Concerted dispersion of Staphylococcus aureus biofilm by bacteriophage and ‘green synthesized’ silver nanoparticles

Staphylococcal biofilms predominantly cause persistent nosocomial infections. The widespread antibiotic resistance followed by its ability to form biofilm in biological and inert surfaces often contributes to major complications in patients and veterinary animals. Strategic importance of bacteriophage therapy against critical staphylococcal infections had been predicted ever since the advent of antibiotic resistant staphylococcal strains. The significance of metal nanoparticles in quenching biofilm associated bacteria was previously reported. In this study, we demonstrate a concerted action of ‘green synthesized’ silver nanoparticles and bacteriophages in removing pre-formed Staphylococcus aureus biofilms from an inert glass surface in a time dependent manner. Our results demonstrate, for the first time, the rapid co-operative dispersion of the bacterial biofilm. In addition, the synergistic activity of the nanoparticles and bacteriophages causes the loss of viability of the biofilm entrapped bacterial cells thus preventing establishment of a new infection and subsequent colonization. This work further opens up a platform for the combinational therapeutic approach with a variety of nanoparticles and bacteriophages against mono or poly bacterial biofilm in environmental, industrial or clinical settings.

Formation of biofilm by Staphylococcus aureus ‘Rumba’ on untreated glass surface and a concerted disruption of the biofilm by silver nanoparticle and phage ϕ44AHJD.     

Bacteriophage Cocktail for the Prevention of Biofilm Formation by Pseudomonas aeruginosa on Catheters in an In Vitro Model System

Microorganisms develop biofilms on indwelling medical devices and are associated with device-related infections, resulting in substantial morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated catheters with Pseudomonas aeruginosa bacteriophages on biofilm formation by P. aeruginosa in an in vitro model. Hydrogel-coated catheters were exposed to a 10 log₁₀ PFU ml⁻¹ lysate of P. aeruginosa phage M4 for 2 h at 37°C prior to bacterial inoculation. The mean viable biofilm count on untreated catheters was 6.87 log₁₀ CFU cm⁻² after 24 h. The pretreatment of catheters with phage reduced this value to 4.03 log₁₀ CFU cm⁻² (P < 0.001). Phage treatment immediately following bacterial inoculation also reduced biofilm viable counts (4.37 log₁₀ CFU cm⁻² reduction; P < 0.001). The regrowth of biofilms on phage-treated catheters occurred between 24 and 48 h, but supplemental treatment with phage at 24 h significantly reduced biofilm regrowth (P < 0.001). Biofilm isolates resistant to phage M4 were recovered from catheters pretreated with phage. The phage susceptibility profiles of these isolates were used to guide the development of a five-phage cocktail from a larger library of P. aeruginosa phages. The pretreatment of catheters with this cocktail reduced the 48-h mean biofilm cell density by 99.9% (from 7.13 to 4.13 log₁₀ CFU cm⁻²; P < 0.001), but fewer biofilm isolates were resistant to these phages. These results suggest the potential of applying phages, especially phage cocktails, to the surfaces of indwelling medical devices for mitigating biofilm formation by clinically relevant bacteria.Indwelling medical devices of various kinds may become colonized with microorganisms, resulting in the formation of microbial biofilms (16). Biofilm-associated organisms are tolerant to antimicrobial agents, can evade the host immune system, and can act as a nidus for infection (16). As a result, device-related infections, such as catheter-associated bloodstream infections, cause substantial morbidity and mortality among specific patient populations (9). Attributable mortality rates for healthcare-associated bloodstream infections have been estimated to be 25% (44).A number of novel strategies have been proposed to more effectively prevent and control device-associated biofilms, either by minimizing microbial attachment to device surfaces or by targeting the biofilm after it has developed. One such strategy is to use bacteriophages (phages) (17). Phages have been used for the treatment of infectious diseases in plants (26), animals (6), and humans (33, 39, 43). The use of phages to control biofilms has potential for several reasons. Phages can replicate at the site of an infection, thereby increasing in numbers where they are most required. During the lytic replication cycle, the infection of a bacterial host cell by a single phage virion will result in the production of dozens or hundreds of progeny phage, depending on the particular phage and host strains. Some phages also have been shown to produce enzymes that degrade the extracellular polymeric substance (EPS) matrix of a biofilm (23, 25). Doolittle et al. (19) showed that progeny phage will propagate radially through a biofilm. At least in theory, a single phage dose should be capable of treating a biofilm infection as progeny phage infect adjacent cells and degrade the biofilm matrix.Curtin and Donlan (13) demonstrated that a phage that is active against Staphylococcus epidermidis could be incorporated into a hydrogel coating on a catheter and significantly reduce biofilm formation by this organism in an in vitro model system. Based on those studies with S. epidermidis, we have investigated whether phages specific for Pseudomonas aeruginosa also can reduce biofilm formation by this organism in a similar in vitro model. Catheters were treated with a single phage or a combination of phages prior to, immediately following, or 24 h after inoculation with the test P. aeruginosa culture, and the effects of the phage treatments on biofilm formation and maintenance were characterized.(Portions of this paper were presented as poster no. A-011 at the 2007 American Society for Microbiology General Meeting in Toronto, Canada [20a].)     

