In Vitro Management of Hospital <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Biofilm Using Indigenous T7-Like Lytic Phage

Pseudomonas aeruginosa, a human pathogen capable of forming biofilm and contaminating medical settings, is responsible for 65% mortality in the hospitals all over the world. This study was undertaken to isolate lytic phages against biofilm forming Ps. aeruginosa hospital isolates and to use them for in vitro management of biofilms in the microtiter plate. Multidrug resistant strains of Ps. aeruginosa were isolated from the hospital environment in and around Pimpri-Chinchwad, Maharashtra by standard microbiological methods. Lytic phages against these strains were isolated from the Pavana river water by double agar layer plaque assay method. A wide host range phage bacterial virus Ps. aeruginosa phage (BVPaP-3) was selected. Electron microscopy revealed that BVPaP-3 phage is a T7-like phage and is a relative of phage species gh-1. A phage at MOI-0.001 could prevent biofilm formation by Ps. aeruginosa hospital strain-6(HS6) on the pegs within 24 h. It could also disperse pre-formed biofilms of all hospital isolates (HS1–HS6) on the pegs within 24 h. Dispersion of biofilm was studied by monitoring log percent reduction in cfu and log percent increase in pfu of respective bacterium and phage on the peg as well as in the well. Scanning electron microscopy confirmed that phage BVPaP-3 indeed causes biofilm reduction and bacterial cell killing. Laboratory studies prove that BVPaP-3 is a highly efficient phage in preventing and dispersing biofilms of Ps. aeruginosa. Phage BVPaP-3 can be used as biological disinfectant to control biofilm problem in medical devices.     

The Natural Antimicrobial Carvacrol Inhibits Quorum Sensing in Chromobacterium violaceum and Reduces Bacterial Biofilm Formation at Sub-Lethal Concentrations

The formation of biofilm by bacteria confers resistance to biocides and presents problems in medical and veterinary clinical settings. Here we report the effect of carvacrol, one of the major antimicrobial components of oregano oil, on the formation of biofilms and its activity on existing biofilms. Assays were carried out in polystyrene microplates to observe (a) the effect of 0–0.8 mM carvacrol on the formation of biofilms by selected bacterial pathogens over 24 h and (b) the effect of 0–8 mM carvacrol on the stability of pre-formed biofilms. Carvacrol was able to inhibit the formation of biofilms of Chromobacterium violaceum ATCC 12472, Salmonella enterica subsp. Typhimurium DT104, and Staphylococcus aureus 0074, while it showed no effect on formation of Pseudomonas aeruginosa (field isolate) biofilms. This inhibitory effect of carvacrol was observed at sub-lethal concentrations (<0.5 mM) where no effect was seen on total bacterial numbers, indicating that carvacrol''s bactericidal effect was not causing the observed inhibition of biofilm formation. In contrast, carvacrol had (up to 8 mM) very little or no activity against existing biofilms of the bacteria described, showing that formation of the biofilm also confers protection against this compound. Since quorum sensing is an essential part of biofilm formation, the effect of carvacrol on quorum sensing of C. violaceum was also studied. Sub-MIC concentrations of carvacrol reduced expression of cviI (a gene coding for the N-acyl-L-homoserine lactone synthase), production of violacein (pigmentation) and chitinase activity (both regulated by quorum sensing) at concentrations coinciding with carvacrol''s inhibiting effect on biofilm formation. These results indicate that carvacrol''s activity in inhibition of biofilm formation may be related to the disruption of quorum sensing.     

Prevention of Staphylococcus aureus biofilm formation and reduction in established biofilm density using a combination of phage K and modified derivatives

Aims: To investigate the ability of a mixture of phage K and six of its modified derivatives to prevent biofilm formation by Staphylococcus aureus and also to reduce the established biofilm density. Methods and Results: The bioluminescence‐producing Staph. aureus Xen29 strain was used in the study, and incubation of this strain in static microtitre plates at 37°C for 48 h confirmed its strong biofilm‐forming capacity. Subsequently, removal of established biofilms of Staph. aureus Xen29 with the high‐titre phage combination was investigated over time periods of 24 h, 48 h and 72 h. Results suggested that these biofilms were eliminated in a time‐dependant manner, with biofilm biomass reduction significantly greater after 72 h than after 24–48 h. In addition, initial challenge of Staph. aureus Xen29 with the phage cocktail resulted in the complete inhibition of biofilm formation over a 48‐h period with no appearance of phage resistance. Conclusions: In general, our findings demonstrate the potential use of a modified phage combination for the prevention and successful treatment of Staph. aureus biofilms, which are implicated in several antibiotic‐resistant infections. Significance and Impact of the Study: This study highlights the first use of phage K for the successful removal and prevention of biofilms of Staph. aureus.     

