growth media on biofilm eradication in this study. The commonly used biofilm model Calgary biofilm device was used to grow 24 h and 72 h biofilms of Staphylococcus aureus and Pseudomonas aeruginosa , which were treated with time-dependent vancomycin and concentration-dependent tobramycin, respectively. Two common bacteriological growth media Tryptic Soy Broth (TSB) and Cation-adjusted Mueller Hinton Broth (CaMHB) were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24 h) were easier to eradicate than old biofilms (72 h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24 h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h P. aeruginosa biofilms. This study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotics treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments.

scholarly journals Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro

2020 ◽  
Author(s):  
Xiaofeng Chen ◽  
Trine Rolighed Thomsen ◽  
Heinz Winkler ◽  
Yijuan Xu
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Treatment Duration ◽  
Time Dependent ◽  
Growth Media ◽  
Antibiotic Concentration ◽  
Biofilm Growth ◽  
Biofilm Removal ◽  
Experimental Parameters

Abstract Background: Biofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported Minimum Biofilm Eradication Concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic concentration and treatment duration, and growth media on biofilm eradication. Additionally, OSTEOmycinTM, a clinically used antibiotic containing allograft bone product, was tested for antibiofilm efficacy. Results: The commonly used Calgary biofilm device was used to grow 24 h and 72 h biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, which were treated with time-dependent vancomycin (up to 3000 mg/L) and concentration-dependent tobramycin (up to 80 mg/L), respectively. Two common bacteriological growth media Tryptic Soy Broth (TSB) and Cation-adjusted Mueller Hinton Broth (CaMHB) were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24 h) were easier to eradicate than old biofilms (72 h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24 h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h P. aeruginosa biofilms. Treatment with tobramycin containing OSTEOmycin TTM removed 72 h and 168 h P. aeruginosa biofilms after one day treatment, while few 72h S. aureus biofilms survived after two days treatment with vancomycin containing OSTEOmycin VTM. Conclusions: This study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotic concentration and treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments. The results of OSTEOmycin products indicated that simple in vitro biofilm test could be used for initial screening of antibiofilm products. For clinical application, a more clinically relevant biofilm model for the specific biofilm infection in question should be developed to guide the amount of antibiotics used for local antibiofilm treatment.

scholarly journals Influence of biofilm growth age, media, antibiotic concentration and exposure time on Staphylococcus aureus and Pseudomonas aeruginosa biofilm removal in vitro

2020 ◽  
Author(s):  
Xiaofeng Chen ◽  
Trine Rolighed Thomsen ◽  
Heinz Winkler ◽  
Yijuan Xu
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Treatment Duration ◽  
Time Dependent ◽  
Growth Media ◽  
Antibiotic Concentration ◽  
Biofilm Growth ◽  
Biofilm Removal ◽  
Experimental Parameters

Abstract Background: Biofilm is known to be tolerant towards antibiotics and difficult to eradicate. Numerous studies have reported minimum biofilm eradication concentration (MBEC) values of antibiotics for many known biofilm pathogens. However, the experimental parameters applied in these studies differ considerably, and often the rationale behind the experimental design are not well described. This makes it difficult to compare the findings. To demonstrate the importance of experimental parameters, we investigated the influence of biofilm growth age, antibiotic concentration and treatment duration, and growth media on biofilm eradication. Additionally, OSTEOmycinTM, a clinically used antibiotic containing allograft bone product, was tested for antibiofilm efficacy. Results: The commonly used Calgary biofilm device was used to grow 24 h and 72 h biofilms of Staphylococcus aureus and Pseudomonas aeruginosa, which were treated with time-dependent vancomycin (up to 3000 mg L-1) and concentration-dependent tobramycin (up to 80 mg L-1), respectively. Two common bacteriological growth media tryptic soy broth (TSB) and cation-adjusted Mueller Hinton broth (CaMHB) were tested. We found for both species that biofilms were more difficult to kill in TSB than in CaMHB. Furthermore, young biofilms (24 h) were easier to eradicate than old biofilms (72 h). In agreement with vancomycin being time-dependent, extension of the vancomycin exposure increased killing of S. aureus biofilms. Tobramycin treatment of 24 h P. aeruginosa biofilms was found concentration-dependent and time-independent, however, increasing killing was indicated for 72 h P. aeruginosa biofilms. Treatment with tobramycin containing OSTEOmycin TTM removed 72 h and 168 h P. aeruginosa biofilms after one day treatment, while few 72h S. aureus biofilms survived after two days treatment with vancomycin containing OSTEOmycin VTM. Conclusions: This study demonstrated biofilm removal efficacy was influenced by media, biofilm age and antibiotic concentration and treatment duration. It is therefore necessary to taking these parameters into consideration when designing experiments. The results of OSTEOmycinTM products indicated that simple in vitro biofilm test could be used for initial screening of antibiofilm products. For clinical application, a more clinically relevant biofilm model for the specific biofilm infection in question should be developed to guide the amount of antibiotics used for local antibiofilm treatment.

