In vitro actinomycete biofilm development and inhibition by the polyene antibiotic, nystatin, on IUD copper surfaces

The presence of intrauterine contraceptive devices (IUDs) gives a solid surface for attachment and an ideal niche for biofilm to form and flourish. Pelvic actinomycosis is often associated with the use of IUDs. Treatment of IUD-associated pelvic actinomycosis requires the immediate removal of the IUD. Therefore, this article presents in vitro evidence to support the use of novel antibiotics in the treatment of actinomycete biofilms. Twenty one clinical actinomycetes isolates from endocervical swabs of IUD wearers were assessed for their biofilm forming ability. An in vitro biofilm model with three isolates, Streptomyces strain A4, Nocardia strain C15 and Nocardia strain C17 was subjected to treatment with nystatin. Inhibition of biofilm formation by nystatin was found to be concentration dependent, with MBIC(50) values in the range 0.08-0.16 mg ml(-1). Furthermore, at a concentration of 0.16 mg ml(-1), nystatin inhibited the twitching motility of the isolates, providing evidence for a possible mechanism of biofilm inhibition.     

The effect of gold and silver nanoparticles,chitosan and their combinations on bacterial biofilms of food-borne pathogens

Abstract

The antimicrobial activity of gold and silver nanoparticles (AuNPs, AgNPs), chitosan (CS) and their combinations was established by determining the minimum inhibitory concentration for planktonic (MICPC₈₀) and biofilm growth (MICBC₈₀), for biofilm formation (MICBF₈₀), metabolic activity (MICBM₈₀) and reduction (MICBR₈₀), and for the metabolic activity of preformed biofilm (MICMPB₈₀). Biofilms were quantified in microtitre plates by crystal violet staining and metabolic activity was evaluated by the MTT assay. Chitosan effectively suppressed biofilm formation (0.31–5?mg ml^?1) in all the tested strains, except Salmonella enterica Infantis (0.16–2.5?mg ml^?1) where CS and its combination with AgNPs induced biofilm formation. Nanoparticles inhibited biofilm growth only when the highest concentrations were used. Even though AuNPs, AgNPs and CS were not able to remove biofilm mass, they reduced its metabolic activity by at least 80%. The combinations of nanoparticles with CS did not show any significant positive synergistic effect on the tested target properties.     

In vitro and in vivo antibiofilm activity of a coral associated actinomycete against drug resistant Staphylococcus aureus biofilms

Staphylococcus aureus is now amongst the most important pathogenic bacteria responsible for bloodstream nosocomial infections and for biofilm formation on indwelling medical devices. Its increasing resistance to common antibiotics, partly attributed to its ability to form biofilms, is a challenge for the development of new antimicrobial agents. Accordingly, the goal of this study was to evaluate the effect of a coral associated actinomycete (CAA) - 3 on S. aureus biofilms both in vitro and in vivo. Methanolic extracts of CAA-3 showed a reduction in in vitro biofilm formation by S. aureus ATCC 11632, methicillin resistant S. aureus ATCC 33591 and clinical isolates of S. aureus at the biofilm inhibitory concentration (BIC) of 0.1 mg ml^?1. Furthermore, confocal laser scanning microscope (CLSM) studies provide evidence of CAA-3 inhibiting intestinal colonisation of S. aureus in the nematode Caenorhabditis elegans. To conclude, this study for the first time, reports CAA as a promising source of anti-biofilm compounds, for developing novel drugs against highly resistant staphylococcal biofilms.     

In vitro and ex vivo biofilms of dermatophytes: a new panorama for the study of antifungal drugs

Abstract

This study describes an ex vivo model that creates an environment for dermatophyte biofilm growth, with features that resemble those of in vivo conditions, designing a new panorama for the study of antifungal susceptibility. Regarding planktonic susceptibility, MIC ranges were 0.125-1?µg ml^?1 for griseofulvin and 0.000097-0.25?µg ml^?1 for itraconazole and terbinafine. sMIC50 ranges were 2->512?µg ml^?1 for griseofulvin and 0.25->64?µg ml^?1 for itraconazole and terbinafine. CLSM images demonstrated a reduction in the amount of cells within the biofilm, but hyphae and conidia were still observed and biofilm biomass was maintained. SEM analysis demonstrated a retraction in the biofilm matrix, but fungal structures and water channels were preserved. These results show that ex vivo biofilms are more tolerant to antifungal drugs than in vitro biofilms, suggesting that environmental and nutritional conditions created by this ex vivo model favor biofilm growth and robustness, and hence drug tolerance.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

