Biofilm Cell Counts Research Articles

Targeted enhancement of bacteriophage activity against antibiotic-resistant Staphylococcus aureus biofilms through an evolutionary assay.

Staphylococcus aureus´ biofilm-forming ability and rapid resistance development pose a significant challenge to successful treatment, particularly in postoperative complications, emphasizing the need for enhanced therapeutic strategies. Bacteriophage (phage) therapy has reemerged as a promising and safe option to combat multidrug-resistant bacteria. However, questions regarding the efficacy of phages against biofilms and the development of phage resistance require further evaluation. Expanding on the adaptable and evolutionary characteristics of phages, we introduce an evolutionary approach to enhance the activity of S. aureus phages against biofilms. Unlike other in vitro directed evolution methods performed in planktonic cultures, we employed pre-stablished biofilms to do a serial-passage assay to evolve phages monitored by real-time isothermal microcalorimetry (IMC). The evolved phages demonstrated an expanded host range, with the CUB_MRSA-COL_R9 phage infecting 83% of strains in the collection (n = 72), surpassing the ISP phage, which represented the widest host range (44%) among the ancestral phages. In terms of antimicrobial efficacy, IMC data revealed superior suppression of bacterial growth by the evolved phages compared to the ancestral CUB-M and/or ISP phages against the respective bacterial strain. The phage cocktail exhibited higher efficacy, achieving over 90% suppression relative to the growth control even after 72 h of monitoring. Biofilm cell-counts, determined by RT-qPCR, confirmed the enhanced antibiofilm performance of evolved phages with no biofilm regrowth up to 48 h in treated MRSA15 and MRSA-COL strains. Overall, our results underscore the potential of biofilm-adapted phage cocktails to improve clinical outcomes in biofilm-associated infections, minimizing the emergence of resistance and lowering the risk of infection relapse. However, further investigation is necessary to evaluate the translatability of our results from in vitro to in vivo models, especially in the context of combination therapy with the current standard of care treatment.

Comparative evaluation of disinfectant efficacy against biofilm-residing microorganisms

Introduction. Bacteria in biofilms (BFs) have increased resistance to antibacterial agents, including disinfectants; however, the efficacy level varies depending on the chosen treatment. Therefore, evaluation of efficacy of main disinfectants against BF-residing microorganisms is of scientific and practical interest.
 The purpose of the study was to explore the effect of disinfectants from various chemical groups on gram-positive and gram-negative bacteria residing in BFs.
 Materials and methods. The effect of the following disinfectants has been evaluated: alkyldimethylbenzylammonium chloride (ADBAC), tertiary amine (TA), polyhexamethylene guanidine chloride (PHMG), hydrogen peroxide (HP), chloramine (CA), dichloroisocyanuric acid sodium salt (Na DCC), sodium hypochlorite (HC), ethyl alcohol (EA), glutaraldehyde (GA)) against Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538-P BFs. BFs were grown in 96-well plates at 37ºC for 24 hours and then exposed to biocide solutions. The efficacy of disinfectants was evaluated by the number of remaining viable cells and BF relative density.
 Results. The analyzed bacterial strains formed moderate BFs; the average number of viable cells in BFs was 6.51 ± 0.19 lg. The viable bacterial cell counts in BFs reduced by more than 4 lg when exposed to HP solutions at a concentration of 6%, Na DCC solution — 0.1% (by active chlorine), HC — 1% (by active chlorine), CA – 1% (by product), PHMG — 0.05%, TA — 1.0 %. The BF density decreased by more than 70%. ADBAC solutions at concentrations of 0.1–1.0%, TA — 0.05%, HP — 3%, Na DCC solution — 0.05% (by active chlorine) caused a 2-lg reduction in viable cell counts in BFs. The efficacy of chlorine-active compounds and HP increased when 0.5% sulfonol was added. GA (0.25–1.00%) and EA (40–70%) solutions were ineffective against BF microorganisms.
 Conclusion. A promising potential in combating microbial biofilms is demonstrated by disinfectants from the group of oxidizing agents (chlorine-active and oxygen-containing), TA and PHMG; using ADBAC as an individual compound is ineffective; aldehydes and alcohols are unable to destroy BFs and eliminate microorganisms in them.

