Biofilm-associated Bacteria Research Articles

Cells released from S. epidermidis biofilms present increased antibiotic tolerance to multiple antibiotics.

Biofilm released cells (Brc) are thought to present an intermediary phenotype between biofilm and planktonic cells and this has the potential of affecting their antimicrobial tolerance.AimCompare the antimicrobial tolerance profiles of Brc, planktonic or biofilm cultures of S. epidermidis.MethodologyPlanktonic, biofilm cultures or Brc from 11 isolates were exposed to peak serum concentrations (PSC) of antibiotics. The antimicrobial killing effect in the three populations was determined by CFU.ResultsIncreased Brc tolerance to vancomycin, teicoplanin, rifampicin, erythromycin, and tetracycline was confirmed in model strain 9142. Furthermore, significant differences in the susceptibility of Brc to vancomycin were further found in 10 other clinical isolates.ConclusionsBrc from distinct clinical isolates presented a decreased susceptibility to most antibiotics tested and maintained that enhanced tolerance despite growing planktonically for up to 6 h. Our data suggest that Brc maintain the typical enhanced antibiotic tolerance of biofilm populations, further suggesting that addressing antimicrobial susceptibility in planktonic cultures might not reflect the full potential of biofilm-associated bacteria to survive therapy.

Potential Role of Biofilm Formation in the Development of Digestive Tract Cancer With Special Reference to Helicobacter pylori Infection.

Bacteria are highly social organisms that communicate via signaling molecules and can assume a multicellular lifestyle to build biofilm communities. Until recently, complications from biofilm-associated infection have been primarily ascribed to increased bacterial resistance to antibiotics and host immune evasion, leading to persistent infection. In this theory and hypothesis article we present a relatively new argument that biofilm formation has potential etiological role in the development of digestive tract cancer. First, we summarize recent new findings suggesting the potential link between bacterial biofilm and various types of cancer to build the foundation of our hypothesis. To date, evidence has been particularly convincing for colorectal cancer and its precursor, i.e., polyps, pointing to several key individual bacterial species, such as Bacteroides fragilis, Fusobacterium nucleatum, and Streptococcus gallolyticus subsp. Gallolyticus. Then, we further extend this hypothesis to one of the most common bacterial infection in humans, Helicobacter pylori (Hp), which is considered a major cause of gastric cancer. Thus far, there has been no direct evidence linking in vivo Hp gastric biofilm formation to gastric carcinogenesis. Yet, we synthesize the information to support an argument that biofilm associated-Hp is potentially more carcinogenic, summarizing biological characteristics of biofilm-associated bacteria. We also discuss mechanistic pathways as to how Hp or other biofilm-associated bacteria control biofilm formation and highlight recent findings on Hp genes that influence biofilm formation, which may lead to strain variability in biofilm formation. This knowledge may open a possibility of developing targeted intervention. We conclude, however, that this field is still in its infancy. To test the hypothesis rigorously and to link it ultimately to gastric pathologies (e.g., premalignant lesions and cancer), studies are needed to learn more about Hp biofilms, such as compositions and biological properties of extracellular polymeric substance (EPS), presence of non-Hp microbiome and geographical distribution of biofilms in relation to gastric gland types and structures. Identification of specific Hp strains with enhanced biofilm formation would be helpful not only for screening patients at high risk for sequelae from Hp infection, but also for development of new antibiotics to avoid resistance, regardless of its association with gastric cancer.

The Antimicrobial Peptide lin-SB056-1 and Its Dendrimeric Derivative Prevent Pseudomonas aeruginosa Biofilm Formation in Physiologically Relevant Models of Chronic Infections.