ATIVIDADE ANTIMICROBIANA IN VITRO DO EXTRATO AQUOSO, HIDROALCOÓLICO E ALCOÓLICO DE FOLHAS DE ESPÉCIES DA FAMÍLIA LAMIACEAE

2018 ◽  
Vol 4 ◽  
Author(s):  
Karlynne Freire Mendonça ◽  
José Klauber Roger Carneiro ◽  
Maria Auxiliadora Silva Oliveira
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Enterococcus Faecalis ◽  
Streptococcus Pyogenes ◽  
Mentha Arvensis ◽  
Ocimum Gratissimum ◽  
Mueller Hinton ◽  
Plectranthus Amboinicus

Objetivos: avaliar a atividade antimicrobiana em extrato aquoso, hidroalcoólico e alcoólico das folhas de espécies da família Lamiaceae frente a bactérias de interesse. Método: Foram escolhidas quatro espécies: Ocimum gratissimum, Plectranthus amboinicus, Mentha arvensis e Plectranthus barbatus. A partir das folhas foram confeccionados os extratos aquoso, hidroalcoólico e alcoólico nas concentrações 100mg/mL, 50mg/mL e 25mg/mL. Foram selecionadas as bactérias Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus aureus e Pseudomonas aeruginosa para os ensaios de antibiose em Ágar Mueller-Hinton. Resultados: P. barbatus, em seu extrato hidroalcoólico mostrou ativo nas três concentrações para bactéria S. aureus, e ainda foi ativo para P. aeruginosa, demonstrando no extrato alcoólico atividade frente as bactérias. Para M. arvensis e P. amboinicus, seus extratos hidroalcoólico e alcoólico apresentaram atividade para S. aureus. Conclusão: Sugere-se que as espécies em questão apresentem boa atividade antimicrobiana, sendo necessária a realização de mais estudos para melhor entender esse mecanismo.

scholarly journals Avaliação da atividade antimicrobiana in vitro do extrato de Tabernaemontana catharinensis A. DC

2011 ◽  
Vol 13 (2) ◽  
pp. 197-202 ◽  
Author(s):  
D.M Gonçalves ◽  
J.H.B Araújo ◽  
M.S Francisco ◽  
M.A Coelho ◽  
J.M Franco
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Mueller Hinton