A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static and flow conditions

Pseudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities – biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P. aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P. aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P. aeruginosa PAO1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension (MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P. aeruginosa infections, which are predominantly biofilm centred.     

Extracellular DNA Inhibits Salmonella enterica Serovar Typhimurium and S. enterica Serovar Typhi Biofilm Development on Abiotic Surfaces

Extracellular DNA (eDNA) was identified and characterized in a 2-day-old biofilms developed by Salmonella enterica ser. Typhimurium SR-11 and S. enterica ser. Typhi ST6 using confocal laser scanning microscopy (CLSM) and enzymatic extraction methods. Results of microtitre plate assay and CLSM analysis showed both Salmonella strains formed significantly more biofilms in the presence of DNase I; Furthermore, a remarkable decrease of biofilm formation was observed when eDNA was added in the inoculation. However, for the pre-established biofilms on polystyrene and glass, no significant difference was observed between the DNase I treated biofilm and the corresponding non-treated controls. In conclusion, these results demonstrate that eDNA is a novel matrix component of Salmonella biofilms. This is the first evidence for the presence of eDNA and its inhibitive and destabilizing effect during biofilm development of S. enterica ser. Typhimurium and S. enterica ser. Typhi on abiotic surfaces.     

The ionic liquid 1-alkyl-3-methylimidazolium demonstrates comparable antimicrobial and antibiofilm behavior to a cationic surfactant

Biofilms are problematic in health and industry because they are resistant to various antimicrobial treatments. Ionic liquids are a novel class of low temperature liquid salts consisting of discrete anions and cations, and have attracted considerable interest as safer alternatives to organic solvents. Ionic liquids have interesting antimicrobial properties and some could find use in the development of novel antiseptics, biocides and antifouling agents. The antimicrobial and antibiofilm activity of 1-dodecyl-3-methylimiazolium iodide ([C₁₂MIM]I) was studied using the clinically important bacterial pathogens, Staphylococcus aureus SAV329 and Pseudomonas aeruginosa PAO1. The ionic liquid increased cell membrane permeability in both S. aureus and P. aeruginosa cells and impaired their growth, attachment and biofilm development. The ionic liquid exhibited superior antimicrobial and antibiofilm activity against the Gram-positive S. aureus compared to the Gram-negative P. aeruginosa cells. BacLight? staining and confocal microscope imaging confirmed that the ionic liquid treatment increased the cell membrane permeability of both the Gram-positive and Gram-negative bacteria. In addition, the antimicrobial and antibiofilm properties of [C₁₂MIM]I were similar or superior to those of cetyltrimethylammonium bromide (CTAB), a well-known cationic surfactant. It is concluded that the ionic liquid induced damage to bacterial cells by disrupting cell membrane, leading to inhibition of growth and biofilm formation. Overall, the results indicate that the ionic liquid 1-dodecyl-3-methylimiazolium iodide was effective in preventing S. aureus and P. aeruginosa biofilms and could have applications in the control of bacterial biofilms.     

Bacteriophage-mediated interference of the c-di-GMP signalling pathway in Pseudomonas aeruginosa

C-di-GMP is a key signalling molecule which impacts bacterial motility and biofilm formation and is formed by the condensation of two GTP molecules by a diguanylate cyclase. We here describe the identification and characterization of a family of bacteriophage-encoded peptides that directly impact c-di-GMP signalling in Pseudomonas aeruginosa. These phage proteins target Pseudomonas diguanylate cyclase YfiN by direct protein interaction (termed YIPs, YfiN Interacting Peptides). YIPs induce an increase of c-di-GMP production in the host cell, resulting in a decrease in motility and an increase in biofilm mass in P. aeruginosa. A dynamic analysis of the biofilm morphology indicates a denser biofilm structure after induction of the phage protein. This intracellular signalling interference strategy by a lytic phage constitutes an unexplored phage-based mechanism of metabolic regulation and could potentially serve as inspiration for the development of molecules that interfere with biofilm formation in P. aeruginosa and other pathogens.     

Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments

The biofilm formation on abiotic surfaces in food and medical sectors constitutes a great public health concerns. In fact, biofilms present a persistent source for pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which lead to severe infections such as foodborne and nosocomial infections. Such biofilms are also a source of material deterioration and failure. The environmental conditions, commonly met in food and medical area, seem also to enhance the biofilm formation and their resistance to disinfectant agents. In this regard, this review highlights the effect of environmental conditions on bacterial adhesion and biofilm formation on abiotic surfaces in the context of food and medical environment. It also describes the current and emergent strategies used to study the biofilm formation and its eradication. The mechanisms of biofilm resistance to commercialized disinfectants are also discussed, since this phenomenon remains unclear to date.     

Spontaneous and transient defence against bacteriophage by phase‐variable glucosylation of O‐antigen in Salmonella enterica serovar Typhimurium

As natural killers of bacteria, bacteriophages have forced bacteria to develop a variety of defence mechanisms. The alteration of host receptors is one of the most common bacterial defence strategies against phage infection, which completely blocks phage attachment but comes at a potential fitness cost to the bacteria. Here, we report the cost‐free, transient emergence of phage resistance in Salmonella enterica subspecies enterica serovar Typhimurium through a phase‐variable modification of the O‐antigen. Phage SPC35 typically requires BtuB as a host receptor but also uses the Salmonella O12‐antigen as an adsorption‐assisting apparatus for the successful infection of S. Typhimurium. The α‐1,4‐glucosylation of galactose residues in the O12‐antigen by phase variably expressed O‐antigen glucosylating genes, designated the ^{LT 2} gtrABC1 cluster, blocks the adsorption‐assisting function of the O12‐antigen. Consequently, it confers transient SPC35 resistance to Salmonella without any mutations to the btuB gene. This temporal switch‐off of phage adsorption through phase‐variable antigenic modification may be widespread among Gram‐negative bacteria‐phage systems.     

Biofilm formation by enteric pathogens and its role in plant colonization and persistence

The significant increase in foodborne outbreaks caused by contaminated fresh produce, such as alfalfa sprouts, lettuce, melons, tomatoes and spinach, during the last 30 years stimulated investigation of the mechanisms of persistence of human pathogens on plants. Emerging evidence suggests that Salmonella enterica and Escherichia coli, which cause the vast majority of fresh produce outbreaks, are able to adhere to and to form biofilms on plants leading to persistence and resistance to disinfection treatments, which subsequently can cause human infections and major outbreaks. In this review, we present the current knowledge about host, bacterial and environmental factors that affect the attachment to plant tissue and the process of biofilm formation by S. enterica and E. coli, and discuss how biofilm formation assists in persistence of pathogens on the plants. Mechanisms used by S. enterica and E. coli to adhere and persist on abiotic surfaces and mammalian cells are partially similar and also used by plant pathogens and symbionts. For example, amyloid curli fimbriae, part of the extracellular matrix of biofilms, frequently contribute to adherence and are upregulated upon adherence and colonization of plant material. Also the major exopolysaccharide of the biofilm matrix, cellulose, is an adherence factor not only of S. enterica and E. coli, but also of plant symbionts and pathogens. Plants, on the other hand, respond to colonization by enteric pathogens with a variety of defence mechanisms, some of which can effectively inhibit biofilm formation. Consequently, plant compounds might be investigated for promising novel antibiofilm strategies.     

Extracellular protease in <Emphasis Type="Italic">Actinomycetes</Emphasis> culture supernatants inhibits and detaches <Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> biofilm formation

Bacterial biofilms are associated with chronic infections due to their resistance to antimicrobial agents. Staphylococcus aureus is a versatile human pathogen and can form biofilms on human tissues and diverse medical devices. To identify novel biofilm inhibitors of S. aureus, the supernatants from a library of 458 Actinomycetes strains were screened. The culture supernatants (1% v/v) of more than 10 Actinomycetes strains inhibited S. aureus biofilm formation by more than 80% without affecting the growth. The culture supernatants of these biofilm-reducing Actinomycetes strains contained a protease (equivalent to 0.1 μg proteinase K ml⁻¹), which both inhibited S. aureus biofilm formation and detached pre-existing S. aureus biofilms. This study suggests that protease treatment could be a feasible tool to reduce and eradicate S. aureus biofilms.     