Antimicrobial peptides (AMPs) are promising templates for the development of novel antibiofilm drugs. Despite the large number of studies on screening and optimization of AMPs, only a few of these evaluated the antibiofilm activity in physiologically relevant model systems. Potent in vitro activity of AMPs often does not translate into in vivo effectiveness due to the interference of the host microenvironment with peptide stability/availability. Hence, mimicking the complex environment found in biofilm-associated infections is essential to predict the clinical potential of novel AMP-based antimicrobials. In the present study, we examined the antibiofilm activity of the semi-synthetic peptide lin-SB056-1 and its dendrimeric derivative (lin-SB056-1)2-K against Pseudomonas aeruginosa in an in vivo-like three-dimensional (3-D) lung epithelial cell model and an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels). Although moderately active when tested alone, lin-SB056-1 was effective in reducing P. aeruginosa biofilm formation in association with 3-D lung epithelial cells in combination with the chelating agent EDTA. The dimeric derivative (lin-SB056-1)2-K demonstrated an enhanced biofilm-inhibitory activity as compared to both lin-SB056-1 and the lin-SB056-1/EDTA combination, reducing the number of biofilm-associated bacteria up to 3-Log units at concentrations causing less than 20% cell death. Biofilm inhibition by (lin-SB056-1)2-K was reported both for the reference strain PAO1 and cystic fibrosis lung isolates of P. aeruginosa. In addition, using fluorescence microscopy, a significant decrease in biofilm-like structures associated with 3-D cells was observed after peptide exposure. Interestingly, effectiveness of (lin-SB056-1)2-K was also demonstrated in the wound model with a reduction of up to 1-Log unit in biofilm formation by P. aeruginosa PAO1 and wound isolates. Overall, combination treatment and peptide dendrimerization emerged as promising strategies to improve the efficacy of AMPs, especially under challenging host-mimicking conditions. Furthermore, the results of the present study underlined the importance of evaluating the biological properties of novel AMPs in in vivo-like model systems representative of specific infectious sites in order to make a more realistic prediction of their therapeutic success, and avoid the inclusion of unpromising peptides in animal studies and clinical trials.

Pseudomonas Infection Responsive Liquid Crystals for Glycoside Hydrolase and Antibiotic Combination.

Bacterial biofilms account for up to 80% of all community-acquired infections for which bacterial eradication is currently not achievable using conventional antimicrobial treatments. The protective matrix that engulfs biofilm-associated bacteria frequently renders antibiotics ineffective. Glycoside hydrolases are a class of enzymes that break down the biofilm matrix, thereby increasing the effectiveness of antibiotics. Herein, nanostructured liquid crystals composed of glyceryl monooleate (GMO) were investigated as an infection responsive delivery system for alginate lyase (glycoside hydrolase) and gentamicin (antibiotic) to treat Pseudomonas biofilms. The presence of Pseudomonas lipase triggered the release of alginate lyase and gentamicin from the GMO liquid crystals. Treatment with the liquid crystals containing alginate lyase and gentamicin resulted in a greater than 2-log reduction in mucoid Pseudomonas aeruginosa (clinical isolate) biofilm. The anti-biofilm activity of alginate lyase and gentamicin from the liquid crystals was sustained for 2 days and equivalent to the respective unformulated solution treatments. Accordingly, GMO based liquid crystals are a promising responsive delivery system for alginate lyase and gentamicin to combat topicalPseudomonas infections.

Comparison of the Antimicrobial Activities of Four Honeys From Three Countries (New Zealand, Cuba, and Kenya).

Skin and chronic wound infections are an increasing and urgent health problem worldwide. Their management is difficult and the development of antibiotic resistance by both planktonic and biofilm-associated bacteria necessitates the use of alternative treatments. The purpose of this study was to compare the antimicrobial activity of four honeys from different floral and geographical origins: Melipona beecheii honey (Cuba) and three Apis mellifera honeys [Manuka honey (New Zealand), A. mellifera honey (Cuba), and African honey (Kenya)]. The physicochemical parameters were within the ranges reported for these honeys and M. beecheii honey stood out due to its acidic character. An agar incorporation technique was used to determine the minimum active dilution of each honey against 52 clinical isolates (34 Gram-positive, 17 Gram-negative, and 1 Candida albicans). The antibiofilm activity of honeys was tested by assessing their ability to inhibit biofilm formation and to disrupt preformed biofilms. Overall, M. beecheii honey had the highest antimicrobial and antibiofilm activity, although a marked disruption in preformed biofilms was shared by all tested honeys. Structural changes induced by M. beecheii honey on Staphylococcus aureus and Pseudomonas aeruginosa cells were observed by transmission electron microscopy suggesting that this honey has a potent antimicrobial action and may be an excellent candidate for the development of topical preparations for the treatment of infected wounds.