Diversas espécies de Tabernaemontana têm sido estudadas devido a diversidade de alcalóides com atividade farmacológica. O objetivo desse trabalho foi avaliar a capacidade antimicrobiana in vitro do extrato das cascas do caule de Tabernaemontana catharinensis A. DC.em cepas de Staphylococcus aureus e Pseudomonas aeruginosa, microrganismos causadores de diversas infecções. Os testes de susceptibilidade bacteriana foram realizados usando o método de Kirby Bauer, consistindo na difusão em disco do antibiótico em meio de cultivo Mueller Hinton. Os testes de inibição foram realizados com soluções do extrato bruto seco de T. catharinensis dissolvido em etanol 70% (v/v) na concentração 1,0 mg mL-1, que aplicada nos discos de área 20 mm², apresentaram concentração de 0,005 mg mm-2. Como controle negativo, realizou-se ensaios com placas contendo P. aeruginosa, e discos com etanol 70% (v/v), e como controle positivo, discos com os antibióticos ceftriaxona sódica (0,25 mg mm-2 de área do disco), tetraciclina (0,005 mg mm-2) e cefalexina (0,005 mg mm-2). A solução do extrato na concentração de 0,005 mg mm-2 inibiu o Staphylococcus aureus, com diâmetro médio do halo de 0,6 cm. O halo de inibição para o Pseudomonas aeruginosa foi em média 1,2 cm. A tetraciclina, a cefalexina, e o controle negativo (etanol 70% v/v) não demonstraram ação antimicrobiana. O halo de inibição usando ceftriaxona foi em média 2,2 cm para P. aeruginosa e 1,0 cm para Staphylococcus aureus.

scholarly journals Efeito antimicrobiano do extrato de Cranberry sobre micro-organismos causadores de infecção urinária

2016 ◽  
Vol 11 (31) ◽  
pp. 113-122
Author(s):  
Carla Franco Porto Belmont Souza ◽  
Luiz Eduardo Souza da Silva Irineu ◽  
Renan Silva De Souza ◽  
Renato da Silva Teixeira ◽  
Ivina Sanches Pereira ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Enterococcus Faecalis ◽  
Proteus Mirabilis ◽  
Enterococcus Faecium ◽  
Vaccinium Macrocarpon ◽  
Mueller Hinton

A resistência microbiana tem se mostrado um problema de proporções mundiais, causando estado de morbidade e mortalidade em diversos pacientes. Em vista disso, tem crescido a busca por métodos alternativos naturais de profilaxia. A investigação clínica sugere que o Extrato de Cranberry está entre as melhores propostas de prevenção natural. O Cranberry (Vaccinium macrocarpon) é um fruto que tem crescido comercialmente pelo sabor e propriedades benéficas à saúde. Dentre as formas comercializadas estão: o suco, o chá e as cápsulas contendo o extrato seco. A ação desta planta está relacionada ao tratamento de doenças do trato urinário, por possuir substâncias que inibem a adesão bacteriana ao epitélio do trato urinário, dificultando sua proliferação e reprodução. Dentre todas as infecções relacionadas à assistência a saúde, a Infecção do Trato Urinário é a mais frequentemente associada a procedimentos invasivos. Se não for tratada, pode resultar em complicações como pielonefrite aguda, bacteremia e pionefrose. Portanto, cranberry pode ser uma nova alternativa para o combate das infecções uroepiteliais, por ser um produto natural de preço acessível, e com formas de comercialização diversificada, ao contrário dos antimicrobianos convencionais, que por sua vez são caros e podem acabar causando resistência nos micro-organismos. Este trabalho teve como objetivo avaliar in vitro a atividade antimicrobiana do extrato de Cranberry, adquirido em farmácia de manipulação, sobre 8 micro-organismos isolados de infecções urinárias. As cepas utilizadas, adquiridas da coleção da FIOCRUZ, foram: Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus mirabilis, Serratia marscecens, Staphylococcus aureus, Enterococcus faecalis e Enterococcus faecium. No estudo, foram utilizados o caldo Mueller Hinton (MH), Extrato de Cranberry e as bactérias patogênicas. O ensaio foi realizado em triplicata, com o uso de um controle de crescimento dos micro-organismos e o experimento para avaliação do crescimento bacteriano na presença do extrato. A turbidez foi medida com o auxílio de um espectrofotômetro, no comprimento de onda de 600 nm, antes e após 24 horas de incubação à 37 ºC. O procedimento forneceu a Densidade Ótica, do qual possibilitou a identificação da inibição microbiana. Para análise estatística foi utilizado o Teste t de Student. O Extrato de Cranberry apresentou atividade antimicrobiana sobre as bactérias Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Serratia marscecens e Enterococcus faecalis (p < 0,05), confirmando seu efeito benéfico em infecções urinárias. No entanto, não teve efeito inibitório significativo sobre Pseudomonas aeruginosa, Proteus mirabilis e Enterococcus faecium (p > 0,05).

