Biofilms Of Clinical Isolates Research Articles

Maxitrol as an antibiofilm agent with potential applications in Otolaryngology‐Head and Neck Surgery

AbstractObjectivesMaxitrol (Novartis) is a topical ophthalmic medication that contains polymyxin B, neomycin, and dexamethasone. If it possesses antibiofilm activity, it may be useful for treating diseases of the head and neck in which biofilms are implicated, including chronic rhinosinusitis, chronic suppurative otitis media and osteoradionecrosis. We investigated the in vitro efficacy of Maxitrol against Staphylococcus aureus and Pseudomonas aeruginosa biofilms.MethodsMinimum biofilm eradication concentration (MBEC) assays were performed using biofilms of P. aeruginosa ATCC 27853 and S. aureus ATCC 6538 type strains, grown on 96‐pin lids and treated in Maxitrol for 30 min, 1 h, or 6 h. Isolates of both species were collected from the middle meatuses of patients with cystic fibrosis. Biofilms of clinical isolates were grown and treated in vitro for 6 h with Maxitrol, both undiluted at full concentration and at the identified MBEC, then cultured to identify bacterial survival.ResultsNeither type strain was eradicated at 30 min nor S. aureus at 1 h at any tested concentration. P. aeruginosa was eradicated by a median of 90% and 5.6% Maxitrol at 1 and 6 h, respectively, and S. aureus with 90% Maxitrol at 6 h. Undiluted Maxitrol reliably eradicated all clinical isolates of P. aeruginosa but only one of five S. aureus isolates.ConclusionsMaxitrol reliably eradicates P. aeruginosa biofilm but not S. aureus biofilm in vitro. It may have a therapeutic role against biofilms in which P. aeruginosa is the dominant pathogen.Level of EvidenceN/A.

Biofilm analyses and exoproduct release by clinical and environmental isolates of Burkholderia pseudomallei from Brazil

Objective: To characterize biofilm production by clinical (n=21) and environmental (n=11) isolates of Burkholderia pseudomallei and evaluate the production of proteases, hemolysins and siderophores. Methods: Initially, the 32 strains were evaluated for biofilm production in Müller-Hinton broth-1% glucose (MH-1% glucose) and BHI broth-1% glucose, using the crystal violet staining technique. Subsequently, growing (48 h) and mature (72 h) biofilms were evaluated by confocal microscopy. Finally, the production of proteases, hemolysins and siderophores by planktonic aggregates, growing biofilms and mature biofilms was evaluated. Results: All isolates produced biofilms, but clinical isolates had significantly higher biomass in both MH-1% glucose (P<0.001) and BHI-glucose 1% (P=0.005). The structural analyses by confocal microscopy showed thick biofilms, composed of multiple layers of cells, homogeneously arranged, with mature biofilms of clinical isolates presenting higher biomass (P=0.019) and thickness of the entire area (P=0.029), and lower roughness coefficient (P=0.007) than those of environmental isolates. Protease production by growing biofilms was significantly greater than that of planktonic (P<0.001) and mature biofilms (P<0.001). Hemolysin release by planktonic aggregates was higher than that of biofilms (P<0.001). Regarding siderophores, mature biofilms presented higher production than growing biofilms (P<0.001) and planktonic aggregates (P<0.001). Conclusions: Clinical isolates have higher production of biofilms than their environmental counterparts; protease and siderophores seem important for growth and maintenance of Burkholderia pseudomallei biofilms.