Bacteriophage-Mediated Control of a Two-Species Biofilm Formed by Microorganisms Causing Catheter-Associated Urinary Tract Infections in an In Vitro Urinary Catheter Model

Microorganisms from a patient or their environment may colonize indwelling urinary catheters, forming biofilm communities on catheter surfaces and increasing patient morbidity and mortality. This study investigated the effect of pretreating hydrogel-coated silicone catheters with mixtures of Pseudomonas aeruginosa and Proteus mirabilis bacteriophages on the development of single- and two-species biofilms in a multiday continuous-flow in vitro model using artificial urine. Novel phages were purified from sewage, characterized, and screened for their abilities to reduce biofilm development by clinical isolates of their respective hosts. Our screening data showed that artificial urine medium (AUM) is a valid substitute for human urine for the purpose of evaluating uropathogen biofilm control by these bacteriophages. Defined phage cocktails targeting P. aeruginosa and P. mirabilis were designed based on the biofilm inhibition screens. Hydrogel-coated catheters were pretreated with one or both cocktails and challenged with approximately 1 × 10³ CFU/ml of the corresponding pathogen(s). The biofilm growth on the catheter surfaces in AUM was monitored over 72 to 96 h. Phage pretreatment reduced P. aeruginosa biofilm counts by 4 log₁₀ CFU/cm² (P ≤ 0.01) and P. mirabilis biofilm counts by >2 log₁₀ CFU/cm² (P ≤ 0.01) over 48 h. The presence of P. mirabilis was always associated with an increase in lumen pH from 7.5 to 9.5 and with eventual blockage of the reactor lines. The results of this study suggest that pretreatment of a hydrogel urinary catheter with a phage cocktail can significantly reduce mixed-species biofilm formation by clinically relevant bacteria.     

大肠埃希菌裂解性噬菌体vB_EcoP-R1的特性及其抗细菌生物膜活性分析

摘要:目的从医 院污水中分离大肠埃希茵特异性噬菌体,分析其生物学特性,并检测其对细菌生物膜的裂解作用。方法. 通过双层琼脂平板法分离纯化噬菌体,透射电镜观察其形态,并测定其一步生长曲线、体外裂解曲线、温度及酸碱稳定性。全 基因测序分析噬菌体基因组组成及特性,结晶紫染色法评估噬菌体对宿主菌生物膜的清除效果。结果从医院污水中新分 离1株大肠埃希菌特异性噬菌体,命名为vB_ EoP-R1;透射电镜下显示形态与尾病毒目足病毒科噬菌体成员相似,在-20~ 50 C的温度范围内以及在pH值5.0~11.0酸碱条件下能稳定存活。vB_ EcoP-R1 基因组全长为40 875 bp,GC含量为49.94%, 不含毒力基因和耐药基因。vB_ _EcoP-R1 能够裂解临床分离的大肠埃希菌,且显示高度种属特异性。噬菌体作用于宿主菌预 成形生物膜4h后,对生物膜的清除率达到80.16%。结论vB_ EcoP-R1 是1株新的大肠埃希茵特异性噬菌体,裂解性强且种 属特异性高,可裂解宿主菌已形成的生物膜,有望用于大肠埃希菌感染的替代治疗。     