Anti-biofilm activity of Quercus infectoria G. Olivier against methicillin-resistant Staphylococcus aureus

Aims: To establish the effect of Quercus infectoria G. Olivier extract and its main constituent, tannic acid, on staphylococcal biofilm and their anti‐biofilm mechanisms. Methods and Results: Anti‐biofilm activity of the plant materials on clinical isolated of methicillin‐resistant Staphylococcus aureus and methicillin‐susceptible Staph. aureus was employed using a crystal violet‐stained microtiter plate method. The extract at minimum inhibitory concentration (MIC; 0·25 mg ml^?1) was significantly reduced the biofilm formation of the isolates (P < 0·05). The effect on staphylococcal cell surface hydrophobicity (CSH) of the test compounds was investigated as a possible mode of action of the anti‐biofilm activity. The hydrophobicity index of all the bacterial isolates increased following treatment with supra‐MIC, MIC and sub‐MIC of the extract and tannic acid. Observation of the treated bacterial cells by electron microscopy revealed that the test compounds caused clumps of partly divided cocci with thickened and slightly rough cell wall. Conclusions: The results indicated that Q. infectoria extract and tannic acid affected staphylococcal biofilm formation and their effect on bacterial CSH and cell wall may involve in the anti‐biofilm activity. Significance and Impact of the Study: This evidence highlighted the anti‐biofilm potency of the natural products and clarified their anti‐biofilm mechanisms.     

Inhibitory effect of α-mangostin on Acinetobacter baumannii biofilms – an in vitro study

The present study was designed to investigate the anti-biofilm potential of alpha-mangostin (α-MG) against Acinetobacter baumannii (AB). The biofilm inhibitory concentration (BIC) of α-MG against AB was found to be 2 μg ml^?1. α-MG (0.5, 1 and 2 μg ml^?1) exhibited non-bactericidal concentration-dependent anti-biofilm activities against AB. However, α-MG failed to disintegrate the mature biofilms of AB even at a 10-fold increased concentration from its BIC. Results from qRT-PCR and in vitro bioassays further demonstrated that α-MG downregulated the expression of bfmR, pgaA, pgaC, csuA/B, ompA, bap, katE, and sodB genes, which correspondingly affects biofilm formation and its associated virulence traits. The present study suggests that α-MG exerts its anti-biofilm property by interrupting initial biofilm formation and the cell-to-cell signaling mechanism of AB. Additional studies are required to understand the mode of action responsible for the anti-biofilm property.     

Inhibitory effects of single-walled carbon nanotubes on biofilm formation from Bacillus anthracis spores

This study reports the inhibitory effect of single walled carbon nanotubes (SWCNTs) on biofilm formation from Bacillus anthracis spores. Although the presence of 50 to 100 μg ml^?1 of SWCNTs in the suspension increased spore attachment in the wells of 96-well plates, the presence of 200 μg ml^?1 of SWCNTs in the germination solution decreased the germination percentage of the attached spores by 93.14%, completely inhibiting subsequent biofilm formation. The inhibition kinetics of 50 μg ml^?1 SWCNTs on biofilm formation showed that this concentration inhibited biofilm formation by 81.2% after incubation for 48 h. SWCNT treatment in the earlier stages of biofilm formation was more effective compared to treatment at later stages. Mature biofilms were highly resistant to SWCNT treatment.     

