Evaluating Biocide Efficacy in Mixed-Species Biofilms: Insights from a Dual Anaerobic Biofilm Reactor: Insights from a Dual Anaerobic Biofilm Reactor
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Early childhood caries, a virulent-form of dental caries, is painful, difficult, and costly to treat that has been associated with high levels of Streptococcus mutans (Sm) and Candida albicans (Ca) in plaque-biofilms on teeth. These microorganisms appear to develop a symbiotic cross-kingdom interaction that amplifies the virulence of plaque-biofilms. Although biofilm studies reveal synergistic bacterial-fungal association, how these organisms modulate cross-kingdom biofilm formation and enhance its virulence in the presence of saliva remain largely unknown. Here, we compared the properties of Sm and Sm-Ca biofilms cultured in saliva by examining the biofilm structural organization and capability to sustain an acidic pH environment conducive to enamel demineralization. Intriguingly, Sm-Ca biofilm is rapidly matured and maintained acidic pH-values (~4.3), while Sm biofilm development was retarded and failed to create an acidic environment when cultured in saliva. In turn, the human enamel slab surface was severely demineralized by Sm-Ca biofilms, while there was minimal damage to the enamel surface by Sm biofilm. Interestingly, Sm-Ca biofilms exhibited an acidic environment regardless of their hyphal formation ability. Our data reveal the critical role of symbiotic interaction between S. mutans and C. albicans in human saliva in the context of pathogenesis of dental caries, which may explain how the cross-kingdom interaction contributes to enhanced virulence of plaque-biofilm in the oral cavity.
Dental plaque
Tooth surface
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Mycobacterium avium subsp. hominissuis is an opportunistic pathogen that is associated with biofilm-related infections of the respiratory tract and is difficult to treat. In recent years, extracellular DNA (eDNA) has been found to be a major component of bacterial biofilms, including many pathogens involved in biofilm-associated infections. To date, eDNA has not been described as a component of mycobacterial biofilms. In this study, we identified and characterized eDNA in a high biofilm-producing strain of Mycobacterium avium subsp. hominissuis (MAH). In addition, we surveyed for presence of eDNA in various MAH strains and other nontuberculous mycobacteria. Biofilms of MAH A5 (high biofilm-producing strain) and MAH 104 (reference strain) were established at 22°C and 37°C on abiotic surfaces. Acellular biofilm matrix and supernatant from MAH A5 7 day-old biofilms both possess abundant eDNA, however very little eDNA was found in MAH 104 biofilms. A survey of MAH clinical isolates and other clinically relevant nontuberculous mycobacterial species revealed many species and strains that also produce eDNA. RAPD analysis demonstrated that eDNA resembles genomic DNA. Treatment with DNase I reduced the biomass of MAH A5 biofilms when added upon biofilm formation or to an already established biofilm both on abiotic surfaces and on top of human pharyngeal epithelial cells. Furthermore, co-treatment of an established biofilm with DNase 1 and either moxifloxacin or clarithromycin significantly increased the susceptibility of the bacteria within the biofilm to these clinically used antimicrobials. Collectively, our results describe an additional matrix component of mycobacterial biofilms and a potential new target to help treat biofilm-associated nontuberculous mycobacterial infections.
Nontuberculous Mycobacteria
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It is clear that biofilm formation causes many serious health-care problems. Interestingly, sub minimum inhibitory concentrations (sub-MICs) of some biocides can induce biofilm formation in bacteria. We investigated whether sub-MICs of Savlon, chlorhexidine and deconex®, as biocidal products, can induce biofilm formation in clinical isolates of Pseudomonas aeruginosa. To determine MICs and biofilm formation, we performed microtitre plate assays. All three biocides induced biofilm formation at sub-MICs; Savlon was the most successful antiseptic agent to induce biofilm formation among P. aeruginosa isolates. Deconex had the best inhibition effect on planktonic cultures of P. aeruginosa isolates. We concluded that sub-MICs of Savlon and deconex could significantly induce biofilm formation.
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Antiseptic
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Staphylococcus aureus forms pathogenic biofilms. Previous studies have indicated that ethanol supplementation during S. aureus biofilm formation results in increased biofilm formation and changes in gene expression. However, the impact of alcohols on preformed S. aureus biofilms has not been studied. In this study, we formed S. aureus biofilms on PVC plastic plates and then treated these preformed biofilms with five different alcohols. We observed that alcohol treatment of preformed S. aureus biofilms led to significant increases in biofilm levels after 24 h of treatment. Many bacteria within these biofilms were found to be alive and metabolically active. Alcohol treatment also resulted in increased transcription of the biofilm-promoting genes icaA and icaD, as well as several antibiotic resistance genes. These results demonstrate that treatment of S. aureus preformed biofilms with alcohols enhances biofilm levels if maintained for extended periods. Thus, alcohols might be of limited usefulness for the eradication of preformed S. aureus biofilms.