Helicobacter pylori Biofilm Formation and Its Potential Role in Pathogenesis.

Despite decades of effort, Helicobacter pylori infections remain difficult to treat. Over half of the world's population is infected by H. pylori, which is a major cause of duodenal and gastric ulcers as well as gastric cancer. During chronic infection, H. pylori localizes within the gastric mucosal layer, including deep within invaginations called glands; thanks to its impressive ability to survive despite the harsh acidic environment, it can persist for the host's lifetime. This ability to survive and persist in the stomach is associated with urease production, chemotactic motility, and the ability to adapt to the fluctuating environment. Additionally, biofilm formation has recently been suggested to play a role in colonization. Biofilms are surface-associated communities of bacteria that are embedded in a hydrated matrix of extracellular polymeric substances. Biofilms pose a substantial health risk and are key contributors to many chronic and recurrent infections. This link between biofilm-associated bacteria and chronic infections likely results from an increased tolerance to conventional antibiotic treatments as well as immune system action. The role of this biofilm mode in antimicrobial treatment failure and H. pylori survival has yet to be determined. Furthermore, relatively little is known about the H. pylori biofilm structure or the genes associated with this mode of growth. In this review, therefore, we aim to highlight recent findings concerning H. pylori biofilms and the molecular mechanism of their formation. Additionally, we discuss the potential roles of biofilms in the failure of antibiotic treatment and in infection recurrence.

Use of the Phytochemical Neem as a Component of Combination Treatment Against Pseudomonas aeruginosa Biofilm

According to the NIH, 80% of chronic infections are caused by biofilm‐associated bacteria. Biofilm communities contribute to antimicrobic resistance by providing a physical extracellular barrier and exhibit metabolic diversity within bacterial subpopulations. Clinically, combination antibiotic strategies are used to provide high dosage treatments to successfully combat biofilm‐associated infections. Microscopically, biofilms of Pseudomonas aeruginosa have been visualized as mushroom‐like structures with two distinct subpopulations: a high‐metabolic “mushroom cap” outer population and a low‐metabolic “stalk” inner population. Further, the inner subpopulation consists of persister cells that are not killed by many therapies and contribute to chronicity of biofilm‐associated infections. It has been proposed that complete eradication of biofilm is achieved by targeting specific biofilm populations with antimicrobic combination of colistin and ciprofloxacin that target inner and outer subpopulations of biofilm respectively. However, colistin is sparingly used clinically because of high renal toxicity. Use of phytochemical alternatives instead of antibiotics is under assessment for treatment of biofilm‐associated infections. Extract from Azadirachta indica, or neem tree, is reported to have antimicrobic and antibiofilm effects on P. aeruginosa with low human toxicity. For these reasons, P. aeruginosa was grown (24 h) and microplates washed to remove non‐adherent cells. Remaining biofilm and cells were treated overnight with neem, colistin, ciprofloxacin or dual combinations. Finally, biofilm was removed from the substratum by scraping and aspiration, and bacterial viability was determined by colony forming unit (CFU/mL) enumeration. Results indicated that neem and ciprofloxacin in combination had an inhibitory effect (≤103 CFU/ml) similar to colistin and ciprofloxacin combination (≤102 CFU/ml) compared to growth and vehicle controls (both of which were 1012 CFU/ml). Colistin and neem combination showed no combined effect (107 CFU/ml) different from single treatment effects (108 and 1010 CFU/ml, respectively). These results suggest that neem extract targets the inner, low‐metabolic subpopulation of bacteria in biofilms and, for this reason, neem has potential to be a non‐toxic alternative to colistin for use therapeutically. When colistin or neem (that target the inner, low‐metabolic bacterial subpopulation) are used in combination with ciprofloxacin (that targets the outer, high‐metabolic subpopulation), the entirety of the biofilm may be targeted and prevent chronicity in biofilm‐associated infections.Support or Funding InformationFinancial support for BPB was received from the DeNardo Education and Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanistic studies of the antibiofilm activity and synergy with antibiotics of isosorbide mononitrate