Nanoscale Plasma Coating Inhibits Formation of Staphylococcus aureus Biofilm

Staphylococcus aureus commonly infects medical implants or devices, with devastating consequences for the patient. The infection begins with bacterial attachment to the device, followed by bacterial multiplication over the surface of the device, generating an adherent sheet of bacteria known as a biofilm. Biofilms resist antimicrobial therapy and promote persistent infection, making management difficult to futile. Infections might be prevented by engineering the surface of the device to discourage bacterial attachment and multiplication; however, progress in this area has been limited. We have developed a novel nanoscale plasma coating technology to inhibit the formation of Staphylococcus aureus biofilms. We used monomeric trimethylsilane (TMS) and oxygen to coat the surfaces of silicone rubber, a material often used in the fabrication of implantable medical devices. By quantitative and qualitative analysis, the TMS/O₂ coating significantly decreased the in vitro formation of S. aureus biofilms; it also significantly decreased in vivo biofilm formation in a mouse model of foreign-body infection. Further analysis demonstrated TMS/O₂ coating significantly changed the protein adsorption, which could lead to reduced bacterial adhesion and biofilm formation. These results suggest that TMS/O₂ coating can be used to effectively prevent medical implant-related infections.     

New In Vitro Model To Study the Effect of Human Simulated Antibiotic Concentrations on Bacterial Biofilms

A new in vitro pharmacokinetic/pharmacodynamic simulator for bacterial biofilms utilizing flow cell technology and confocal laser scanning microscopy is described. The device has the ability to simulate the changing antibiotic concentrations in humans associated with intravenous dosing on bacterial biofilms grown under continuous culture conditions. The free drug concentrations of a single 2-g meropenem intravenous bolus dose and first-order elimination utilizing a half-life of 0.895 h (elimination rate constant, 0.776 h⁻¹) were simulated. The antibacterial activity of meropenem against biofilms of Pseudomonas aeruginosa PAO1 and three clinical strains isolated from patients with cystic fibrosis was investigated. Additionally, the effect of meropenem on PAO1 biofilms cultured for 24 h versus that on biofilms cultured for 72 h was examined. Using confocal laser scanning microscopy, rapid biofilm killing was observed in the first hour of the dosing interval for all biofilms. However, for PAO1 biofilms cultured for 72 h, only bacterial subpopulations at the periphery of the biofilm were affected, with subpopulations at the substratum remaining viable, even at the conclusion of the dosing interval. The described model is a novel method to investigate antimicrobial killing of bacterial biofilms using human simulated concentrations.     

Role of Mutation in Pseudomonas aeruginosa Biofilm Development

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.     

Inhibition of Salmonella enterica Biofilm Formation Using Small-Molecule Adenosine Mimetics

Biofilms have been widely implicated in chronic infections and environmental persistence of Salmonella enterica, facilitating enhanced colonization of surfaces and increasing the ability of the bacteria to be transmitted to new hosts. Salmonella enterica serovar Typhi biofilm formation on gallstones from humans and mice enhances gallbladder colonization and bacterial shedding, while Salmonella enterica serovar Typhimurium biofilms facilitate long-term persistence in a number of environments important to food, medical, and farming industries. Salmonella regulates expression of many virulence- and biofilm-related processes using kinase-driven pathways. Kinases play pivotal roles in phosphorylation and energy transfer in cellular processes and possess an ATP-binding pocket required for their functions. Many other cellular proteins also require ATP for their activity. Here we test the hypothesis that pharmacological interference with ATP-requiring enzymes utilizing adenosine mimetic compounds would decrease or inhibit bacterial biofilm formation. Through the screening of a 3,000-member ATP mimetic library, we identified a single compound (compound 7955004) capable of significantly reducing biofilm formation by S. Typhimurium and S. Typhi. The compound was not bactericidal or bacteriostatic toward S. Typhimurium or cytotoxic to mammalian cells. An ATP-Sepharose affinity matrix technique was used to discover potential protein-binding targets of the compound and identified GroEL and DeoD. Compound 7955004 was screened against other known biofilm-forming bacterial species and was found to potently inhibit biofilms of Acinetobacter baumannii as well. The identification of a lead compound with biofilm-inhibiting capabilities toward Salmonella provides a potential new avenue of therapeutic intervention against Salmonella biofilm formation, with applicability to biofilms of other bacterial pathogens.     