Adsorptive Immobilization of a Pseudomonas Strain on Solid Carriers for Augmented Decolourization in a Chemostat Bioreactor

Pseudomonas GM3, a highly efficient strain in cleavage of azo bonds of synthetic dyes under anoxic conditions, was immobilized via adsorption on two types of carriers, porous glass beads and solid PVA particles. The cells were cultivated in a nutrient medium, adsorbed on sterile carriers, stabilized as biofilms in repeated batch cultures, and introduced into a chemostat activated sludge reactor for augmented decolourization. The microbial cells were quickly adsorbed and fixed on the PVA surface, compared to a slow and linear immobilization on the glass surface. The porous structure of glass beads provided shelter for the embedded cells, giving a high biomass loading or thick biofilm (13.3 mg VS ml^?1 carrier) in comparison with PVA particles (4.8 mg VS ml^?1 carrier), but the mass transfer of substrate in the biofilm became a significant limiting factor in the thicker biofilms (effectiveness factor η = 0.31). The microbial decolourization rate per volume of carriers was 0.15 and 0.17 mg dye ml^?1 of glass beads and PVA particles, respectively. In augmented decomposition of a recalcitrant azo dye (60 mg l^?1), the immobilized Pseudomonas cells in porous glass beads gave a stable decolourization efficiency (80 - 81%), but cells fixed on solid PVA particles showed an initial high colour removal of 90% which then declined to a stable removal efficiency of 81%. In both cases, the colour removal efficiency of the chemostat bioreactor was increased from < 10% by an activated sludge to ～80% by the augmented system.     

Evaluation of Paenibacillus spp. isolates for the biological control of black rot in Brassica oleracea var. capitata (cabbage)

The ability of isolates of Paenibacillus spp. to protect Brassica oleracea var. capitata (cabbage) against the black rot pathogen, Xanthomonas campestris pv. campestris (Xcc),was evaluated. Twenty-four isolates of Paenibacillus spp., isolated from New Zealand-grown brassica hosts or soil, were evaluated for in vitro antagonism towards six Xcc isolates. Seven Paenibacillus spp. isolates with different levels of in vitro suppressive activity against Xcc were screened in pot experiments for their capacity to reduce black rot symptoms on cabbage. Two Paenibacillus isolates (P10 and P16) exhibited biocontrol activity against Xcc, and four isolates (P1, P6, P9, and P24) reduced cabbage seed germination and seedling emergence. The dependence of bioactivity on inoculum rate was investigated with three Paenibacillus isolates (P6, P10, and P16) at three different concentrations (5?×?10⁸, 5?×?10⁹, and 5?×?10¹⁰?CFU?ml^?1). Negative effects on seedling emergence were detected with isolate P6 at concentrations?≥5?×?10⁹?CFU?ml^?1. All three isolates applied at the three concentrations reduced black rot symptoms on the cotyledons and true leaves. There was poor or no relationship between the inhibitory effect of Paenibacillus spp. isolates on the growth of Xcc in vitro, and their biocontrol activity in vivo. Paenibacillus isolate P16 was identified as a potential biological control of black rot in cabbage.     

Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) biofilm by cationic poly (D,L-lactide-co-glycolide) nanoparticles

Abstract

The emergent need for new treatment methods for multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) has focused attention on novel potential tools like nanoparticles (NPs). In the present study, a drug-free cationic nanoparticles (CNPs) system was developed and its anti-MRSA effects were firstly investigated. The results showed that CNPs (261.7?nm, 26.1?mv) showed time- and concentration-dependent activity against MRSA growth, killing ～ 90% of planktonic bacterial cells in 3?h at 400?μg ml^?1, and completely inhibiting biofilm formation at 1000?μg ml^?1. Moreover, CNPs at 400?μg ml^?1 reduced the minimum inhibitory concentration (MIC) of vancomycin on inhibition of planktonic MRSA growth (～ 25%) and biofilm formation (～ 50%). The CNPs–bacteria interaction force was up to 22 nN. Overall, these data suggest that CNPs have a good potential in clinical applications for the prevention and treatment of MRSA infection.     