The use of nitric oxide (NO), a naturally occurring antimicrobial agent, as an alternative strategy to combat bacterial biofilms has recently gained considerable momentum in light of the global threat of emerging antibiotic resistance. While previous NO-based anti-biofilm approaches were aimed at killing bacterial cells within biofilms, NO has also been recently identified as a key mediator of biofilm dispersal, causing the release of cells from the biofilm community. This is of great interest towards the design of more effective anti-biofilm strategies but further studies are warranted to validate this concept. Therefore, in the present study we investigated whether a NO precursor, isosorbide mononitrate (ISMN) or its analogue D-isosorbide can induce bacteria cell dispersal from Staphylococcus aureus (S. aureus) biofilms and explored the potential synergy of ISMN and the antimicrobial compounds mupirocin and ciprofloxacin in biofilm eradication. This study demonstrate that ISMN causes dispersal of S. aureus biofilm bacteria, particularly when exposed to high levels of drug. ISMN at 60mg/mL increased the number of colony forming units (CFU) (~3log10 and ~5log10) of planktonic bacteria after 6 and 24-h exposure respectively, compared to control biofilms. This suggests that ISMN induces the transition of sessile biofilm cells to free-swimming planktonic cells. In addition, ISMN exhibits synergistic effects against S. aureus biofilms with ciprofloxacin when tested above its minimum inhibitory concentration (MIC). Specifically, exposure to ISMN significantly enhanced the efficacy of ciprofloxacin by reducing the number of CFU (~3log10 or ~2log10) of biofilm-associated and planktonic bacteria respectively, compared to drug alone. Combined exposure to both ISMN and certain antimicrobial agents may therefore offer an innovative approach to control persistent biofilm and biofilm-associated infections.

Inhibition of Hafnia alvei H4 Biofilm Formation by the Food Additive Dihydrocoumarin

Quorum sensing (QS) is an intercellular signaling and gene regulatory mechanism that is implicated in food spoilage caused by bacteria. Thus, blocking QS may suppress QS-controlled phenotypes of these bacteria that are responsible for food spoilage. Biofilm formation is closely related to bacterial infection, and it is also a major mechanism responsible for the increased resistance of biofilm-associated bacteria to antimicrobial drugs. Food spoilage and biofilm formation caused by food-related bacteria have posed a significant problem for the food industry. Thus, adopting an antibiofilm approach would provide an alternative to an antibiotic strategy. Dihydrocoumarin is a compound that is derived from coumarin, a known natural QS inhibitor that has been used as an additive in food. Hafnia alvei is a spoilage bacterium; H. alvei H4 was isolated from ready-to-eat sea cucumber. Considering that QS and biofilm are often closely linked, this research aimed to detect the effect of dihydrocoumarin on the production of violacein by Chromobacterium violaceum 026 and to evaluate the inhibitory effect of dihydrocoumarin on the formation of biofilm by H. alvei H4 by using violacein and crystal violet assays. C. violaceum 026 treated with dihydrocoumarin showed as much as 70.1% reduction in QS-mediated production of violacein compared with untreated cells, while exhibiting no significant change in growth. H. alvei H4 treated with dihydrocoumarin displayed 75.8% reduction in swimming motility, and as much as 89.4% reduction in biofilm formation compared with the nontreated cells, with the reduction in both cases being dependent on the concentration of dihydrocoumarin. Scanning electron microscopy showed that dihydrocoumarin could effectively destroy the biofilm structure of H. alvei H4 and decrease biofilm density. These findings indicate that dihydrocoumarin can be developed into a new QS inhibitor or antibiofilm agent for controlling food spoilage and potentially investigated to increase food safety.

Staphylococcal Biofilms and Immune Polarization During Prosthetic Joint Infection.

Staphylococcal species are a leading cause of community- and nosocomial-acquired infections, where the placement of foreign materials increases infection risk. Indwelling medical devices and prosthetic implants are targets for staphylococcal cell adherence and biofilm formation. Biofilm products actively suppress proinflammatory microbicidal responses, as evident by macrophage polarization toward an anti-inflammatory phenotype and the recruitment of myeloid-derived suppressor cells. With the rise in prosthetic hip and knee arthroplasty procedures, together with the recalcitrance of biofilm infections to antibiotic therapy, it is imperative to better understand the mechanism of crosstalk between biofilm-associated bacteria and host immune cells. This review describes the current understanding of how staphylococcal biofilms evade immune-mediated clearance to establish persistent infections. The findings described herein may facilitate the identification of novel treatments for these devastating biofilm-mediated infections.