PLGA/xylitol nanoparticles enhance antibiofilm activity via penetration into biofilm extracellular polymeric substances

Biofilms are gelatinous masses of microorganisms attached to wound surfaces. Previous studies suggest that biofilms generate resistance towards antibiotic treatments. It was reported that hydrogels containing xylitol and antibiotic combinations produced additive antibiofilm inhibition. However, hydrogel formulations lack specificity, due to which xylitol cannot penetrate into the biofilm matrix and gets easily degraded by bacterial beta lactamase enzymes. It was hypothesized that the incorporation of xylitol in PLGA (polylactic-co-glycolic acid) nanoparticles will enhance penetration into the EPS (extra polymeric substance) component of the biofilm matrix and potentially overcome the antibiotic resistance associated with the biofilms. The purpose of this study was to develop PLGA nanoparticles loaded with xylitol, which will enhance bacterial biofilm penetration. The nanoparticles were loaded with different amounts of xylitol (0.5–5% w/w) and characterized for physiochemical and drug release properties. The metabolic antibiofilm activity of the PLGA nanoparticles containing xylitol was demonstrated by an XTT assay using as references the cultures of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) and the polymicrobial biofilms of both bacterial strains. Live/dead viability staining was also performed to investigate the viability ratio of bacterial cells present in the biofilms. The biofilm penetration study of the PLGA nanoparticles was assessed by combining the nanoparticles with conjugated concanavalin A (Con A)-fluorescein isothiocyanate (FITC) and by viewing using confocal laser scanning electron microscopy (CLSM). In conclusion, the PLGA nanoparticles loaded with xylitol were successfully developed and were found to promote the antibiofilm activity of xylitol in infected wounds.

Biofilms are gelatinous masses of microorganisms attached to wound surfaces.     

Synergy of Silver Nanoparticles and Aztreonam against Pseudomonas aeruginosa PAO1 Biofilms

Pathogenic bacterial biofilms, such as those found in the lungs of patients with cystic fibrosis (CF), exhibit increased antimicrobial resistance, due in part to the inherent architecture of the biofilm community. The protection provided by the biofilm limits antimicrobial dispersion and penetration and reduces the efficacy of antibiotics that normally inhibit planktonic cell growth. Thus, alternative antimicrobial strategies are required to combat persistent infections. The antimicrobial properties of silver have been known for decades, but silver and silver-containing compounds have recently seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the efficacy of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the monobactam antibiotic aztreonam, to inhibit Pseudomonas aeruginosa PAO1 biofilms. Among the different sizes of AgNPs examined, 10-nm nanoparticles were most effective in inhibiting the recovery of P. aeruginosa biofilm cultures and showed synergy of inhibition when combined with sub-MIC levels of aztreonam. Visualization of biofilms treated with combinations of 10-nm AgNPs and aztreonam indicated that the synergistic bactericidal effects are likely caused by better penetration of the small AgNPs into the biofilm matrix, which enhances the deleterious effects of aztreonam against the cell envelope of P. aeruginosa within the biofilms. These data suggest that small AgNPs synergistically enhance the antimicrobial effects of aztreonam against P. aeruginosa in vitro, and they reveal a potential role for combinations of small AgNPs and antibiotics in treating patients with chronic infections.     

Respiratory quinolones can eradicate amoxicillin-induced mature biofilms and nontypeable Haemophilus influenzae in biofilms

ObjectivesBiofilm is thought to be involved in the persistent bacterial infections caused by nontypeable Haemophilus influenzae (NTHi). This study aims to evaluate the efficacy of antibiotics against NTHi biofilms.MethodsA 96-wells pin replicator assay was applied for evaluation of antimicrobial efficacies against NTHi biofilms. The NTHi IH-202 strain for the standard and 10 clinical strains were evaluated, as well as the viability of NTHi in biofilms after antimicrobial exposures.ResultsBiofilms formed by IH-202 strain accumulated during incubation. AMPC if not high concentrations, neither reduce or inhibit biofilm formation, nor eradicate matured NTHi biofilms. The NTHi in matured biofilm were alive after exposure to amoxicillin (AMPC). Even high concentration of AMPC produced live NTHi after suspension of exposure, while tosufloxacin and garenoxacin inhibited biofilm formation of NTHi and eradicated matured biofilms. The respiratory quinolones, but not AMPC, killed NTHi in biofilms even at sub-MIC.ConclusionsNTHi persists in biofilms, even after exposure to AMPC. These findings may eventually lead to a better understanding of effective use of antibiotics to eradicate NTHi growing as biofilms, or even to the development of novel therapeutic agents for treating patients with mucosal NTHi biofilm infections. Meanwhile, respiratory quinolones are attractive agents in reducing NTHi biofilm formation and destroying established biofilm.     

