605 research outputs found
Antimicrobial activity of biogenically produced spherical Se-nanomaterials embedded in organic material against Pseudomonas aeruginosa and Staphylococcus aureus strains on hydroxyapatite-coated surfaces
In an effort to prevent the formation of pathogenic biofilms on hydroxyapatite (HA)-based clinical devices and surfaces, we present a study evaluating the antimicrobial efficacy of Spherical biogenic Se-Nanostructures Embedded in Organic material (Bio Se-NEMO-S) produced by Bacillus mycoides SelTE01 in comparison with two different chemical selenium nanoparticle (SeNP) classes. These nanomaterials have been studied as potential antimicrobials for eradication of established HA-grown biofilms, for preventing biofilm formation on HA-coated surfaces and for inhibition of planktonic cell growth of Pseudomonas aeruginosa NCTC 12934 and Staphylococcus aureus ATCC 25923. Bio Se-NEMO resulted more efficacious than those chemically produced in all tested scenarios. Bio Se-NEMO produced by B. mycoides SelTE01 after 6 or 24 h of Na 2 SeO 3 exposure show the same effective antibiofilm activity towards both P. aeruginosa and S. aureus strains at 0.078 mg ml −1 (Bio Se-NEMO 6 ) and 0.3125 mg ml −1 (Bio Se-NEMO 24 ). Meanwhile, chemically synthesized SeNPs at the highest tested concentration (2.5 mg ml −1 ) have moderate antimicrobial activity. The confocal laser scanning micrographs demonstrate that the majority of the P. aeruginosa and S. aureus cells exposed to biogenic SeNPs within the biofilm are killed or eradicated. Bio Se-NEMO therefore displayed good antimicrobial activity towards HA-grown biofilms and planktonic cells, becoming possible candidates as new antimicrobials
Characterization of Antimicrobial Susceptibility of Bacterial Biofilms on Biological Tissues
abstract: Prosthetic joint infection (PJI) is a devastating complication associated with total joint arthroplasty that results in high cost and patient morbidity. There are approximately 50,000 PJIs per year in the US, imposing a burden of about $5 billion on the healthcare system. PJI is especially difficult to treat because of the presence of bacteria in biofilm, often highly tolerant to antimicrobials. Treatment of PJI requires surgical debridement of infected tissues, and local, sustained delivery of antimicrobials at high concentrations to eradicate residual biofilm bacteria. However, the antimicrobial concentrations required to eradicate biofilm bacteria grown in vivo or on tissue surfaces have not been measured. In this study, an experimental rabbit femur infection model was established by introducing a variety of pathogens representative of those found in PJIs [Staphylococcus Aureus (ATCC 49230, ATCC BAA-1556, ATCC BAA-1680), Staphylococcus Epidermidis (ATCC 35984, ATCC 12228), Enterococcus Faecalis (ATCC 29212), Pseudomonas Aeruginosa (ATCC 27853), Escherichia Coli (ATCC 25922)]. Biofilms of the same pathogens were grown in vitro on biologic surfaces (bone and muscle). The ex vivo and in vitro tissue minimum biofilm eradication concentration (MBEC; the level required to eradicate biofilm bacteria) and minimum inhibitory concentration (MIC; the level required to inhibit planktonic, non-biofilm bacteria) were measured using microbiological susceptibility assays against tobramycin (TOB) and vancomycin (VANC) alone or in 1:1 weight combination of both (TOB+VANC) over three exposure durations (6 hour, 24 hour, 72 hour). MBECs for all treatment combinations (pathogen, antimicrobial used, exposure time, and tissue) were compared against the corresponding MIC values to compare the relative susceptibility increase due to biofilm formation. Our data showed median in vitro MBEC to be 100-1000 times greater than the median MIC demonstrating the administration of local antimicrobial doses at MIC level would not kill the persisting bacteria in biofilm. Also, administering dual agent (TOB+VANC) showed median MBEC values to be comparable or lower than the single agents (TOB or VANC)Dissertation/ThesisMasters Thesis Bioengineering 201
High-throughput metal susceptibility testing of microbial biofilms