L-tyrosine modulates biofilm formation of Bacillus cereus ATCC 14579

Bacillus cereus is a food-borne pathogen capable of producing biofilms. Following analysis of biofilm formation by B. cereus ATCC 14579 transposon mutants in defined medium (DM), a deletion mutant of bc2939 (Δbc2939) was constructed that showed decreased crystal violet biofilm staining and biofilm cell counts. In addition, Δbc2939 also produced smaller colony biofilms with lower cell counts and loss of wrinkly morphology. The bc2939 gene encodes for Prephenate dehydrogenase, which converts Prephenate to 4-Hydroxy-phenylpyruvate (4-HPPA) in the l-tyrosine branch of the Shikimate pathway. While growth of the mutant and WT in DM was similar, addition of l-tyrosine was required to restore WT-like (colony) biofilm formation. Comparative proteomics showed reduced expression of Tyrosine-protein kinase/phosphatase regulators and extracellular polysaccharide cluster 1 (EPS1) proteins, aerobic electron transfer chain cytochrome aa3/d quinol oxidases, and iso-chorismate synthase involved in menaquinone synthesis in DM grown mutant biofilm cells, while multiple oxidative stress-related catalases and superoxide dismutases were upregulated. Performance in shaking cultures showed a 100-fold lower concentration of menaquinone-7 and reduction in cell counts of DM grown Δbc2939 indicating increased oxygen sensitivity. Combining all results, points to an important role of Tyrosine-modulated EPS1 production and menaquinone-dependent aerobic respiration in B. cereus ATCC 14579 (colony) biofilm formation.

Biofilm formation, sodium hypochlorite susceptibility and genetic diversity of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a marine oriented pathogen; and biofilm formation enables its survival and persistence on seafood processing plant, complicating the hygienic practice. The objectives of this study are to assess the ability of V. parahaemolyticus isolated from seafood related environments to form biofilms, to determine the effective sodium hypochlorite concentrations required to inactivate planktonic and biofilm cells, and to evaluate the genetic diversity required for strong biofilm formation. Among nine isolates, PFR30J09 and PFR34B02 isolates were identified as strong biofilm forming strains, with biofilm cell counts of 7.20, 7.08 log10 CFU/cm2, respectively, on stainless steel coupons after incubation at 25 °C. Free available chlorine of 1176 mg/L and 4704 mg/L was required to eliminate biofilm cells of 1.74–2.28 log10 CFU/cm2 and > 7 log10 CFU/cm2, respectively, whereas 63 mg/L for planktonic cells, indicating the ineffectiveness of sodium hypochlorite in eliminating V. parahaemolyticus biofilm cells at recommended concentration in the food industry. These strong biofilm-forming isolates produced more polysaccharides and were less susceptible to sodium hypochlorite, implying a possible correlation between polysaccharide production and sodium hypochlorite susceptibility. Genetic diversity in mshA, mshC and mshD contributed to the observed variation in biofilm formation between isolates. This study identified strong biofilm-forming V. parahaemolyticus strains of new multilocus sequence typing (MLST) types, showed a relationship between polysaccharide production and sodium hypochlorite resistance.

Influence of Different Stainless Steel Finishes on Biofilm Formation by Listeria monocytogenes

Biofilm formation of Listeria monocytogenes on stainless steel, a widely used abiotic surface in the food processing industry, was investigated by focusing on the attachment tendency and behavior of L. monocytogenes 08-5578 on eight different stainless steel surfaces: glass bead blasted (rough and fine), deburred (Timesaver), drum deburred, pickled, pickled and drum polished, electrolytic polished, and cold rolled (untreated control). The aim was to see whether there are finishes with significantly lower bacterial attachment. Surface roughness data (measured via four roughness parameters), determined by interferometry, was also compared with the number of adhering cells to detect possible correlations. Cultivation of L. monocytogenes biofilms was carried out using a CDC biofilm reactor with 1% tryptic soy broth set at 20°C for 4, 8, and 24 h. In addition, a cultivation trial was run with continuous nutrient flow (1% tryptic soy broth, 6.2 mL/min) for 24 h. Eight-hour results showed a significant difference (P < 0.05) in biofilm cell counts in biofilms between the glass bead-blasted surfaces (3.23 and 3.26 log CFU/cm2 for the fine and rough, respectively) and deburred (Timesaver) surface (2.57 log CFU/cm2), between drum deburred and deburred (Timesaver) surface (3.41 versus 2.57 log CFU/cm2), and between drum deburred and pickled surface (3.41 versus 2.77 log CFU/cm2). Data gained after 4-h, 24-h, and 24-h plus an additional 24-h continuous flow cultivation showed no significant difference in attachment among surfaces. No correlation between roughness data and attachment was found after all four incubation times, suggesting that roughness values, at these ranges, are insufficient in determining the surfaces' affinity to bacteria. Overall, this study suggests that roughness values cannot be used to predict the degree of L. monocytogenes attachment to a specific stainless steel surface.