Antibiofilm and antimicrobial activity of curcumin-chitosan nanocomplexes and trimethoprim-sulfamethoxazole on Achromobacter, Burkholderia, and Stenotrophomonas isolates

ABSTRACT Background Non-fermenting Gram-negative Achromobacter xylosoxidans, Burkholderia cepacia complex, and Stenotrophomonas maltophilia species cause healthcare-associated infections, often showing resistance to first-line drugs such as trimethoprim-sulfamethoxazole (TMP-SXT). The aim of this study was to determine the effect of curcumin-chitosan nanocomplexes on biofilm-producing clinical isolates of non-fermenting Gram-negative bacilli. Methods A. xylosoxidans, B. cepacia complex, and S. maltophilia clinical isolates were identified by MALDI-TOF mass spectrometry. Antimicrobial susceptibility was determined by broth microdilution. Curcumin (Cur), chitosan (Chi), and sodium tripolyphosphate (TPP) were encapsulated by ionotropic gelation in magnetic nanoparticles (MNP) and were assessed by scanning electron microscopy (SEM) and Fourier-transform infrared (FTIR). Biofilm inhibition and eradication by Cur-Chi-TPP-MNP with TMP-SXT was assessed. Results Cur-Chi-TPP-MNP in combination with TMP-SXT showed biofilm inhibition activity in A. xylosoxidans (37.5 µg/mL), B. cepacia (18.75 µg/mL), and S. maltophilia (4.69–18.75 µg/mL) and low biofilm eradication activity in all three strains (150 − 300 µg/mL). Conclusions Cur-Chi-TPP-MNP in combination with TMP-SXT was able to inhibit biofilm and in lower effect to eradicate established biofilms of clinical isolates of A. xylosoxidans, B. cepacia complex, and S. maltophilia species. Our results highlight the need to assess these potential treatment options to be used clinically in biofilm-associated infections.

Comparison of doses of paracetamol or ibuprofen to inhibit the formation of biofilms Pseudomonas aeruginosa bacteria

Background: Some antimicrobials are not effective for bacterial therapy Multiple drug resistance and that form biofilms so strategies of looking for alternative therapies began to be developed. Paracetamol and ibuprofen as non-antibiotic drugs that have the potential to inhibit the formation of biofilms. In this study, a trial was carried out comparing exposure to doses of paracetamol and ibuprofen to inhibit the formation of biofilms of clinical isolates stored Pseudomonas aeruginosa. Method: This study is a true experimental study. Results: The results of the study obtained paracetamol and ibuprofen were able to inhibit the formation of biofilms. A dose of paracetamol 1000mg is able to inhibit the formation of Pseudomonas aeruginosa biofilm up to 68.15%. A dose of ibuprofen 800mg is able to inhibit up to 49.13%. The results of statistical tests comparing doses capable of inhibiting, paracetamol and ibuprofen showed significant differences for all doses, except ibuprofen doses of 600mg and 800mg were not obtained significantly different from p=0.422 values. Conclusion: Paracetamol and ibuprofen are able to inhibit the formation of biofilm of Pseudomonas aeruginosa bacteria with concentration-dependent activity through optical density value measurement tests using ELISA reader.

Antibiofilm potential of Psidium guajava and Passiflora edulis pulp extracts against Staphylococcus aureus, cytotoxicity, and interference on the activity of antimicrobial drugs

BackgroundPathogenic strains of Staphylococcus aureus can cause several diseases including septicemia and endocarditis, in spite of being a commensal species of the human microbiota. The current drug resistance of S. aureus raises the need for new antimicrobials, and natural products represent a feasible source for prospection of such compounds, due to features including the diversity of structures and mechanisms of action. Here, we provide evidence of the antimicrobial activity of methanolic of Psidium guajava and Passiflora edulis pulps against planktonic cells and biofilms of clinical isolates of S. aureus.ResultsThe extracts were effective against the strains in concentrations up to 7.81 and 250 μg/mL for planktonic cells and biofilms, respectively. Antagonistic interactions of the extracts to antimicrobial drugs were observed. The pulps caused no cytotoxic effects on BGM cells. GC-MS analysis found relevant molecules, and UPLC analysis suggested the presence of flavonoids. To the best of our knowledge, this is the first antibiofilm evidence of such extracts.ConclusionThe extracts seem to be safe and effective enough for more studies aiming at exploring isolated antimicrobial molecules using in vivo models for the treatment of staphylococcal diseases.