scholarly journals Atividade antimicrobiana in vitro dos extratos aquosos, hidroalcoólicos e alcoólicos das folhas de espécies da família Myrtaceae frente à cepas de bactérias de interesse

2017 ◽  
Vol 16 (2) ◽  
pp. 139
Author(s):  
Francisca Hérica Chaves Albuquerque ◽  
Karina Da Silva Soares ◽  
Maria Auxiliadora Silva Oliveira
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Psidium Guajava ◽  
Syzygium Cumini ◽  
Eugenia Uniflora ◽  
Mueller Hinton

<p><strong>Introdução</strong>: a Organização Mundial da Saúde (OMS) tem incentivado o estudo de plantas tradicionalmente conhecidas como medicinais, tendo como objetivo avaliar cientificamente os benefícios da utilização de medicamentos fitoterápicos e de conhecer, ao mesmo tempo, a segurança de seu uso indevido. <strong>Objetivos</strong>: este trabalho teve como objetivo avaliar a atividade antimicrobiana do extrato aquoso, hidroalcoólico e alcoólico de folhas de espécies da família Myrtaceae frente às cepas de bactérias de interesse. <strong>Metodologia</strong>: a análise da atividade antibacteriana dos extratos das folhas das plantas, jambo, azeitona preta, pitanga e goiaba foram verificados a partir do teste de difusão em Ágar Mueller-Hinton. As cepas utilizadas, após reativação, foram diluídas em solução salina estéril a 0,85%. Sendo consideradas com potencial antimicrobiano aqueles extratos que geraram halos ≥ 7 mm (sete milímetros). <strong>Resultados</strong>: os resultados encontrados na análise dos dados do extrato aquoso demonstraram que Eugenia uniflora, Syzygium cumini e Psidium guajava apresentaram atividade antimicrobiana sobre Staphylococcus aureus, na concentração de 100 mg/mL. Na análise dos dados dos extratos hidroalcoólico, Syzygium cumini demonstrou atividade antimicrobiana na concentração de 100 mg/mL sobre Pseudomonas aeruginosa, diante de Staphylococcus aureus, Eugenia uniflora, Syzygium malaccense, Syzygium cumini e Psidium guajava demonstraram atividade na concentração de 100 mg/mL cada. Observando os resultados dos extratos alcoólicos, Psidium guajava e Syzygium cumini demonstraram atividade antimicrobiana diante das quatro cepas estudadas. <strong>Conclusão</strong>: o presente estudo demonstrou que os extratos alcoólicos das espécies em estudo apresentaram melhor atividade antimicrobiana e maior espectro de ação quando comparado ao extrato aquoso e hidroalcoólico.</p>

scholarly journals Increased rates of genomic mutation in a biofilm co-culture model of Pseudomonas aeruginosa and Staphylococcus aureus

2018 ◽  
Author(s):  
C.J. Frapwell ◽  
R.P. Howlin ◽  
O. Soren ◽  
B.T. McDonagh ◽  
C.M. Duignan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Mutation Rates ◽  
Interspecies Interactions ◽  
Short Term ◽  
Biofilm Growth ◽  
Biofilm Model ◽  
Adaptive Processes ◽  
Cystic Fibrosis Isolate