BACKGROUND: Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxicity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated. RESULTS: This high-throughput method generated 96-statistically equivalent biofilms in a single device and thus allowed for comparative and combinatorial experiments of media, microbial strains, exposure times and metals. By adjusting growth conditions, it was possible to examine biofilms of different microorganisms that had similar cell densities. In one example, Pseudomonas aeruginosa ATCC 27853 was up to 80 times more resistant to heavy metalloid oxyanions than Escherichia coli TG1. Further, biofilms were up to 133 times more tolerant to tellurite (TeO(3)(2-)) than corresponding planktonic cultures. Regardless of the growth medium, the tolerance of biofilm and planktonic cell E. coli JM109 to metals was time-dependent. CONCLUSION: This method results in accurate, easily reproducible comparisons between the susceptibility of planktonic cells and biofilms to metals. Further, it was possible to make direct comparisons of the ability of different microbial strains to withstand metal toxicity. The data presented here also indicate that exposure time is an important variable in metal susceptibility testing of bacteria
Novel therapeutic strategies for the management of diabetic foot infections : the evaluation of selected antimicrobial peptides against clinically isolated bacterial pathogens
Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e BiomédicasDiabetic foot infections (DFIs) are a frequent complication of Diabetes mellitus. These ulcers are prone to be colonized by Staphylococcus aureus and Pseudomonas aeruginosa, including multidrug resistant and biofilm-producing strains, possibly leading to DFI chronicity and amputation. New therapeutic strategies for DFI management are urgent and the antimicrobial peptides (AMPs) nisin and pexiganan are potential candidates. This project aimed to evaluate the activity of these AMPs, incorporated in a guar gum biogel, against selected DFI clinical isolates.
Firstly, nisin’s activity against a collection of S. aureus DFI clinical isolates was determined. Results showed that nisin was able to inhibit and eradicate S. aureus planktonic and biofilm cells at concentrations below its acceptable daily intake. When incorporated in the biogel, nisin kept its antimicrobial activity. This work also evaluated the potential of nisin to complement the activity of conventional antiseptics and antibiotics against established biofilms formed by these isolates. An in vitro antimicrobial schematic protocol was developed to mimetize DFI management guidelines. Fifteen antimicrobial combinations, including nisin-biogel, chlorhexidine, clindamycin, gentamicin and vancomycin, were tested. Results showed that the higher levels of biofilm inhibitory effects were presented by therapeutic combinations that included the nisin-biogel formulation.
Nisin-biogel ideal storage conditions and cytotoxicity were also evaluated. Results demonstrate that if stored at temperatures between -20 and 22ºC, nisin-biogel is able to maintain its antimicrobial activity up to 24 months. Moreover, after 24 h of exposition, the nisin-biogel presented no significant levels of toxicity regarding the human keratinocytes under study. Lastly, to cover the complex microbiota present in DFIs, a combination of AMPs with different action spectra was developed, based on the simultaneous incorporation of nisin and pexiganan in the biogel. The activity of this dual-AMPs formulation was tested against two S. aureus and P. aeruginosa strains isolated from the same DFI. Acting together, these AMPs were able to diffuse from the biogel and inhibit and eradicate biofilms formed by these DFI isolates.
The effectiveness of AMPs, particularly nisin and pexiganan, as novel antimicrobial strategies for the management of DFIs is still an unknown territory that merits investigation. In vitro biofilm models are the basis of preliminary research; however, they underrepresent the complex microbiota present in DFIs and their interaction with the immune system and skin cells constituents. Further research is necessary to understand the AMPs full potential regarding the clinical management of biofilm-related diseases, such as DFIs.RESUMO - As infecções do pé diabético (IPDs) são uma complicação frequente da Diabetes mellitus. Estas úlceras tendem a ser colonizadas por Staphylococcus aureus e Pseudomonas aeruginosa, incluindo estirpes multirresistentes e produtoras de biofilme, possivelmente causando cronicidade da IPD e amputação. É urgente criar novas estratégias para o tratamento das IPD e os péptidos antimicrobianos (PAMs) nisina e pexiganan são potenciais candidatos. Este projecto avaliou a actividade destes PAM, incorporados num biogel de goma de guar, contra isolados de IPD.
