Titik Nuryastuti

Effect of Cinnamon Oil on icaA Expression and Biofilm Formation by Staphylococcus epidermidis

ABSTRACT Staphylococcus epidermidis is notorious for its biofilm formation on medical devices, and novel approaches to prevent and kill S. epidermidis biofilms are desired. In this study, the effect of cinnamon oil on planktonic and biofilm cultures of clinical S. epidermidis isolates was evaluated. Initially, susceptibility to cinnamon oil in planktonic cultures was compared to the commonly used antimicrobial agents chlorhexidine, triclosan, and gentamicin. The MIC of cinnamon oil, defined as the lowest concentration able to inhibit visible microbial growth, and the minimal bactericidal concentration, the lowest concentration required to kill 99.9% of the bacteria, were determined using the broth microdilution method and plating on agar. A checkerboard assay was used to evaluate the possible synergy between cinnamon oil and the other antimicrobial agents. The effect of cinnamon oil on biofilm growth was studied in 96-well plates and with confocal laser-scanning microscopy (CLSM). Biofilm susceptibility was determined using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Real-time PCR analysis was performed to determine the effect of sub-MIC concentrations of cinnamon oil on expression of the biofilm-related gene, icaA. Cinnamon oil showed antimicrobial activity against both planktonic and biofilm cultures of clinical S. epidermidis strains. There was only a small difference between planktonic and biofilm MICs, ranging from 0.5 to 1% and 1 to 2%, respectively. CLSM images indicated that cinnamon oil is able to detach and kill existing biofilms. Thus, cinnamon oil is an effective antimicrobial agent to combat S. epidermidis biofilms.

Ica-expression and gentamicin susceptibility of Staphylococcus epidermidis biofilm on orthopedic implant biomaterials.

Ica-expression by Staphylococcus epidermidis and slime production depends on environmental conditions such as implant material and presence of antibiotics. Here, we evaluate biofilm formation and ica-expression of S. epidermidis strains on biomaterials involved in total hip- and knee arthroplasty [polyethylene (PE), polymethylmethacrylate (PMMA), stainless steel (SS)]. Ica-expression, assayed using real-time RT-PCR, was highest on PE as confirmed using confocal laser scanning microscopy. Yet biofilm formation by S. epidermidis was most extensive on SS, with less slime production. Ica-expression and slime production were minimal on PMMA. After 3 h of continued growth of 24 h old biofilms in the presence of gentamicin, biofilms on PE showed lower susceptibility to gentamicin, relative to the other materials, presumably as a result of the stronger ica-expression. A higher gentamicin concentration further decreased metabolic activity on all biomaterials. It is concluded that the level of biomaterial-induced ica-expression does not correlate with the amount of biofilm formed, but initially aids bacteria in surviving antibiotic attacks. Once antibiotic treatment has started however, also the antibiotic itself induces slime production and only if its concentration is high enough, killing results. Results suggest that biomaterial-associated infections in orthopedics by S. epidermidis on PE may be more difficult to eradicate than on PMMA or SS.

recA mediated spontaneous deletions of the icaADBC operon of clinical Staphylococcus epidermidis isolates : a new mechanism of phenotypic variations

Phenotypic variation of Staphylococcus epidermidis involving the slime related ica operon results in heterogeneity in surface characteristics of individual bacteria in axenic cultures. Five clinical S. epidermidis isolates demonstrated phenotypic variation, i.e. both black and red colonies on Congo Red agar. Black colonies displayed bi-modal electrophoretic mobility distributions at pH 2, but such phenotypic variation was absent in red colonies of the same strain as well as in control strains without phenotypic variation. All red colonies had lost ica and the ability to form biofilms, in contrast to black colonies of the same strain. Real time PCR targeting icaA indicated a reduction in gene copy number within cultures exhibiting phenotypic variation, which correlated with phenotypic variations in biofilm formation and electrophoretic mobility distribution of cells within a culture. Loss of ica was irreversible and independent of the mobile element IS256. Instead, in high frequency switching strains, spontaneous mutations in lexA were found which resulted in deregulation of recA expression, as shown by real time PCR. RecA is involved in genetic deletions and rearrangements and we postulate a model representing a new mechanism of phenotypic variation in clinical isolates of S. epidermidis. This is the first report of S. epidermidis strains irreversibly switching from biofilm-positive to biofilm-negative phenotype by spontaneous deletion of icaADBC.