YmdB-mediated down-regulation of sucA inhibits biofilm formation and induces apramycin susceptibility in Escherichia coli

Antibiotic resistance associated with biofilm formation is a major concern when treating bacterial infections with drugs. The genes and pathways involved in biofilm formation have been extensively studied and are also involved in antibiotic resistance. Recent studies show that overexpression of Escherichia coli (E. coli) YmdB protein alters gene expression profiles and inhibits biofilm formation. Therefore, it is expected that YmdB and its regulated genes play a key role in development of biofilm and antibiotic resistance phenotypes. The present study screened antibiotics to identify those whose susceptibility profiles were regulated by YmdB levels. This protocol identified apramycin. Additional screening for genes negatively regulated by inactivation of RNase III activity via YmdB overexpression revealed that a gene associated with the tricarboxylic acid cycle gene, sucA, was necessary for the YmdB-like phenotype. Taken together, these data suggest that regulation of RNase III activity by trans-acting factors may be the key to identifying genes or pathways connecting biofilm and antibiotic resistance phenotypes. This information could be used to reduce the emergence of biofilm-associated multidrug-resistant bacteria.

Evaluation of the Impact of Methicillin-Resistant Staphylococcus pseudintermedius Biofilm Formation on Antimicrobial Susceptibility.

To compare the minimum inhibitory concentration (MIC) of four antimicrobials in planktonic vs. biofilm-associated Staphylococcus pseudintermedius. In vitro study. 78 isolates from dogs colonized or infected with methicillin-resistant S. pseudintermedius (MRSP, n=39) or methicillin-susceptible S. pseudintermedius (MSSP, n=39). Agar dilution was used to determine the MIC of amikacin, cefazolin, enrofloxacin, and gentamicin for planktonic bacteria. A modified broth microdilution assay was used to assess the MIC of biofilm-associated bacteria. MIC were significantly higher in biofilm-associated vs. planktonic bacteria for all antimicrobials; amikacin (median MIC: biofilm >2,000 μg/mL vs. planktonic 3 μg/mL, P<.0001), cefazolin (>1,000 vs. 0.5 μg/mL, P<.0001), enrofloxacin (>1,000 vs. 0.25 μg/mL, P<.0001), and gentamicin (>1,000 vs. 0.3 μg/mL, P<.001). For all antimicrobials, there were significant differences in planktonic MIC for MRSP and MSSP (all P<.0001) but no differences between biofilm MIC for MRSP and MSSP (P=.08-1.0). The MIC for biofilm-associated S. pseudintermedius are significantly higher than for planktonic bacteria. Standard methods for determining MIC are not appropriate for biofilm-associated infections. This must be considered when determining treatment regimens for infections that potentially involve biofilms, and further study of methods to control biofilm-associated infections is needed.

Sub-Optimal Treatment of Bacterial Biofilms.

Bacterial biofilm is an emerging clinical problem recognized in the treatment of infectious diseases within the last two decades. The appearance of microbial biofilm in clinical settings is steadily increasing due to several reasons including the increased use of quality of life-improving artificial devices. In contrast to infections caused by planktonic bacteria that respond relatively well to standard antibiotic therapy, biofilm-forming bacteria tend to cause chronic infections whereby infections persist despite seemingly adequate antibiotic therapy. This review briefly describes the responses of biofilm matrix components and biofilm-associated bacteria towards sub-lethal concentrations of antimicrobial agents, which may include the generation of genetic and phenotypic variabilities. Clinical implications of bacterial biofilms in relation to antibiotic treatments are also discussed.

Antimicrobial Peptide P60.4Ac-Containing Creams and Gel for Eradication of Methicillin-Resistant Staphylococcus aureus from Cultured Skin and Airway Epithelial Surfaces.