Hydrogen-peroxide generating electrochemical bandage is active in vitro against mono- and dual-species biofilms

Biofilms formed by antibiotic-resistant bacteria in wound beds present unique challenges in terms of treating chronic wound infections; biofilms formed by one or more than one bacterial species are often involved. In this work, the in vitro anti-biofilm activity of a novel electrochemical bandage (e-bandage) composed of carbon fabric and controlled by a wearable potentiostat, designed to continuously deliver low amounts of hydrogen peroxide (H2O2) was evaluated against 34 mono-species and 12 dual-species membrane bacterial biofilms formed by Staphylococcus aureus, S. epidermidis, Enterococcus faecium, E. faecalis, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Cutibacterium acnes, and Bacteroides fragilis. Biofilms were grown on polycarbonate membranes placed atop agar plates. An e-bandage, which electrochemically reduces dissolved oxygen to H2O2 when polarized at −0.6 VAg/AgCl, was then placed atop each membrane biofilm and polarized continuously for 12, 24, and 48 h using a wearable potentiostat. Time-dependent decreases in viable CFU counts of all mono- and dual-species biofilms were observed after e-bandage treatment. 48 h of e-bandage treatment resulted in an average reduction of 8.17 ± 0.40 and 7.99 ± 0.32 log10 CFU/cm2 for mono- and dual-species biofilms, respectively. Results suggest that the described H2O2 producing e-bandage can reduce in vitro viable cell counts of biofilms grown either in mono- or dual-species forms, and should be further developed as a potential antibiotic-free treatment strategy for treating chronic wound infections.

Trans-cinnamaldehyde potently kills Enterococcus faecalis biofilm cells and prevents biofilm recovery

Enterococcus faecalis is a biofilm-forming, nosocomial pathogen that is frequently isolated from failed root canal treatments. Contemporary root canal disinfectants are ineffective in eliminating these biofilms and preventing reinfection. As a result, there is a pressing need to identify novel and safe antibiofilm molecules. The effect of short-term (5 and 15 min) and long-term (24 h) treatments of trans-cinnamaldehyde (TC) on the viability of E. faecalis biofilms was compared with currently used root canal disinfectants. Treatment for 15 min with TC reduced biofilm metabolic activity as effective as 1% sodium hypochlorite and 2% chlorhexidine. Treatment with TC for 24 h was significantly more effective than 2% chlorhexidine in reducing the viable cell counts of biofilms. This serendipitous effect of TC was sustained for 10 days under growth-favoring conditions. For the first time, our study highlights the strong antibacterial activity of TC against E. faecalis biofilms, and notably, its ability to prevent biofilm recovery after treatment.

Biofilm formation of Listeria monocytogenes and its resistance to quaternary ammonium compounds in a simulated salmon processing environment