Polymicrobial antibiofilm activity of the membranotropic peptide gH625 and its analogue

This work illustrates a new role for the membranotropic peptide gH625 and its derivative gH625-GCGKKK in impairing formation of polymicrobial biofilms. Mixed biofilms composed of Candida and bacterial species cause frequently infections and failure of medical silicone devices and also show a major drug resistance than single-species biofilms. Inhibition and eradication of biofilms were evaluated by complementary methods: XTT-reduction, and crystal violet staining (CV). Our results indicate that gH625-GCGKKKK, better than the native peptide, strongly inhibited formation of mixed biofilms of clinical isolates of C. tropicalis/S. marcescens and C. tropicalis/S. aureus and reduced the biofilm architecture, interfering with cell adhesion and polymeric matrix, as well as eradicated the long-term polymicrobial biofilms on silicone surface.

Enhancing antibiofilm activity with functional chitosan nanoparticles targeting biofilm cells and biofilm matrix

Bacterial biofilms play a key role during infections, which are associated with an increased morbidity and mortality. The classical antibiotic therapy cannot eradicate biofilm-related infections because biofilm bacteria display high drug resistance due to biofilm matrix. Thus, novel drug delivery to overcome biofilm resistance and eliminate biofilm-protected bacteria is needed to be developed. In this study, positively charged chitosan nanoparticles (CSNP) loaded with oxacillin (Oxa) and Deoxyribonuclease I (CSNP-DNase-Oxa) were fabricated. The antibiofilm activity was evaluated against Staphylococcus aureus biofilms. Biofilm architecture on silicone surfaces was investigated by scanning electron microscopy (SEM). Confocal laser scanning microscopy (CLSM) was used to examine live/dead organisms within biofilm. CSNP-DNase-Oxa exhibited higher antibiofilm activity than Oxa-loaded nanoparticles without DNase (CSNP-Oxa) and free Oxa (Oxa and Oxa + DNase) at each concentration in all in-vitro tests. CSNP-DNase-Oxa inhibited biofilm formation in-vitro and eradicated mature biofilm effectively. CSNP-DNase-Oxa could disrupt the biofilm formation through degradation of eDNA, reduced biofilm thickness and the amount of viable cells on silicone. Repeated treatment with CSNP-DNase-Oxa for two days resulted in 98.4% biofilm reduction. Moreover, CSNP-DNase-Oxa was not only able to affect the biofilm of a standard S. aureus strain, but also showed the highest eradication of biofilms of clinical isolates compared with control groups. These results suggest the potential applicability of NPs for the treatment of biofilm-related infections and provide a platform for designing novel drug delivery with more functions.

Sodium hypochlorite is more effective than 70% ethanol against biofilms of clinical isolates of Staphylococcus aureus

Although disinfectants are used for eradication of bacteria from environmental surfaces, their antibiofilm efficacy is often not considered in determining the choice of disinfectant. This study aimed to compare the effectiveness of 2 commonly used disinfectants, sodium hypochlorite and ethanol, against the planktonic and biofilm state of Staphylococcus aureus clinical isolates. Effect of 0.6% sodium hypochlorite and 70% ethanol was determined on the planktonic and biofilm states of 10 strong and weak biofilm formers through estimation of changes in colony forming unit counts and absorbance values. The morphologic changes were observed by scanning electron microscopy. Significant difference in the efficacy of sodium hypochlorite and ethanol was observed against the biofilm (P = .004) as well as planktonic (P = .000) states of S aureus. However, no significant difference was observed in their activity against strong and weak biofilm formers. On electron microscopy, sodium hypochlorite was found to induce significant formation of craters and irregular depressions on the surface of strong biofilm formers. Sodium hypochlorite demonstrated superior efficacy in controlling both planktonic and biofilm states of growth in S aureus. Furthermore, the characteristic morphologic changes observed in strong biofilm formers hint at its biofilm-specific activity.