Combined Use of Bacteriophage K and a Novel Bacteriophage To Reduce Staphylococcus aureus Biofilm Formation

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-resistant S. aureus (MRSA). Bacteriophages are obligate parasites of bacteria. They multiply intracellularly and lyse their bacterial host, releasing their progeny. We isolated a novel phage, DRA88, which has a broad host range among S. aureus bacteria. Morphologically, the phage belongs to the Myoviridae family and comprises a large double-stranded DNA (dsDNA) genome of 141,907 bp. DRA88 was mixed with phage K to produce a high-titer mixture that showed strong lytic activity against a wide range of S. aureus isolates, including representatives of the major international MRSA clones and coagulase-negative Staphylococcus. Its efficacy was assessed both in planktonic cultures and when treating established biofilms produced by three different biofilm-producing S. aureus isolates. A significant reduction of biofilm biomass over 48 h of treatment was recorded in all cases. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus biofilms.     

Linoleic acid inhibits Pseudomonas aeruginosa biofilm formation by activating diffusible signal factor‐mediated quorum sensing

Bacterial biofilm formation causes serious problems in various fields of medical, clinical, and industrial settings. Antibiotics and biocide treatments are typical methods used to remove bacterial biofilms, but biofilms are difficult to remove effectively from surfaces due to their increased resistance. An alternative approach to treatment with antimicrobial agents is using biofilm inhibitors that regulate biofilm development without inhibiting bacterial growth. In the present study, we found that linoleic acid (LA), a plant unsaturated fatty acid, inhibits biofilm formation under static and continuous conditions without inhibiting the growth of Pseudomonas aeruginosa. LA also influenced the bacterial motility, extracellular polymeric substance production, and biofilm dispersion by decreasing the intracellular cyclic diguanylate concentration through increased phosphodiesterase activity. Furthermore, quantitative gene expression analysis demonstrated that LA induced the expression of genes associated with diffusible signaling factor‐mediated quorum sensing that can inhibit or induce the dispersion of P. aeruginosa biofilms. These results suggest that LA is functionally and structurally similar to a P. aeruginosa diffusible signaling factor (cis‐2‐decenoic acid) and, in turn, act as an agonist molecule in biofilm dispersion.     

Antimicrobial Action of Carvacrol at Different Stages of Dual-Species Biofilm Development by Staphylococcus aureus and Salmonella enterica Serovar Typhimurium

The effects of carvacrol, a natural biocide, on dual-species biofilms formed by Staphylococcus aureus and Salmonella enterica serovar Typhimurium were investigated with a constant-depth film fermentor. Biofilm development reached a quasi-steady state in 12 days at 25°C with S. aureus predominance (≈99%). Cryosectional analysis detected viable S. aureus and S. enterica serovar Typhimurium at depths of 320 and 180 μm from the film surface, respectively. Carvacrol pulses (1.0 mmol/h) inhibited S. aureus by 2.5 log CFU/biofilm during the early stages of film formation, ultimately causing a significant reduction (P < 0.001) of the staphylococcal population at quasi-steady state. Initial carvacrol pulsing elicited a 3 log CFU/biofilm reduction in viable S. enterica serovar Typhimurium, and additional periodic carvacrol pulses instigated significant inhibition of salmonellae (1 to 2 log CFU/biofilm) during biofilm development. Carvacrol pulsing reduced protein levels fivefold (P < 0.001) during initial biofilm development. Comparative studies with a peroxide-based commercial sanitizer (Spor-Klenz RTU) revealed that this commercial sanitizer was more biocidal than carvacrol during early biofilm development. When the biofilm reached quasi-steady state, however, periodic pulses with 1 mmol of carvacrol per h (P = 0.021) elicited a significantly higher inhibition than Spor-Klenz RTU (P = 0.772). Dual-species microcolonies formed under the influence of continuously fed low carvacrol concentrations (1.0 mmol/h) but failed to develop into a mature quasi-steady-state biofilm and did not reach any stage of film formation in the presence of high concentrations (5.0 mmol/h). These data show that carvacrol is an effective natural intervention to control dual-species biofilm formation.     