Identification ofStreptomyces sp. Tc022, an endophyte inAlpinia galanga, and the isolation of actinomycin D

Some endophytic actinomycetes (120) were isolated from the roots ofAlpinia galanga. Identification of these endophytes was based on their morphology and amino acid composition of the whole-cell extract. Most isolates were classified aStreptomyces sp. (82), with the remainder belonging toNocardia sp. (11),Microbispora sp. (3) andMicromonospora sp. (2). Eight isolates were unclassified and 14 were lost during subculture. The strain identified as endophyticStreptomyces sp. Tc022 strongly inhibitedColletotrichum musae andCandida albicans. This endophyte was cultured, the agar was extracted with organic solvent and the extract was purified on a column of silica gel to give a major component, which was identified to be actinomycin D on the basis of spectroscopic dat Actinomycin D showed antifungal activity againstColletotrichum musae andCandida albicans with the MIC of 10 and 20 mg ml^?1, respectively.     

In vitro effect of subinhibitory concentrations of antibiotics on biofilm formation by clinical strains of Salmonella enterica serovar Typhimurium isolated in Slovakia

Aims: In this study, we examined the biofilm formation of 75 Salmonella enterica serovar Typhimurium (Salm. Typhimurium) human clinical isolates and the effect of subinhibitory concentrations (sub-MICs) of gentamicin, ciprofloxacin and cefotaxime on biofilm formation and exopolysaccharides (EPS) production. Methods and Results: Quantification of biofilm formation and EPS production were carried out using a modified microtitre plate assay and spectrophotometric method, respectively. The results indicate that 38 isolates (50·7%), which are predominantly of DT104 phage type, presented as the strong biofilm producers in vitro on plastic surface. When strains with the highest biofilm-forming capacity were grown in the presence of sub-MICs of gentamicin and ciprofloxacin, the inhibition of biofilm formation and EPS production was observed. In contrast, cefotaxime at 1/2 MIC (0·039 μg ml⁻¹) was able to significantly induce the production of biofilm as well as EPS in three isolates with nontypable and DT104 phage type, respectively. Conclusions: These results clearly indicate that all the three antibiotics tested are able to interfere with biofilm formation and EPS production by Salm. Typhimurium isolates. Significance and Impact of the Study: The current study demonstrated that cefotaxime at sub-MIC can be beneficial for the behaviour of pathogen Salm. Typhimurium in vitro.     

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth,biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 μg ml^?1) inhibited Trichosporon growth. RIT (100 μg ml^?1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 μg ml^?1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.     

Effects of extracellular DNA and DNA‐binding protein on the development of a Streptococcus intermedius biofilm

Aims

The aim of this study was to clarify the effects of homologous and heterologous extracellular DNAs (eDNAs) and histone‐like DNA‐binding protein (HLP) on Streptococcus intermedius biofilm development and rigidity.

Methods and Results

Formed biofilm mass was measured with 0·1% crystal violet staining method and observed with a scanning electron microscope. The localizations of eDNA and extracellular HLP (eHLP) in formed biofilm were detected by staining with 7‐hydoxyl‐9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one) and anti‐HLP antibody without fixation, respectively. DNase I treatment (200 U ml^?1) markedly decreased biofilm formation and cell density in biofilms. Colocalization of eHLP and eDNA in biofilm was confirmed. The addition of eDNA (up to 1 μg ml^?1) purified from Strep. intermedius, other Gram‐positive bacteria, Gram‐negative bacteria, or human KB cells into the Strep. intermedius culture increased the biofilm mass of all tested strains of Strep. intermedius, wild‐type, HLP‐downregulated strain and control strains. In contrast, the addition of eDNA (>1 μg ml^?1) decreased the biofilm mass of all Strep. intermedius strains.

Conclusions

These findings demonstrated that eDNA and eHLP play crucial roles in biofilm development and its rigidity.

Significance and Impact of the Study

eDNA‐ and HLP‐targeting strategies may be applicable to novel treatments for bacterial biofilm‐related infectious diseases.     