L. monocytogenes is able to form biofilms as survival strategy in food processing environments. This study aimed to evaluate biofilm formation and sanitizer resistance of L. monocytogenes under a simulated salmon processing environment, involving low temperature, presence of organic matter and cultivable indigenous microorganisms (CIMs) in fresh salmon. Biofilms of L. monocytogenes in single- and mixed-species with CIMs were formed on stainless steel coupons by incubating in diluted trypticase soy broth (dTSB, 1:20) or salmon broth at 4 and 15 °C for 14 days. Biofilm cell counts before and after exposure to Whisper™ V (quaternary ammonium compounds, 200 ppm) were determined on day 1, 4, 7, and 14. Results showed that L. monocytogenes single-species biofilms developed at 15 °C had higher populations compared with those formed at 4 °C from day 1 to day 7. The cell populations of the 4 °C-grown biofilms increased by 1–2 log CFU/cm2 after 14 days, whereas the cell density of 15 °C-grown biofilms on day 14 was not significantly (P > 0.05) different from that on day 1. The presence of CIMs led to lower L. monocytogenes populations in mixed-species biofilms on day 7 and day 14, whereas it did not significantly (P > 0.05) influence the sanitizer sensitivity of L. monocytogenes. By PCA analysis, culture medium was identified a dominant factor influencing biofilm formation and QAC resistance compared with other factors. Specifically, salmon broth promoted L. monocytogenes biofilm formation and diminished the efficacy of sanitizer compared with dTSB. Confocal images showed thick matrix of biofilms cultured in salmon broth, and a visible coating layer can be observed on the surface. This conditioning layer might explain for the lower sanitizer susceptibility of L. monocytogenes in salmon broth. Thus, these results could advance our understanding towards persistence of L. monocytogenes in salmon processing environments, with emphasize on food-related broth in increasing its biofilm formation and sanitizer resistance. Our study suggests that food-related broth should be applied in biofilm study instead of chemical culture medium, which might lead to underestimation of L. monocytogenes survival at food premises.

Sensitizing bacterial cells to antibiotics by shape recovery triggered biofilm dispersion

Microbial biofilms are a leading cause of chronic infections in humans and persistent biofouling in industries due to extremely high-level tolerance of biofilm cells to antimicrobial agents. Eradicating mature biofilms is especially challenging because of the protection of the extracellular matrix and slow growth of biofilm cells. Recently, we reported that established biofilms can be effectively removed (e.g. 99.9% dispersion of 48 h Pseudomonas aeruginosa PAO1 biofilms) by shape memory polymer-based dynamic changes in surface topography. Here, we demonstrate that such biofilm dispersion also sensitizes biofilm cells to conventional antibiotics. For example, shape recovery in the presence of 50 µg/mL tobramycin reduced biofilm cell counts by more than 3 logs (2,479-fold) compared to the static flat control. The observed effects were attributed to the disruption of biofilm structure and increase in cellular activities as evidenced by an 11.8-fold increase in intracellular level of adenosine triphosphate (ATP), and 4.1-fold increase in expression of the rrnB gene in detached cells. These results can help guide the design of new control methods to better combat biofilm associated antibiotic-resistant infections. Statement of SignificanceMicrobial infections are challenging due to high-level antibiotic resistance of biofilm cells. The protection of an extracellular matrix and slow growth of biofilm cells render conventional antibiotics ineffective. Thus, it is important to develop new technologies that can remove mature biofilms and sensitize biofilm cells to antibiotics. Recently, we demonstrated that dynamic change in surface topography can remove 48 h Pseudomonas aeruginosa PAO1 biofilms by 99.9%. In this study, we investigated how shape recovery triggered dispersion affect the physiology of biofilm cells and associated antibiotic susceptibility. These results are helpful for understanding biofilm dispersion and developing more effective control methods.

Evaluation of Antimicrobial Durability and Anti-Biofilm Effects in Urinary Catheters Against Enterococcus faecalis Clinical Isolates and Reference Strains

Background:Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans biofilms are major causes of catheter-associated urinary tract infections. Antimicrobial-coated or impregnated urinary catheters are seen as a possible way to prevent these infections.Aims:To determine the biofilm-forming ability of 89 E. faecalis isolates from urinary tract infections and to compare several urinary catheters for antimicrobial durability and the inhibitory effects on biofilm formation of different laboratory strains and clinical isolates of E. faecalis.Study Design:In vitro experimental study.Methods:The biofilm forming ability of E. faecalis isolates was determined by the crystal violet staining and plate counting methods. For comparison of urinary catheters, biofilms of 45 E. faecalis isolates from the catheter samples of hospitalized patients and five laboratory strains of E. coli ATCC25922, S. epidermidis ATCC35984, P. aeruginosa ATCC27853, E. faecalis ATCC29212 and C. albicans ATCC90028 were formed on the catheters in 24-well tissue culture plates. Scanning electron microscopy analysis was performed to observe biofilms.Results:All 89 E. faecalis isolates were found to be biofilm positive. Nitrofurazone-impregnated catheters significantly reduced the cell counts of E. faecalis isolates and completely inhibited the formation of P. aeruginosa and S. epidermidis biofilms compared with the others. Regarding reduction of biofilm cell counts, a hydrophilic-coated catheter was more effective against P. aeruginosa, whereas a silver-coated catheter was found to be more effective against S. epidermidis. The nitrofurazone-impregnated catheter had the best antimicrobial durability.Conclusion:Urine isolates of E. faecalis had considerable ability with respect to biofilm formation. The nitrofurazone-impregnated catheter was the most effective against all tested bacteria; however, the effect of a hydrophilic or silver-coated catheter depends on the species present in it.