Aeromonas hydrophila biofilm,exoprotease, and quorum sensing responses to co-cultivation with diverse foodborne pathogens and food spoilage bacteria on crab surfaces

Abstract

The effects of dual species interactions on biofilm formation by Aeromonas hydrophila in the presence of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pectobacterium carotovorum, Salmonella Typhimurium, and Listeria monocytogenes were examined. High-performance liquid chromatography and liquid-chromatography-mass spectrometry were performed to identify N-acyl homoserine lactone (AHL) molecules secreted by monocultures and dual cultures grown in crab broth. Field emission scanning electron microscopy was performed to observe attachment and biofilm formation. P. aeruginosa and P. fluorescens inhibited biofilm formation by A. hydrophila on the crab surface, without affecting their own biofilm-forming abilities. Dual biofilms of S. Typhimurium, L. monocytogenes, or P. carotovorum did not affect A. hydrophila biofilm formation. Exoprotease, AHL, and AI-2 levels were significantly reduced in dual cultures of P. aeruginosa and P. fluorescens with A. hydrophila, supporting the relationship between quorum sensing and biofilm formation. Dual-species biofilms were studied in their natural environment and in the laboratory.     

Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing Pseudomonas aeruginosa and Escherichia coli resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings.     

Effects of Norspermidine and Spermidine on Biofilm Formation by Potentially Pathogenic Escherichia coli and Salmonella enterica Wild-Type Strains

Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.     

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.     

A refined technique for extraction of extracellular matrices from bacterial biofilms and its applicability

Biofilm‐forming bacteria embedded in polymeric extracellular matrices (ECMs) that consist of polysaccharides, proteins and/or extracellular DNAs (eDNAs) acquire high resistance to antimicrobial agents and host immune systems. To understand molecular mechanisms of biofilm formation and maintenance and to develop therapeutic countermeasures against chronic biofilm‐associated infections, reliable methods to isolate ECMs are inevitable. In this study, we refined the ECM extraction method recently reported and evaluated its applicability. Using three Staphylococcus aureus biofilms in which proteins, polysaccharides or eDNAs are major contributors to their integrity, ECMs were extracted using salts and detergents. We found that extraction with 1.5 M sodium chloride (NaCl) could be optimum for not only ECM proteins but also polysaccharides and eDNAs. In addition, long‐time incubation was not necessary for efficient ECM isolation. Lithium chloride (LiCl) was comparative to NaCl but is more expensive. In contrast to SDS, NaCl hardly caused leakage of intracellular proteins and did not affect viability of bacterial cells within biofilms. Furthermore, this method is applicable to other bacteria such as Gram‐positive Staphylococcus epidermidis and Gram‐negative Escherichia coli and Pseudomonas aeruginosa. Thus, this refined method is very simple, rapid, low cost and non‐invasive and could be used for a broad range of applications.     

Modelling antisepsis using defined populations of facultative and anaerobic wound pathogens grown in a basally perfused biofilm model

An in vitro model was developed to assess the effects of topical antimicrobials on taxonomically defined wound biofilms. Biofilms were exposed over seven days to povidone-iodine, silver acetate or polyhexamethylene biguanide (PHMB) at concentrations used in wound dressings. The rank order of tolerance in multi-species biofilms, based on an analysis of the average bacterial counts over time was P. aeruginosa > methicillin-resistant Staphylococcus aureus (MRSA) > B. fragilis > S. pyogenes. The rank order of effectiveness for the antimicrobials in the biofilm model was povidone-iodine > PHMB > silver acetate. None of the test compounds eradicated P. aeruginosa or MRSA from the biofilms although all compounds except silver acetate eliminated S. pyogenes. Antimicrobial effectiveness against bacteria grown in multi-species biofilms did not correlate with planktonic susceptibility. Defined biofilm populations of mixed-species wound pathogens could be maintained in the basal perfusion model, facilitating the efficacy testing of treatments regimens and potential dressings against multi-species biofilms composed of wound isolates.