AbstractBiofilms are major contributors to disease chronicity and are typically multi-species in nature. Pseudomonas aeruginosa and Staphylococcus aureus are leading causes of morbidity and mortality in a variety of chronic diseases but current in vitro dual-species biofilms models involving these pathogens are limited by short co-culture times (24 to 48 hours). Here, we describe the establishment of a stable (240 hour) co-culture biofilm model of P. aeruginosa and S. aureus that is reproducible and more representative of chronic disease.The ability of two P. aeruginosa strains, (PAO1 and a cystic fibrosis isolate, PA21), to form co-culture biofilms with S. aureus was investigated. Co-culture was stable for longer periods using P. aeruginosa PA21 and S. aureus viability within the model improved in the presence of exogenous hemin. Biofilm co-culture was associated with increased tolerance of P. aeruginosa to tobramycin and increased susceptibility of S. aureus to tobramycin and a novel antimicrobial, HT61, previously shown to be more effective against non-dividing cultures of Staphylococcal spp. Biofilm growth was also associated with increased short-term mutation rates; 10-fold for P. aeruginosa and 500-fold for S. aureus.By describing a reproducible 240 hour co-culture biofilm model of P. aeruginosa and S. aureus, we have shown that interspecies interactions between these organisms may influence short-term mutation rates and evolution, which could be of importance in understanding the adaptive processes that lead to the development of antimicrobial resistance.

scholarly journals Aminoglycoside inhibition of Staphylococcus aureus biofilm formation is nutrient dependent

2014 ◽  
Vol 63 (6) ◽  
pp. 861-869 ◽  
Author(s):  
Michelle J. Henry-Stanley ◽  
Donavon J. Hess ◽  
Carol L. Wells
Keyword(s):  
Staphylococcus Aureus ◽  
Antibiotic Resistance ◽  
Growth Medium ◽  
Antibiotic Susceptibility ◽  
Biofilm Formation ◽  
Nutrient Concentration ◽  
Growth Media ◽  
Biofilm Growth

Biofilms represent microbial communities, encased in a self-produced matrix or extracellular polymeric substance. Microbial biofilms are likely responsible for a large proportion of clinically significant infections and the multicellular nature of biofilm existence has been repeatedly associated with antibiotic resistance. Classical in vitro antibiotic-susceptibility testing utilizes artificial growth media and planktonic microbes, but this method may not account for the variability inherent in environments subject to biofilm growth in vivo. Experiments were designed to test the hypothesis that nutrient concentration can modulate the antibiotic susceptibility of Staphylococcus aureus biofilms. Developing S. aureus biofilms initiated on surgical sutures, and in selected experiments planktonic cultures, were incubated for 16 h in 66 % tryptic soy broth, 0.2 % glucose (1× TSBg), supplemented with bactericidal concentrations of gentamicin, streptomycin, ampicillin or vancomycin. In parallel experiments, antibiotics were added to growth medium diluted one-third (1/3× TSBg) or concentrated threefold (3× TSBg). Following incubation, viable bacteria were enumerated from planktonic cultures or suture sonicates, and biofilm biomass was assayed using spectrophotometry. Interestingly, bactericidal concentrations of gentamicin (5 µg gentamicin ml−1) and streptomycin (32 µg streptomycin ml−1) inhibited biofilm formation in samples incubated in 1/3× or 1× TSBg, but not in samples incubated in 3× TSBg. The nutrient dependence of aminoglycoside susceptibility is not only associated with biofilm formation, as planktonic cultures incubated in 3× TSBg in the presence of gentamicin also showed antibiotic resistance. These findings appeared specific for aminoglycosides because biofilm formation was inhibited in all three growth media supplemented with bactericidal concentrations of the cell wall-active antibiotics, ampicillin and vancomycin. Additional experiments showed that the ability of 3× TSBg to overcome the antibacterial effects of gentamicin was associated with decreased uptake of gentamicin by S. aureus. Uptake is known to be decreased at low pH, and the kinetic change in pH of growth medium from biofilms incubated in 5 µg gentamicin ml−1 in the presence of 3× TSBg was decreased when compared with pH determinations from biofilms formed in 1/3× or 1× TSBg. These studies underscore the importance of environmental factors, including nutrient concentration and pH, on the antibiotic susceptibility of S. aureus planktonic and biofilm bacteria.

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