The impact of culture medium on the development and physiology of biofilms of Pseudomonas fluorescens formed on polyurethane paint

Microbial biofilms cause the deterioration of polymeric coatings such as polyurethanes (PUs). In many cases, microbes have been shown to use the PU as a nutrient source. The interaction between biofilms and nutritive substrata is complex, since both the medium and the substratum can provide nutrients that affect biofilm formation and biodeterioration. Historically, studies of PU biodeterioration have monitored the planktonic cells in the medium surrounding the material, not the biofilm. This study monitored planktonic and biofilm cell counts, and biofilm morphology, in long-term growth experiments conducted with Pseudomonas fluorescens under different nutrient conditions. Nutrients affected planktonic and biofilm cell numbers differently, and neither was representative of the system as a whole. Microscopic examination of the biofilm revealed the presence of intracellular storage granules in biofilms grown in M9 but not yeast extract salts medium. These granules are indicative of nutrient limitation and/or entry into stationary phase, which may impact the biodegradative capability of the biofilm.

Efficacy of natural biocide on control of microbial induced corrosion in oil pipelines mediated by Desulfovibrio vulgaris and Desulfovibrio gigas

We compared the efficacy of a natural biocide with four chemical tetrakishydroxymethyl phosphonium sulfonate, benzyl trimethyl ammonium chloride, and formaldehyde, glutaraldehyde, to control microbial induced corrosion in oil pipelines. The efficacy of biocides were monitored against Desulfovibrio vulgaris and Desulfovibrio gigas in experimental pipes by measuring cell counts, H 2S production, Fe(II) production, production of extracellular polymeric substances and structure of biofilm. The treatment with cow urine had minimum planktonic cell counts of 3 × 10 2 CFU/mL as well as biofilm cell counts of 9 × 10 1 CFU/mL as compared with tetraki shy droxy 1 methyl phosphonium sulfonate, benzyl trimethyl ammonium chloride, formaldehyde and glutaraldehyde. Sulfide production was the lowest with cow urine (0.08 mmol/L), followed by tetraki shy droxy methyl phosphonium sulfonate 0.72 mmol/L. On day 90 of treatment, Fe(II) production was also found to be the lowest with cow urine. The scanning electron microscopic studies indicated that the biofilm bacteria were killed by cow urine. These results demonstrate the cow urine mediated control of microbially induced corrosion, and this is indicative of its potential as a viable substitute of toxic biocides. To the best of our knowledge, this seems to be the first report which screens possible biocidal activity by cow urine as compared to the most common biocides which oil industry is currently using.

Autoinducer‐2 Activity of Gram‐Negative Foodborne Pathogenic Bacteria and Its Influence on Biofilm Formation

This study evaluated whether autoinducer-2 (AI-2) activity would be associated with biofilm formation by Salmonella and Escherichia coli O157:H7 strains on food contact surfaces. In study I, a Salmonella Typhimurium DT104 strain and an E. coli O157:H7 strain, both AI-2 positive, were individually inoculated into 50 mL of Luria-Bertani (LB) or LB + 0.5% glucose (LBG) broth, without or with stainless steel or polypropylene (Salmonella) coupons. At 0, 14 (Salmonella), 24, 48, and 72 h of storage (25 degrees C), cells in suspension and detached cells from the coupons, obtained by vortexing, were enumerated on tryptic soy agar. In study II, a Salmonella Thompson AI-2-positive strain and an AI-2-negative strain, and an E. coli O157:H7 AI-2-positive strain and an AI-2-negative strain were inoculated into LB broth with stainless steel coupons. Cells were enumerated as in study I. In both studies, AI-2 activity was determined in cell-free supernatants. Cell numbers of S. Typhimurium DT104 on biofilms were higher (P < 0.05) in LB than those in LBG, while the E. coli O157:H7 strain showed no difference (P>or= 0.05) in biofilm cell counts between LB and LBG after storage for 72 h. Both S. Typhimurium DT104 and E. coli O157:H7 strains produced higher (P < 0.05) AI-2 activity in LBG than LB cell suspensions. Cell counts of AI-2-positive and-negative S. Thompson and E. coli O157:H7 strains were not different (P>or= 0.05) within suspensions or coupons (study II). The results indicated that, under the conditions of this study, AI-2 activity of the pathogen strains tested may not have a major influence on biofilm formation on food contact surfaces, which was similar between AI-2-positive and -negative strains.