We previously found the LL-37-derived peptide P60.4Ac to be effective against methicillin-resistant Staphylococcus aureus (MRSA) on human epidermal models (EMs). The goal of this study was to identify the preferred carrier for this peptide for topical application on skin and mucosal surfaces. We prepared P60.4Ac in three formulations, i.e., a water-in-oil cream with lanolin (Softisan 649), an oil-in-water cream with polyethylene glycol hexadecyl ether (Cetomacrogol), and a hydroxypropyl methylcellulose (hypromellose) 4000 gel. We tested the antimicrobial efficacy of the peptide in these formulations against mupirocin-resistant and -sensitive MRSA strains on EMs and bronchial epithelial models (BEMs). The cytotoxic effects of formulated P60.4Ac on these models were determined using histology and WST-1 and lactate dehydrogenase assays. Moreover, we assessed the stability of the peptide in these formulations with storage for up to 3 months. Killing of MRSA by P60.4Ac in the two creams was less effective than that by P60.4Ac in the hypromellose gel. In agreement with those findings, P60.4Ac in the hypromellose gel was highly effective in eradicating the two MRSA strains from EMs. We found that even 0.1% (wt/wt) P60.4Ac in the hypromellose gel killed >99% of the viable planktonic bacteria and >85% of the biofilm-associated bacteria on EMs. Hypromellose gels containing 0.1% and 0.5% (wt/wt) P60.4Ac effectively reduced the numbers of viable MRSA cells from BEMs by >90%. No cytotoxic effects of P60.4Ac in the hypromellose gel with up to 2% (wt/wt) P60.4Ac on keratinocytes in EMs and in the hypromellose gel with up to 0.5% (wt/wt) P60.4Ac on epithelial cells in BEMs were observed. High-performance liquid chromatography analysis showed that P60.4Ac was stable in the Softisan cream and the hypromellose gel but not in the Cetomacrogol cream. We conclude that P60.4Ac formulated in hypromellose gel is both stable and highly effective in eradicating MRSA from colonized EMs and BEMs.

In Vivo and In Vitro Efficacy of Minocycline-Based Combination Therapy for Minocycline-Resistant Acinetobacter baumannii

Minocycline-based combination therapy has been suggested to be a possible choice for the treatment of infections caused by minocycline-susceptible Acinetobacter baumannii, but its use for the treatment of infections caused by minocycline-resistant A. baumannii is not well established. In this study, we compared the efficacy of minocycline-based combination therapy (with colistin, cefoperazone-sulbactam, or meropenem) to that of colistin in combination with meropenem for the treatment of minocycline-resistant A. baumannii infection. From 2006 to 2010, 191 (17.6%) of 1,083 A. baumannii complex isolates not susceptible to minocycline from the Taiwan Surveillance of Antimicrobial Resistance program were collected. Four representative A. baumannii isolates resistant to minocycline, amikacin, ampicillin-sulbactam, ceftazidime, ciprofloxacin, cefepime, gentamicin, imipenem, levofloxacin, meropenem, and piperacillin-tazobactam were selected on the basis of the diversity of their pulsotypes, collection years, health care setting origins, and geographic areas of origination. All four isolates had tetB and overexpressed adeABC, as revealed by quantitative reverse transcription-PCR. Among all minocycline-based regimens, only the combination with colistin produced a fractional inhibitory concentration index comparable to that achieved with meropenem combined with colistin. Minocycline (4 or 16 μg/ml) in combination with colistin (0.5 μg/ml) also synergistically killed minocycline-resistant isolates in time-kill studies. Minocycline (50 mg/kg of body weight) in combination with colistin (10 mg/kg) significantly improved the survival of mice and reduced the number of bacteria present in the lungs of mice compared to the results of monotherapy. However, minocycline (16 μg/ml)-based therapy was not effective at reducing biofilm-associated bacteria at 24 or 48 h when its effectiveness was compared to that of colistin (0.5 μg/ml) and meropenem (8 μg/ml). The clinical use of minocycline in combination with colistin for the treatment of minocycline-resistant A. baumannii may warrant further investigation.