Antibiotic-Loaded Synthetic Calcium Sulfate Beads for Prevention of Bacterial Colonization and Biofilm Formation in Periprosthetic Infections

Periprosthetic infection (PI) causes significant morbidity and mortality after fixation and joint arthroplasty and has been extensively linked to the formation of bacterial biofilms. Poly(methyl methacrylate) (PMMA), as a cement or as beads, is commonly used for antibiotic release to the site of infection but displays variable elution kinetics and also represents a potential nidus for infection, therefore requiring surgical removal once antibiotics have eluted. Absorbable cements have shown improved elution of a wider range of antibiotics and, crucially, complete biodegradation, but limited data exist as to their antimicrobial and antibiofilm efficacy. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin, or vancomycin-tobramycin dual treatment (in a 1:0.24 [wt/wt] ratio) were assessed for their abilities to eradicate planktonic methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis relative to that of PMMA beads. The ability of the calcium sulfate beads to prevent biofilm formation over multiple days and to eradicate preformed biofilms was studied using a combination of viable cell counts, confocal microscopy, and scanning electron microscopy of the bead surface. Biofilm bacteria displayed a greater tolerance to the antibiotics than their planktonic counterparts. Antibiotic-loaded beads were able to kill planktonic cultures of 10⁶ CFU/ml, prevent bacterial colonization, and significantly reduce biofilm formation over multiple days. However, established biofilms were harder to eradicate. These data further demonstrate the difficulty in clearing established biofilms; therefore, early preventive measures are key to reducing the risk of PI. Synthetic calcium sulfate loaded with antibiotics has the potential to reduce or eliminate biofilm formation on adjacent periprosthetic tissue and prosthesis material and, thus, to reduce the rates of periprosthetic infection.     

Prevention and Treatment of Virulent Bacterial Biofilms with an Enzymatic Nitric Oxide-Releasing Dressing

The use of percutaneous medical devices often results in nosocomial infections. Attachment of microorganisms to the surfaces of these medical devices triggers biofilm formation, which presents significant complications to the health of a patient and may lead to septicemia, thromboembolism, or endocarditis if not correctly treated. Although several antimicrobials are commonly used for prevention of biofilm formation, they have limited efficacy against formed biofilms. In this study, we report the use of an enzymatic, gaseous nitric oxide (gNO)-releasing dressing for the prevention and treatment of Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa biofilms. Results show that the bactericidal activity against biofilms of the test strains was dependent on time and rate of gNO release from the dressing. Following 6 h of treatment, gNO-releasing dressings significantly inhibited the growth of test strains relative to vehicle control dressings, demonstrating eradication of bacterial concentrations of up to 10⁵ CFU/cm². Complete cell death was observed for both prevention of biofilm formation and treatment of 24-h-grown biofilms after 6 h of treatment with the gNO-releasing dressings. Further, gNO-releasing dressings were more efficient against formed biofilms than other antimicrobial agents currently used. These results demonstrate that the gNO-releasing dressing can produce sufficient levels of gNO over a therapeutically relevant duration for maximal bactericidal effects against virulent bacterial strains known to cause nosocomial infections.     

Antibiofilm Activity of Electrical Current in a Catheter Model

Catheter-associated infections are difficult to treat with available antimicrobial agents because of their biofilm etiology. We examined the effect of low-amperage direct electrical current (DC) exposure on established bacterial and fungal biofilms in a novel experimental in vitro catheter model. Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida parapsilosis biofilms were grown on the inside surfaces of polyvinyl chloride (PVC) catheters, after which 0, 100, 200, or 500 μA of DC was delivered via intraluminally placed platinum electrodes. Catheter biofilms and intraluminal fluid were quantitatively cultured after 24 h and 4 days of DC exposure. Time- and dose-dependent biofilm killing was observed with all amperages and durations of DC administration. Twenty-four hours of 500 μA of DC sterilized the intraluminal fluid for all bacterial species studied; no viable bacteria were detected after treatment of S. epidermidis and S. aureus biofilms with 500 μA of DC for 4 days.     