A combination of ellagic acid and tetracycline inhibits biofilm formation and the associated virulence of Propionibacterium acnes in vitro and in vivo

Propionibacterium acnes is an opportunistic pathogen which has become notorious owing to its ability to form a recalcitrant biofilm and to develop drug resistance. The current study aimed to develop anti-biofilm treatments against clinical isolates of P. acnes under in vitro and in vivo conditions. A combination of ellagic acid and tetracycline (ETC; 250 μg ml^?1 + 0.312 μg ml^?1) was determined to effectively inhibit biofilm formation by P. acnes (80–91%) without affecting its growth, therefore potentially limiting the possibility of the bacterium attaining resistance. In addition, ETC reduced the production of extracellular polymeric substances (EPS) (20–26%), thereby making P. acnes more susceptible to the human immune system and antibiotics. The anti-biofilm potential of ETC was further substantiated under in vivo conditions using Caenorhabditis elegans. This study reports a novel anti-biofilm combination that could be developed as an ideal therapeutic agent with broad cosmeceutical and pharmaceutical applicability in the era of antibiotic resistance.     

Baseline Sensitivity of Populations of Phytophthora capsici from China to Three Carboxylic Acid Amide (CAA) Fungicides and Sequence Analysis of Cholinephosphotranferases from a CAA‐sensitive Isolate and CAA‐resistant Laboratory Mutants

During 2006–2008, 572 isolates of Phytophthora capsici were collected from seven provinces in China, and their sensitivities to three carboxylic acid amides (CAA), dimethomorph, flumorph and pyrimorph were determined. Of these isolates, 90 isolates without a history of exposure to CAA fungicides (CAAs) were used to set up the baseline sensitivity. Baseline EC₅₀ values ranged from 0.122 to 0.203 (mean ± SD, 0.154 ± 0.022) μg ml^?1 for dimethomorph, from 0.301 to 0.487 (mean ± SD, 0.373 ± 0.043) μg ml^?1 for flumorph and from 0.557 to 0.944 (mean ± SD, 0.712 ± 0.082) μg ml^?1 for pyrimorph, respectively. The other 482 isolates were tested with a single discriminatory dose and were completely inhibited at 0.5 μg ml^?1 of dimethomorph. Four CAA‐resistant mutants were generated by repeated exposure to dimethomorph in vitro. As compared to the parental wild‐type isolate, the four CAA‐resistant mutants showed similar fitness in hyphal growth, sporulation in vitro and pathogenicity in vivo. Mutants resistant to CAAs in the in vitro assay caused visible lesions on pepper stems or roots treated with the recommended dose of dimethomorph. Previous studies upon the mode of action of CAAs suggested that these fungicides maybe inhibit phospholipid biosynthesis and that the primary target could be the cholinephosphotranferase (CPT), which is referred to aminoalcoholphosphotransferases (AAPTs). We sequenced and analyzed two CPT (AAPT1 and AAPT2) genes in P. capsici. Based on the cDNA sequence, we found that the AAPT1 and AAPT2 gene span 1538 and 1459 bp and were interrupted by five and three introns, respectively. There was no difference between the parental wild‐type isolate and the four CAA‐resistant mutants in the amino acid sequences of AAPT1 and AAPT2 gene. So, it was assumed that the resistance to dimethomorph was not due to mutations in the amino acid sequence of these two possible target genes.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Antimycotic Activity Potentiation of Allium sativum Extract and Silver Nanoparticles against Trichophyton rubrum

A natural and biocompatible extract of garlic as a support, decorated with silver nanoparticles, is a proposal to generate an effective antifungal agent against dermatophytes at low concentrations. Silver nanoparticles (AgNPs) with a diameter of 26±7 nm were synthesized and their antimycotic activity was examined against Trichophyton rubrum (T. rubrum), inhibiting 94 % of growth at a concentration of 0.08 mg ml^?1. Allium sativum (garlic) extract was also obtained (AsExt), and its MIC was 0.04 mg ml^?1. To increase the antifungal capacity of those systems, AsExt was decorated with AgNPs, obtaining AsExt‐AgNPs. Using an AsExt concentration of 0.04 mg ml^?1 in independent experiments with concentrations from 0.01 to 0.08 mg ml^?1 of AgNPs, it was possible to inhibit T. rubrum at all AgNPs concentrations; it proves a synergistic effect between AgNPs and AsExt. Even if 1 % of the minimum inhibitory concentration of AsExt (0.0004 mg ml^?1) is used, it was possible to inhibit T. rubrum at all concentrations of AgNPs, demonstrating the successful antimycotic activity potentiation when combining AsExt and AgNPs.