Efficacy of antimicrobial impregnated urinary catheter in thwarting foreign body associated urinary tract infections caused by Pseudomonas aeruginosa

Event Abstract Back to Event Efficacy of antimicrobial impregnated urinary catheter in thwarting foreign body associated urinary tract infections caused by Pseudomonas aeruginosa Hina Saini1, Sanjay Chhibber1 and Kusum Harjai1 1 Panjab University, Microbiology, India Introduction: Catheter associated urinary tract infections (CAUTIs) are rising exponentially around the globe; especially in intensive care units. The formation of biofilm on the surface of an indwelling urinary catheter (UC) marks the onset of CAUTI. Treatment of CAUTI/eradication of biofilm poses an intricate challenge to clinicians and researchers since bacteria within biofilm have increased protection against host immune response and resistance to antimicrobials. Biofilm-associated bacteria on the surface of UC produce quorum sensing (QS) signal molecules which regulate the expression of genes required for biofilm formation. Attenuation of QS, which in turn regulates biofilm production, seems to be a novel approach to curb CAUTI. The present study was thus designed with a preventive strategy to generate a biomaterial by impregnating a silicone UC with combination of azithromycin (AZM) and ciprofloxacin (CIP) and testing its efficacy in-vitro against Pseudomonas aeruginosa. Materials and Methods: Medical grade silicone tubing (0.30mm X 0.64mm) was impregnated with antimicrobials: 3% CIP and 6% AZM. In-vitro efficacy was assessed for control and AZM-CIP impregnated UC against P. aeruginosa standard strain PAO1 using serial plate transfer test (SPTT), in-vitro biofilm colonization, durability test, scanning electron microscopy (SEM) to visualise biofilm and shelf-life test. Results and Discussion: SPTT of AZM-CIP impregnated UC revealed prominent zones of inhibition (> 30mm) against P. aeruginosa PAO1 during testing period of one month and no bacterial growth was observed around or under the UC. These results indicate prolonged antimicrobial activity of these catheters with sustained diffusion of drugs and absence of resistance development since no bacteria was found attached to catheters following continuous exposure to heavy inoculum of P. aeruginosa. AZM-CIP impregnated UC showed complete inhibition of bacterial colonization and biofilm formation after 24h exposure to P. aeruginosa PAO1. These findings suggest anti-adherence activity of catheters. AZM-CIP impregnated UC were tested for durability by dipping in sterile human urine for 4 weeks simulating biological conditions. The results showed significant decline in biofilm formation over 4 weeks suggesting longer durability. These findings were further confirmed by SEM which showed limited bacterial cells within disrupted biofilm on the surface of antimicrobial impregnated UC as compared with fully grown intact biofilm on the surface of control catheters. Additionally, AZM-CIP impregnated UC showed complete inhibition of biofilm formation at quarterly intervals displaying stable real-time shelf life for one year. Conclusion: AZM-CIP impregnated UC demonstrated prolonged duration of antimicrobial and antibiofilm activity. The present study bring out the fact that synergistic combination of currently available antibiotics AZM and CIP may be usefully applied to generate a biomaterial by impregnating these drugs into a UC to prevent bacterial colonization and biofilm formation. Such a catheter would be beneficial in clinical situations, especially in long-term foreign body associated urinary tract infections. This work was funded by University Grants Commission (UGC), New Delhi, India (F1-17.1/2013-14/MANF-2013-14-SIK-CHA-22060/(SA-III/Website). We thank Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh for their kind support in Scanning Electron Microscopy

Irrigation waters and pipe-based biofilms as sources for antibiotic-resistant bacteria.