Dispersing biofilms with engineered enzymatic bacteriophage

Synthetic biology involves the engineering of biological organisms by using modular and generalizable designs with the ultimate goal of developing useful solutions to real-world problems. One such problem involves bacterial biofilms, which are crucial in the pathogenesis of many clinically important infections and are difficult to eradicate because they exhibit resistance to antimicrobial treatments and removal by host immune systems. To address this issue, we engineered bacteriophage to express a biofilm-degrading enzyme during infection to simultaneously attack the bacterial cells in the biofilm and the biofilm matrix, which is composed of extracellular polymeric substances. We show that the efficacy of biofilm removal by this two-pronged enzymatic bacteriophage strategy is significantly greater than that of nonenzymatic bacteriophage treatment. Our engineered enzymatic phage substantially reduced bacterial biofilm cell counts by approximately 4.5 orders of magnitude ( approximately 99.997% removal), which was about two orders of magnitude better than that of nonenzymatic phage. This work demonstrates the feasibility and benefits of using engineered enzymatic bacteriophage to reduce bacterial biofilms and the applicability of synthetic biology to an important medical and industrial problem.

Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains

Thirteen Listeria monocytogenes strains were used to grow biofilms on glass surfaces in static conditions at 37°C for up to 4 days. After the initial 3-h adhesion and in subsequent 1-day intervals, cell numbers were determined using standard plate count after swabbing the cells from the glass surface. The three-dimensional structure of in situ biofilms was determined by confocal scanning laser microscopy (CSLM). After 3 h incubation, bacterial cells for all 13 strains of L. monocytogenes were found attached to glass slides and all strains formed biofilms within 24 h. The strains varied significantly in their ability to adhere to the surface and significant differences for cell numbers after 24 h biofilm growth were found. Cell counts in biofilms formed by five L. monocytogenes strains were monitored over 4 days. The counts increased for the first 2 days reaching 10 5cfu/cm 2, except for L. monocytogenes 7148 (10 4 cfu/cm 2). After 2 days, cell counts remained at 10 5cfu/cm 2 for four strains (tested on days 3 and 4), while L. monocytogenes 7148 continued to grow and reached 10 5 cfu/cm 2 on day 4. This difference in biofilm growth was not related to variations in growth rates of planktonic cells suggesting that growth behaviour of Listeria in biofilms may be different from their planktonic growth. CSLM revealed that the biofilms grown under static conditions consisted of two distinct layers with 0.5 log 10 higher cell numbers in the bottom layer as compared to the upper layer.

Electrical enhancement of Streptococcus gordonii biofilm killing by gentamicin

This electrical enhancement was demonstrated in an in vitro model. Streptococcus gordonii biofilms were grown for 6 days in continuous-flow reactors on one-tenth strength trypticase peptone broth. The biofilms attained a mean areal cell density of 2.4×10 8 c.f.u./cm 2 and a thickness of approx. 19 μm. Biofilms exhibited characteristic resistance to killing by an antibiotic. When treated with 2 μg/ml gentamicin for 24 h, they exhibited a 0.84 log reduction in viable cell numbers; a 4.7 log reduction was measured in a planktonic culture. Killing of planktonic bacteria by this treatment was reduced to 1.2 log when an oxygen-scavenging enzyme was added to the medium. When a 2-mA direct current was applied during antibiotic treatment, biofilm killing increased to a 4.3 log reduction. Electrical current alone caused a 1.9 log reduction in biofilm cell counts. It is suggested that gentamicin was less effective against Strep. gordonii under anaerobic conditions than it was under aerobic conditions and that this can explain both the reduced susceptibility of the biofilm (due to oxygen depletion) and electrical enhancement of efficacy (due to oxygen generation by electrolysis).