Soluble factors from biofilms of wound pathogens modulate human bone marrow-derived stromal cell differentiation, migration, angiogenesis, and cytokine secretion.

BackgroundChronic, non-healing wounds are often characterized by the persistence of bacteria within biofilms - aggregations of cells encased within a self-produced polysaccharide matrix. Biofilm bacteria exhibit unique characteristics from planktonic, or culture-grown, bacterial phenotype, including diminished responses to antimicrobial therapy and persistence against host immune responses. Mesenchymal stromal cells (MSCs) are host cells characterized by their multifunctional ability to undergo differentiation into multiple cell types and modulation of host-immune responses by secreting factors that promote wound healing. While these characteristics make MSCs an attractive therapeutic for wounds, these pro-healing activities may be differentially influenced in the context of an infection (i.e., biofilm related infections) within chronic wounds. Herein, we evaluated the effect of soluble factors derived from biofilms of clinical isolates of Staphylococcus aureus and Pseudomonas aeruginosa on the viability, differentiation, and paracrine activity of human MSCs to evaluate the influence of biofilms on MSC activity in vitro.ResultsExposure of MSCs to biofilm-conditioned medias of S. aureus and P. aeruginosa resulted in reductions in cell viability, in part due to activation of apoptosis. Similarly, exposure to soluble factors from biofilms was also observed to diminish the migration ability of cells and to hinder multi-lineage differentiation of MSCs. In contrast to these findings, exposure of MSCs to soluble factors from biofilms resulted in significant increases in the release of paracrine factors involved in inflammation and wound healing.ConclusionsCollectively, these findings demonstrate that factors produced by biofilms can negatively impact the intrinsic properties of MSCs, in particular limiting the migratory and differentiation capacity of MSCs. Consequently, these studies suggest use/application of stem-cell therapies in the context of infection may have a limited therapeutic effect.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0412-x) contains supplementary material, which is available to authorized users.

Effects of local delivery of d-amino acids from biofilm-dispersive scaffolds on infection in contaminated rat segmental defects

Infectious complications of open fractures continue to be a significant factor contributing to non-osseous union and extremity amputation. The persistence of bacteria within biofilms despite meticulous debridement and antibiotic therapy is believed to be a major cause of chronic infection. Considering the difficulties in treating biofilm-associated infections, the use of biofilm dispersal agents as a therapeutic strategy for the prevention of biofilm-associated infections has gained considerable interest. In this study, we investigated whether local delivery of d-Amino Acids (d-AAs), a biofilm dispersal agent, protects scaffolds from contamination and reduces microbial burden within contaminated rat segmental defects in vivo. In vitro testing on biofilms of clinical isolates of Staphylococcus aureus demonstrated that d-Met, d-Phe, d-Pro, and d-Trp were highly effective at dispersing and preventing biofilm formation individually, and the effect was enhanced for an equimolar mixture of d-AAs. Incorporation of d-AAs into polyurethane scaffolds as a mixture (1:1:1 d-Met:d-Pro:d-Trp) significantly reduced bacterial contamination on the scaffold surface in vitro and within bone when implanted into contaminated femoral segmental defects. Our results underscore the potential of local delivery of d-AAs for reducing bacterial contamination by targeting bacteria within biofilms, which may represent a treatment strategy for improving healing outcomes associated with open fractures.