Primariamente, foi determinada a actividade da nisina contra uma colecção de S. aureus isolados de IPD. Os resultados mostraram que a nisina é capaz de inibir e erradicar S. aureus na forma planctónica e de biofilme a concentrações abaixo da dose diária recomendada. Quando incorporada no biogel, a nisina manteve a sua actividade. Foi ainda avaliado o potencial da nisina para complementar a actividade de antissépticos e antibióticos convencionais contra biofilmes formados por estes isolados. Foi criado um protocolo que simula in vitro o tratamento convencional das IPDs. Foram testadas 15 combinações de antimicrobianos, incluindo biogel de nisina, clorohexidina, clindamicina, gentamicina e vancomicina. Os resultados mostraram que o maior efeito inibidor de biofilmes pertencia a combinações que incluam o biogel de nisina.
Foram também avaliadas as condições de armazenamento ideais para o biogel de nisina e a sua citotoxicidade. Quando armazenado a temperaturas entre -20 e 22ºC, o biogel de nisina manteve a sua actividade antimicrobiana durante pelo menos 24 meses. Adicionalmente, após exposição durante 24 horas, o biogel de nisina não apresentou nÃveis significativos de toxicidade relativamente aos queratinócitos humanos em estudo. Por último, para abranger a complexa microbiota presente nas IPDs, foi avaliada uma combinação de PAMs com diferentes espectros de acção, baseada na incorporação simultânea de nisina e pexiganan no biogel. A actividade desta formulação foi testada contra duas estirpes de S. aureus e P. aeruginosa isoladas da mesma IPD. Conjuntamente, estes PAMs foram capazes de se difundir do biogel e inibir e erradicar biofilmes formados por estes isolados.
A eficácia dos PAMs como novas estratégias para o tratamento das IPD é ainda uma área desconhecida. Os modelos in vitro de biofilmes são a base da investigação; contudo, não representam a microbiota presente nas IPD nem a sua interacção com o sistema imunitário e outros constituintes celulares. É essencial continuar a investigar para compreender o potencial dos PAMs na terapêutica de doenças onde haja formação de biofilmes, como é o caso das IPDs.N/
Impact of Delftia tsuruhatensis and Achromobacter xylosoxidans on Escherichia coli dual-species biofilms treated with antibiotic agents
Recently it was demonstrated that for urinary tract infections species with a lower or unproven pathogenic potential, such as Delftia tsuruhatensis and Achromobacter xylosoxidans, might interact with conventional pathogenic agents such as Escherichia coli. Here, single- and dual-species biofilms of these microorganisms were characterized in terms of microbial composition over time, the average fitness of E. coli, the spatial organization and the biofilm antimicrobial profile. The results revealed a positive impact of these species on the fitness of E. coli and a greater tolerance to the antibiotic agents. In dual-species biofilms exposed to antibiotics, E. coli was able to dominate the microbial consortia in spite of being the most sensitive strain. This is the first study demonstrating the protective effect of less common species over E. coli under adverse conditions imposed by the use of antibiotic agents.This work was financially supported by the FCT/MEC with
national funds and when applicable co-funded by FEDER in
the scope of the P2020 Partnership Agreement [Project UID/
EQU/00511/2013-LEPABE]; FEDER funds through the Operational
Programme for Competitiveness Factors – COMPETE,
O Novo Norte–North Portugal Regional Operational
Programme – ON2 and National Funds through Foundation
for Science and Technology – FCT [Project NORTE-07-0124-
FEDER-000025–RL2_ Environment&Health and Project
‘DNAmimics’ PIC/IC/82815/2007]; PhD fellowship [SFRH/
BD/82663/2011]; and postdoctoral fellowship [SFRH/
BPD/74480/2010]
An investigation, using an in-vitro alginate biofilm model, into locally delivered antibiotic combinations to treat staphylococcal prosthetic infection
INTRODUCTION
Joint replacement is a common and effective procedure but unfortunately, a small