Contributions of antibiotic penetration,oxygen limitation,and low metabolic activity to tolerance of Pseudomonas aeruginosa biofilms to ciprofloxacin and tobramycin

The roles of slow antibiotic penetration, oxygen limitation, and low metabolic activity in the tolerance of Pseudomonas aeruginosa in biofilms to killing by antibiotics were investigated in vitro. Tobramycin and ciprofloxacin penetrated biofilms but failed to effectively kill the bacteria. Bacteria in colony biofilms survived prolonged exposure to either 10 micro g of tobramycin ml(-1)or 1.0 micro g of ciprofloxacin ml(-1). After 100 h of antibiotic treatment, during which the colony biofilms were transferred to fresh antibiotic-containing plates every 24 h, the log reduction in viable cell numbers was only 0.49 +/- 0.18 for tobramycin and 1.42 +/- 0.03 for ciprofloxacin. Antibiotic permeation through colony biofilms, indicated by a diffusion cell bioassay, demonstrated that there was no acceleration in bacterial killing once the antibiotics penetrated the biofilms. These results suggested that limited antibiotic diffusion is not the primary protective mechanism for these biofilms. Transmission electron microscopic observations of antibiotic-affected cells showed lysed, vacuolated, and elongated cells exclusively near the air interface in antibiotic-treated biofilms, suggesting a role for oxygen limitation in protecting biofilm bacteria from antibiotics. To test this hypothesis, a microelectrode analysis was performed. The results demonstrated that oxygen penetrated 50 to 90 micro m into the biofilm from the air interface. This oxic zone correlated to the region of the biofilm where an inducible green fluorescent protein was expressed, indicating that this was the active zone of bacterial metabolic activity. These results show that oxygen limitation and low metabolic activity in the interior of the biofilm, not poor antibiotic penetration, are correlated with antibiotic tolerance of this P. aeruginosa biofilm system.     

Microscopic and Spectroscopic Analyses of Chlorhexidine Tolerance in Delftia acidovorans Biofilms

The physicochemical responses of Delftia acidovorans biofilms exposed to the commonly used antimicrobial chlorhexidine (CHX) were examined in this study. A CHX-sensitive mutant (MIC, 1.0 μg ml⁻¹) was derived from a CHX-tolerant (MIC, 15.0 μg ml⁻¹) D. acidovorans parent strain using transposon mutagenesis. D. acidovorans mutant (MT51) and wild-type (WT15) strain biofilms were cultivated in flow cells and then treated with CHX at sub-MIC and inhibitory concentrations and examined by confocal laser scanning microscopy (CLSM), scanning transmission X-ray microscopy (STXM), and infrared (IR) spectroscopy. Specific morphological, structural, and chemical compositional differences between the CHX-treated and -untreated biofilms of both strains were observed. Apart from architectural differences, CLSM revealed a negative effect of CHX on biofilm thickness in the CHX-sensitive MT51 biofilms relative to those of the WT15 strain. STXM analyses showed that the WT15 biofilms contained two morphochemical cell variants, whereas only one type was detected in the MT51 biofilms. The cells in the MT51 biofilms bioaccumulated CHX to a similar extent as one of the cell types found in the WT15 biofilms, whereas the other cell type in the WT15 biofilms did not bioaccumulate CHX. STXM and IR spectral analyses revealed that CHX-sensitive MT51 cells accumulated the highest levels of CHX. Pretreating biofilms with EDTA promoted the accumulation of CHX in all cells. Thus, it is suggested that a subpopulation of cells that do not accumulate CHX appear to be responsible for greater CHX resistance in D. acidovorans WT15 biofilm in conjunction with the possible involvement of bacterial membrane stability.     