Influence of biofilm lubricity on shear-induced transmission of staphylococcal biofilms from stainless steel to silicone rubber

In real‐life situations, bacteria are often transmitted from biofilms growing on donor surfaces to receiver ones. Bacterial transmission is more complex than adhesion, involving bacterial detachment from donor and subsequent adhesion to receiver surfaces. Here, we describe a new device to study shear‐induced bacterial transmission from a (stainless steel) pipe to a (silicone rubber) tube and compare transmission of EPS‐producing and non‐EPS‐producing staphylococci. Transmission of an entire biofilm from the donor to the receiver tube did not occur, indicative of cohesive failure in the biofilm rather than of adhesive failure at the donor‐biofilm interface. Biofilm was gradually transmitted over an increasing length of receiver tube, occurring mostly to the first 50 cm of the receiver tube. Under high‐shearing velocity, transmission of non‐EPS‐producing bacteria to the second half decreased non‐linearly, likely due to rapid thinning of the lowly lubricious biofilm. Oppositely, transmission of EPS‐producing strains to the second tube half was not affected by higher shearing velocity due to the high lubricity and stress relaxation of the EPS‐rich biofilms, ensuring continued contact with the receiver. The non‐linear decrease of ongoing bacterial transmission under high‐shearing velocity is new and of relevance in for instance, high‐speed food slicers and food packaging.

Physico-chemistry of bacterial transmission versus adhesion

Bacterial adhesion is a main problem in many biomedical, domestic, natural and industrial environments and forms the onset of the formation of a biofilm, in which adhering bacteria grow into a multi-layered film while embedding themselves in a matrix of extracellular polymeric substances. It is usually assumed that bacterial adhesion occurs from air or by convective-diffusion from a liquid suspension, but often bacteria adhere by transmission from a bacterially contaminated donor to a receiver surface. Therewith bacterial transmission is mechanistically different from adhesion, as it involves bacterial detachment from a donor surface followed by adhesion to a receiver one. Transmission is further complicated when the donor surface is not covered with a single layer of adhering bacteria but with a multi-layered biofilm, in which case bacteria can be transmitted either by interfacial failure at the biofilm-donor surface or through cohesive failure in the biofilm. Transmission through cohesive failure in a biofilm is more common than interfacial failure. The aim of this review is to oppose surface thermodynamics and adhesion force analyses, as can both be applied towards bacterial adhesion, with their appropriate extensions towards transmission. Opposition of surface thermodynamics and adhesion force analyses, will allow to distinguish between transmission of bacteria from a donor covered with a (sub)monolayer of adhering bacteria or a multi-layered biofilm. Contact angle measurements required for surface thermodynamic analyses of transmission are of an entirely different nature than analyses of adhesion forces, usually measured through atomic force microscopy. Nevertheless, transmission probabilities based on Weibull analyses of adhesion forces between bacteria and donor and receiver surfaces, correspond with the surface thermodynamic preferences of bacteria for either the donor or receiver surface. Surfaces with low adhesion forces such as polymer-brush coated or nanostructured surfaces are thus preferable for use as non-adhesive receiver surfaces, but at the same time should be avoided for use as a donor surface. Since bacterial transmission occurs under a contact pressure between two surfaces, followed by their separation under tensile or shear pressure and ultimately detachment, this will affect biofilm structure. During the compression phase of transmission, biofilms are compacted into a more dense film. After transmission, and depending on the ability of the bacterial strain involved to produce extracellular polymeric substances, biofilm left-behind on a donor or transmitted to a receiver surface will relax to its original, pre-transmission structure owing to the viscoelasticity of the extracellular polymeric substances matrix, when present. Apart from mechanistic differences between bacterial adhesion and transmission, the low numbers of bacteria generally transmitted require careful selection of suitably sensitive enumeration methods, for which culturing and optical coherence tomography are suggested. Opposing adhesion and transmission as done in this review, not only yields a better understanding of bacterial transmission, but may stimulate researchers to more carefully consider whether an adhesion or transmission model is most appropriate in the specific area of application aimed for, rather than routinely relying on adhesion models.

In Vitro Antifungal Activity of (1)-N-2-Methoxybenzyl-1,10-phenanthrolinium Bromide against Candida albicans and Its Effects on Membrane Integrity

Abstract Metal-based drugs, such as 1,10-phenanthroline, have demonstrated anticancer, antifungal and antiplasmodium activities. One of the 1,10-phenanthroline derivatives compounds (1)-N-2-methoxybenzyl-1,10-phenanthrolinium bromide (FEN), which has been demonstrated an inhibitory effect on the growth of Candida spp. This study aimed to explore the in vitro antifungal activity of FEN and its effect on the membrane integrity of Candida albicans. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of FEN against planktonic C. albicans cells were determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute guidelines. Cell membrane integrity was determined with the propidium iodide assay using a flow cytometer and were visualized using scanning electron microscopy (SEM). Planktonic cells growth of C. albicans were inhibited by FEN, with an MIC of 0.39–1.56 μg/mL and a MFC that ranged from 3.125 to 100 μg/mL. When C. albicans was exposed to FEN, the uptake of propidium iodide was increased, which indicated that membrane disruption is the probable mode of action of this compound. There was cells surface changes of C. albicans when observed under SEM.