The presence of antibiotic-resistant bacteria in environmental surface waters has gained recent attention. Wastewater and drinking water distribution systems are known to disseminate antibiotic-resistant bacteria, with the biofilms that form on the inner-surfaces of the pipeline as a hot spot for proliferation and gene exchange. Pipe-based irrigation systems that utilize surface waters may contribute to the dissemination of antibiotic-resistant bacteria in a similar manner. We conducted irrigation events at a perennial stream on a weekly basis for 1 month, and the concentrations of total heterotrophic bacteria, total coliforms, and fecal coliforms, as well as the concentrations of these bacterial groups that were resistant to ampicillin and tetracycline, were monitored at the intake water. Prior to each of the latter three events, residual pipe water was sampled and 6-in. sections of pipeline (coupons) were detached from the system, and biofilm from the inner-wall was removed and analyzed for total protein content and the above bacteria. Isolates of biofilm-associated bacteria were screened for resistance to a panel of seven antibiotics, representing five antibiotic classes. All of the monitored bacteria grew substantially in the residual water between irrigation events, and the biomass of the biofilm steadily increased from week to week. The percentages of biofilm-associated isolates that were resistant to antibiotics on the panel sometimes increased between events. Multiple-drug resistance was observed for all bacterial groups, most often for fecal coliforms, and the distributions of the numbers of antibiotics that the total coliforms and fecal coliforms were resistant to were subject to change from week to week. Results from this study highlight irrigation waters as a potential source for antibiotic-resistant bacteria, which can subsequently become incorporated into and proliferate within irrigation pipe-based biofilms.

Bacterial resistance in biofilm-associated bacteria.

Biofilms are structured groups of different bacterial species that are responsible for most chronic and recurrent infections. Biofilm-related infections reoccur in approximately 65-80% of cases. Bacteria associated with the biofilm are highly resistant to antibiotics. Knowledge of biofilm formation, its propagation and the resistance associated with it is scant and a multidisciplinary approach is followed to understand the science and develop strategies to address this problem. This article discusses the role of various biochemical factors, molecular mechanisms and altered host environment causes associated with bacterial resistance in biofilm. It also reveals the target sites and different multidisciplinary strategies adapted for destroying or preventing the formation of biofilms.

Effects of Carbon Dioxide Aerosols on the Viability of Escherichia coli during Biofilm Dispersal.

A periodic jet of carbon dioxide (CO2) aerosols is a very quick and effective mechanical technique to remove biofilms from various substrate surfaces. However, the impact of the aerosols on the viability of bacteria during treatment has never been evaluated. In this study, the effects of high-speed CO2 aerosols, a mixture of solid and gaseous CO2, on bacteria viability was studied. It was found that when CO2 aerosols were used to disperse biofilms of Escherichia coli, they led to a significant loss of viability, with approximately 50% of the dispersed bacteria killed in the process. By comparison, 75.6% of the biofilm-associated bacteria were viable when gently dispersed using Proteinase K and DNase I. Indirect proof that the aerosols are damaging the bacteria was found using a recombinant E. coli expressing the cyan fluorescent protein, as nearly half of the fluorescence was found in the supernatant after CO2 aerosol treatment, while the rest was associated with the bacterial pellet. In comparison, the supernatant fluorescence was only 9% when the enzymes were used to disperse the biofilm. As such, these CO2 aerosols not only remove biofilm-associated bacteria effectively but also significantly impact their viability by disrupting membrane integrity.

The Natural Surfactant Glycerol Monolaurate Significantly Reduces Development of Staphylococcus aureus and Enterococcus faecalis Biofilms.

Bacterial biofilms are involved in a large proportion of clinical infections, including device-related infections. Unfortunately, biofilm-associated bacteria are typically less susceptible to antibiotics, and infected devices must often be removed. On the basis of a recent observation that lipid-rich biofilm matrix material is present in early biofilm formation and may protect a population of bacteria from interacting with ordinarily diffusible small molecules, we hypothesized that surfactants may be useful in preventing biofilm development. Experimental Staphylococcus aureus or Enterococcus faecalis biofilms were cultivated on surgical suture suspended in a growth medium supplemented with the natural surfactant glycerol monolaurate (GML) or with a component molecule, lauric acid. After 16 h incubation, the numbers of viable biofilm-associated bacteria were measured by standard microbiologic techniques and biofilm biomass was measured using the colorimetric crystal violet assay. Both GML and lauric acid were effective in inhibiting biofilm development as measured by decreased numbers of viable biofilm-associated bacteria as well as decreased biofilm biomass. Compared with lauric acid on a molar basis, GML represented a more effective inhibitor of biofilms formed by either S. aureus or E. faecalis. Because the natural surfactant GML inhibited biofilm development, resulting data were consistent with the hypothesis that lipids may play an important role in biofilm growth, implying that interfering with lipid formation may help control development of clinically relevant biofilms.