Importance of the biofilm matrix for the erosion stability of Bacillus subtilis NCIB 3610 biofilms

Production and secretion of biomolecules can provide new emergent functionalities to the synthesizing organism. In particular, the secretion of extracellular polymeric substances (EPS) by biofilm forming bacteria creates a biofilm matrix that protects the individual bacteria within the biofilm from external stressors such as antibiotics, chemicals and shear flow. Although the main matrix components of biofilms formed by Bacillus subtilis are known, it remains unclear how these matrix components contribute to the erosion stability of B. subtilis biofilms. Here, we combine different biophysical techniques to assess this relation. In particular, we quantify the importance of specific biofilm matrix components on the erosion behavior of biofilms formed by the well-studied Bacillus subtilis NCIB 3610. We find that the absence of biofilm matrix components decreases the erosion stability of NCIB 3610 biofilms in water, largely by abolishing the hydrophobic surface properties of the biofilm and by reducing the biofilm stiffness. However, the erosion resistance of NCIB 3610 biofilms is strongly increased in the presence of metal ions or the antibiotic ciprofloxacin. In the first case, unspecific ionic cross-linking of biofilm components or individual bacteria seems to be responsible for the observed effect, and in the second case there seems to be an unspecific interaction between the antibiotic and the biofilm matrix. Taken together, our results emphasize the importance of the biofilm matrix to reduce biofilm erosion and give insights into how the specific biomolecules interact with certain chemicals to fulfill this task.

Erosion of bacterial biofilms is dependent on the composition of the biofilm matrix and the surrounding chemical environment.     

The Secret Life of the Anthrax Agent Bacillus anthracis: Bacteriophage-Mediated Ecological Adaptations

Ecological and genetic factors that govern the occurrence and persistence of anthrax reservoirs in the environment are obscure. A central tenet, based on limited and often conflicting studies, has long held that growing or vegetative forms of Bacillus anthracis survive poorly outside the mammalian host and must sporulate to survive in the environment. Here, we present evidence of a more dynamic lifecycle, whereby interactions with bacterial viruses, or bacteriophages, elicit phenotypic alterations in B. anthracis and the emergence of infected derivatives, or lysogens, with dramatically altered survival capabilities. Using both laboratory and environmental B. anthracis strains, we show that lysogeny can block or promote sporulation depending on the phage, induce exopolysaccharide expression and biofilm formation, and enable the long-term colonization of both an artificial soil environment and the intestinal tract of the invertebrate redworm, Eisenia fetida. All of the B. anthracis lysogens existed in a pseudolysogenic-like state in both the soil and worm gut, shedding phages that could in turn infect non-lysogenic B. anthracis recipients and confer survival phenotypes in those environments. Finally, the mechanism behind several phenotypic changes was found to require phage-encoded bacterial sigma factors and the expression of at least one host-encoded protein predicted to be involved in the colonization of invertebrate intestines. The results here demonstrate that during its environmental phase, bacteriophages provide B. anthracis with alternatives to sporulation that involve the activation of soil-survival and endosymbiotic capabilities.     

Bacteriophage Can Prevent Encrustation and Blockage of Urinary Catheters by Proteus mirabilis

Proteus mirabilis forms dense crystalline biofilms on catheter surfaces that occlude urine flow, leading to serious clinical complications in long-term catheterized patients, but there are presently no truly effective approaches to control catheter blockage by this organism. This study evaluated the potential for bacteriophage therapy to control P. mirabilis infection and prevent catheter blockage. Representative in vitro models of the catheterized urinary tract, simulating a complete closed drainage system as used in clinical practice, were employed to evaluate the performance of phage therapy in preventing blockage. Models mimicking either an established infection or early colonization of the catheterized urinary tract were treated with a single dose of a 3-phage cocktail, and the impact on time taken for catheters to block, as well as levels of crystalline biofilm formation, was measured. In models of established infection, phage treatment significantly increased time taken for catheters to block (∼3-fold) compared to untreated controls. However, in models simulating early-stage infection, phage treatment eradicated P. mirabilis and prevented blockage entirely. Analysis of catheters from models of established infection 10 h after phage application demonstrated that phage significantly reduced crystalline biofilm formation but did not significantly reduce the level of planktonic cells in the residual bladder urine. Taken together, these results show that bacteriophage constitute a promising strategy for the prevention of catheter blockage but that methods to deliver phage in sufficient numbers and within a key therapeutic window (early infection) will also be important to the successful application of phage to this problem.