Structural changes in S. epidermidis biofilms after transmission between stainless steel surfaces

Abstract Transmission is a main route for bacterial contamination, involving bacterial detachment from a donor and adhesion to receiver surfaces. This work aimed to compare transmission of an extracellular polymeric substance (EPS) producing and a non-EPS producing Staphylococcus epidermidis strain from biofilms on stainless steel. After transmission, donor surfaces remained fully covered with biofilm, indicating transmission through cohesive failure in the biofilm. Counter to the numbers of biofilm bacteria, the donor and receiver biofilm thicknesses did not add up to the pre-transmission donor biofilm thickness, suggesting more compact biofilms after transmission, especially for non-EPS producing staphylococci. Accordingly, staphylococcal density per unit biofilm volume had increased from 0.20 to 0.52 μm–3 for transmission of the non-EPS producing strain under high contact pressure. The EPS producing strain had similar densities before and after transmission (0.17 μm–3). This suggests three phases in biofilm transmission: (1) compression, (2) separation and (3) relaxation of biofilm structure to its pre-transmission density in EPS-rich biofilms.

Transmission of Monospecies and Dual-Species Biofilms from Smooth to Nanopillared Surfaces

Bacterial transmission from biofilm-covered surfaces to surfaces is mechanistically different from bacterial adhesion to surfaces and involves detachment from the donor and adhesion to the receiver surfaces under pressure. Bacterial transmission occurs, for instance, in food processing or packaging, in household situations, or between surfaces in hospitals. Patients admitted to a hospital room previously occupied by a patient with antibiotic-resistant pathogens are at elevated infection risk by the same pathogens through transmission. Nanopillared receiver surfaces did not collect less biofilm from a smooth donor than a smooth receiver, likely because the pressure applied during transmission negated the smaller contact area between bacteria and nanopillared surfaces, generally held responsible for reduced adhesion. Biofilm left behind on smooth donor surfaces of a non-extracellular-polymeric-substance (EPS)-producing strain and dual species had undergone different structural changes than an EPS-producing strain, which is important for their possible further treatment by antimicrobials or disinfectants. ABSTRACT The transmission of bacteria in biofilms from donor to receiver surfaces precedes the formation of biofilms in many applications. Biofilm transmission is different from bacterial adhesion, because it involves biofilm compression in between two surfaces, followed by a separation force leading to the detachment of the biofilm from the donor surface and subsequent adhesion to the receiver surface. Therewith, the transmission depends on a balance between donor and receiver surface properties and the cohesiveness of the biofilm itself. Here, we compare bacterial transmission from biofilms of an extracellular-polymeric-substance (EPS)-producing and a non-EPS-producing staphylococcal strain and a dual-species oral biofilm from smooth silicon (Si) donor surfaces to smooth and nanopillared Si receiver surfaces. Biofilms were fully covering the donor surface before transmission. However, after transmission, the biofilms only partly covered the donor and receiver surfaces regardless of nanopillaring, indicating bacterial transmission through adhesive failure at the interface between biofilms and donor surfaces as well as through cohesive failure in the biofilms. The numbers of bacteria per unit volume in EPS-producing staphylococcal biofilms before transmission were 2-fold smaller than in biofilms of the non-EPS-producing strain and of dual species. This difference increased after transmission in the biofilm left behind on the donor surfaces due to an increased bacterial density for the non-EPS-producing strain and a dual-species biofilm. This suggests that biofilms of the non-EPS-producing strain and dual species remained compressed after transmission, while biofilms of the EPS-producing strain were induced to produce more EPS during transmission and relaxed toward their initial state after transmission due to the viscoelasticity conferred to the biofilm by its EPS. IMPORTANCE Bacterial transmission from biofilm-covered surfaces to surfaces is mechanistically different from bacterial adhesion to surfaces and involves detachment from the donor and adhesion to the receiver surfaces under pressure. Bacterial transmission occurs, for instance, in food processing or packaging, in household situations, or between surfaces in hospitals. Patients admitted to a hospital room previously occupied by a patient with antibiotic-resistant pathogens are at elevated infection risk by the same pathogens through transmission. Nanopillared receiver surfaces did not collect less biofilm from a smooth donor than a smooth receiver, likely because the pressure applied during transmission negated the smaller contact area between bacteria and nanopillared surfaces, generally held responsible for reduced adhesion. Biofilm left behind on smooth donor surfaces of a non-extracellular-polymeric-substance (EPS)-producing strain and dual species had undergone different structural changes than an EPS-producing strain, which is important for their possible further treatment by antimicrobials or disinfectants.