In Vitro Modulation of E. coli Community Behavior and Human Innate Immune System by Lantibiotic Nisin

Biofilms are microbial communities with genetically divergent microorganisms. Such communal behavior is known to provide survival benefit to the unicellular organisms in adverse conditions. Pathogenicity of opportunistic bacterial pathogens largely depends on their success in proper quorum establishment and biofilm formation. Thus molecules causing quorum-sensing attenuation, preventing the biofilm formation or instigating preformed biofilm dislodgement could serve as attractive drugs/drug supplements. Here we investigate the effect of nisin—type A lantibiotic naturally produced by Lactococcus lactis—on laboratory developed Escherichia coli biofilms and on isolated human neutrophils. Activity evaluation was done on the biofilms of clinical isolates of E. coli, developed on glass slides in a simple static bioreactor design. Nisin not only inhibited the formation but also effectively dislodged the preformed E. coli biofilms developed on glass surfaces. Presence of nisin also demonstrated a significant decrease in the expression of E. coli virulence factors viz. hemolysin and curli expression. The microorganisms dislodged from the biofilms and set free in the circulation of infected host might later reassociate to form new biofilms after nisin clearance from circulation. Thus complete eradication of infective bacterium will depend on stimulatory effect of nisin (if any) on human immune system cells. Therefore modulation of human neutrophil activity by nisin was also evaluated. Presence of nisin induced neutrophil extracellular trap (NET) formation or NETosis in a manner similar to that demonstrated by LPS (lipopolysaccharide) in vitro. Our results thus present nisin as a plausible molecule to be used in treatment of chronic bacterial infections as it indicated increased fitness for the same.

Effect of antibiotic co-administration on young and mature biofilms of cystic fibrosis clinical isolates: the importance of the biofilm model

The prognosis of patients with cystic fibrosis (CF) has improved dramatically over the last three decades although the majority of patients still die in early adulthood. Infection with Pseudomonas aeruginosa has generally been associated with declining lung function and increased mortality in patients. This study aimed to investigate the in vitro activity of tobramycin/clarithromycin combination on biofilms of clinical isolates of P. aeruginosa, meticillin-susceptible and -resistant Staphylococcus aureus, and Burkholderia cepacia. First, the impact of antibiotic co-administration on biofilms at different stages of maturation, i.e. during early formation and on 24-h-old and 12-day-old biofilms, was compared. The 24-h-old biofilms were found to behave differently compared with those aged 12 days, which were more resistant to antibiotics. A kinetic study of antibiotic co-administration twice a day for 9 days on 12-day-old P. aeruginosa biofilms was then performed to simulate the effect of treatment of CF patients by inhaled tobramycin through aerosolisation (TOBI ®). The results obtained support a synergistic activity of tobramycin/clarithromycin combination on biofilms of P. aeruginosa PY02 and PA01, with a logarithmic bacterial decrease of 3.37 and 3.96, respectively. On the other hand, increased resistance to each of the antibacterial agents used alone was observed. This study highlights the importance of the biofilm stage for in vitro investigations and enabled the development of an in vitro model of mature biofilm that is more appropriate to mimic in vivo conditions in CF patients.

In vitro activity of antibiotic combinations against Pseudomonas aeruginosa biofilm and planktonic cultures

In order to investigate the hypothesis that the combination of clarithromycin with other antibacterial agents offers a successful treatment in the eradication of Pseudomonas aeruginosa biofilm, we first determined the activity of eight antimicrobial agents against planktonic cultures of P. aeruginosa isolates by the microdilution technique. Second, we determined the in vitro effects of these antimicrobial agents individually and in combination against planktonic cultures and pre-formed biofilms of P. aeruginosa PA01. Drug combinations with marked activity were tested on biofilms of clinical isolates. The percentages of planktonic culture survival and biofilm persistence were determined using spectrophotometry and scanning electron microscopy, respectively. Bacterial enumeration was used as a more quantitative method to assess the viability of bacteria in the biofilm. Among the antibacterial agents tested, tobramycin and polymyxin B had the strongest activity against planktonic cultures when tested alone. Synergistic activity was observed for the combination chitosan/tobramycin against planktonic cultures but not biofilms, and for the combination tobramycin/clarithromycin against biofilms but not planktonic cultures. The results suggest that the combination clarithromycin/tobramycin may be successful for eradicating infections involving bacterial biofilms such as in cystic fibrosis patients chronically infected by P. aeruginosa. Further studies on representative isolates in vivo are warranted to support these results.