proportion of patients develop Prosthetic joint Infection (PJI). The bacteria
responsible for these infections exist within a surface -associated community known
as a biofilm. When this biofilm phenotype is expressed, it allows the organisms to
resist phagocytic host defenses, tolerate the stresses induced by antimicrobials and
colonize peri- prosthetic niches. PJI is invariably refractory to standard therapies
and clinicians are required to use a combination of systemic and local
antimicrobials, repeated debridement and prosthesis exchange to treat the patient
resulting in significant morbidity. Standard antibiotic sensitivity tests offer little
insight into bacterial susceptibilities in the biofilm state. Furthermore, they neither
test at the higher levels, nor in the combinations of antibiotics that are commonly
locally delivered during surgery. The aim of this thesis was to develop and validate
a biofilm model, and to use it to test staphylococcal biofilms with clinically
achievable concentrations of antibiotics in combination.METHODS
Sodium alginate gel was chelated with a calcium chloride solution to form bullet
shaped beads with a surface area of 161mm2 These beads were then removed and
their surface inoculated with either a methicillin sensitive Staphylococcus aureus
(ATCC 29213) or a clinical strain of coagulase negative staphylococcus. After being
incubated aerobically for 20 hours in a 48 -well micro -titre plate, growth controls
were sampled and enumerated. The remaining beads were washed to remove non - adherent bacteria and placed into fresh broth containing antibiotics. After 3 hours
of antibiotic challenge, they were removed, washed and the biofilm detached by
dissolving the bead in a citric acid and Na2CO3 solution. Viable organisms were
enumerated after micro -dilution and Miles Misra plating onto agar.RESULTS
Cryo -scanning electron microscopy demonstrated the model allowed a biofilm to
develop on the surface of alginate beads. Overall the technique performed with
satisfactory resemblance of the control data and acceptable responsiveness after
disinfection. The repeatability of disinfection was found to be most variable around
the level used to define bacterial eradication. The model was used to compare the
minimum inhibitory (MIC) and biofilm eradication concentrations (MBEC) of seven
commonly used antibiotics. Poor correlation was found between the susceptibility
of the standard planktonic cultures to antibiotics and those that were effective
against organisms in biofilm. Gentamicin and daptomycin were found to be the
only mono -therapies that were effective against the biofilm at clinically achievable
levels. Combining antibiotics that were ineffective as single agents did not confer
additional benefit.Interestingly despite gentamicin being effective when tested alone, combining it
with clindamycin, rifampacin or linezolid reduced the bactericidal effect markedly
in both strains. This phenomenon was investigated further by varying the
concentration of antibiotics within the combinations. Combining the bactericidal
antibiotics tested with gentamicin had an additive or synergistic effect. More
importantly, a strong antagonistic effect was observed, with between 8 and 32 times
more gentamicin being required, when it was combined with antibiotics considered
bacteriostatic.CONCLUSION
Standard microbiology laboratory testing is inadequate to guide clinical treatment
of PJI. Testing of biofilm susceptibility to combinations of antimicrobials at high
concentrations should be included in the laboratory testing of PJI. Further research
should be directed towards understanding the mechanisms in which bacteriostatic
antibiotics induce the organisms to become more tolerant to other antibiotics.
If the antagonistic effect is confirmed in -vivo then it is logical to study a two stage
antimicrobial strategy, avoiding potential antagonism by only introducing
bacteriostatic agents when the bactericidal drugs dip below their minimum biofilm
eradication concentration
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