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Biofilm formation in the clinical environment is of increasing concern since a significant part of human infections is associated, and caused by biofilm establishment of (opportunistic) pathogens, for instance Candida albicans and Staphylococcus epidermidis. The rapidly increasing number of antibiotic-resistant biofilms urgently requires the development of novel and effective strategies to prevent biofilm formation ideally targeting a wide range of infectious microorganisms. Both, synthesis of extracellular polymeric substances and quorum sensing are crucial for biofilm formation, and thus potential attractive targets to combat undesirable biofilms.We evaluated the ability of numerous recently identified metagenome-derived bacterial quorum quenching (QQ) proteins to inhibit biofilm formation of C. albicans and S. epidermidis. Here, proteins QQ-5 and QQ-7 interfered with the morphogenesis of C. albicans by inhibiting the yeast-to-hyphae transition, ultimately leading to impaired biofilm formation. Moreover, QQ5 and QQ-7 inhibited biofilm formation of S. epidermidis; in case of QQ7 most likely due to induced expression of the icaR gene encoding the repressor for polysaccharide intercellular adhesin (PIA) synthesis, the main determinant for staphylococcal biofilm formation. Our results indicate that QQ-5 and QQ-7 are attractive potential anti-biofilm agents in the prevention and treatment of C. albicans and S. epidermidis mono-species biofilms, and potentially promising anti-biofilm drugs in also combating multi-species infections.
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Abstract Biofilms are communities of microbes embedded in a matrix of extracellular polymeric substances (EPS). Matrix components can be produced by biofilm organisms and can also originate from the environment and then be incorporated into the biofilm. For example, we have recently shown that collagen, a host-produced protein that is abundant in many different infection sites, can be taken up into the biofilm matrix, altering biofilm mechanics. The biofilm matrix protects bacteria from clearance by the immune system, and some of that protection likely arises from the mechanical properties of the biofilm. Pseudomonas aeruginosa and Staphylococcus aureus are common human pathogens notable for forming biofilm infections in anatomical sites rich in collagen. Here, we show that the incorporation of Type I collagen into P. aeruginosa and S. aureus biofilms significantly hinders phagocytosis of biofilm bacteria by human neutrophils. However, enzymatic treatment with collagenase, which breaks down collagen, can partly or entirely negate the protective effect of collagen and restore the ability of neutrophils to engulf biofilm bacteria. From these findings, we suggest that enzymatic degradation of host materials may be a potential way to compromise biofilm infections and enhance the efficacy of the host immune response without promoting antibiotic resistance. Such an approach might be beneficial both in cases where the infecting species is known and also in cases wherein biofilm components are not readily known, such as multispecies infections or infections by unknown species.
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Monochloramine at 4 mg·l−1 was used to treat biofilms of Pseudomonas aeruginosa grown in annular reactors on various metal substrata, including stainless steel, mild steel and copper. Disinfection of in situ biofilms was most efficient on the stainless steel slides. The treatment reduced viable cells in the biofilm on the stainless steel slides by more than 4 orders of magnitude after 60 min, while the same treatment reduced viable cells in the biofilms on the other slides (mild steel and copper) by only 2 orders of magnitude. Mild steel corrosion products retarded the disinfection of homogenized biofilm cells. The composition of steady state biofilms on the various substrata (before biocide treatment) differed. Biofilm on stainless steel slides had more viable cells but less extracellular polymeric substance (EPS). Biofilms on mild steel and copper slides had fewer viable cells but more EPS. Almost 95% of total cells in the biofilm on the copper slides were dead cells. The lower efficacy of disinfection of in situ biofilms on mild steel and copper is hypothesized to be due to the higher EPS content of biofilms on those substrata.
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Biofouling
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Bacteria may hide in a hydrated polysaccharide matrix known as a biofilm. The structure of the bacterial biofilm renders phagocytosis difficult and increases antibiotic resistance. We hypothesized that repeated doses of antibiotics have an effect on bacteria within the biofilm and that it could inhibit or eradicate biofilm formation. Two clinical biofilm-positive coagulase-negative staphylococcus isolates were evaluated. The effects of antibiotics on preformed and nascent biofilm and on bacterial eradication within the biofilm were determined using different doses of vancomycin, daptomycin, and tigecycline for different durations in an in vitro biofilm model. Vancomycin neither penetrated the biofilm nor had any microbicidal effect on bacteria within the biofilm. Daptomycin had a microbicidal effect on bacteria within the biofilm but had no effect on biofilm inhibition and eradication (independent from dose and treatment time). Tigecycline inhibited and eradicated biofilm formation and had a microbicidal effect on bacteria within the biofilm. In conclusion, (i) biofilm formation appeared to be a major barrier to vancomycin activity, (ii) daptomycin had an antimicrobial effect on the bacteria within the biofilm but not on the biofilm burden, and (iii) tigecycline had effects both on bacteria within the biofilm and on biofilm burden. Thus, both tigecycline and daptomycin might be promising candidates for the treatment of biofilm infections.
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ABSTRACT Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA nuc1 − mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. nuc1 − mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the nuc1 − mutant. In the sloughing assay, nuc1 − mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections. IMPORTANCE Infections caused by antibiotic-resistant bacteria known as methicillin-resistant Staphylococcus aureus (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem.
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Autolysin
Extracellular polymeric substance
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This study sought to identify novel and nontoxic biofilm inhibitors from the Actinomycete library for attenuating biofilm formation by Staphylococcus epidermidis. After investigating the antibiofilm activities of spent media from 185 Actinomycete strains using two S. epidermidis strains (ATCC 35984 and a clinical strain 5‐121‐2) as target bacteria, three strains of tested Actinomycete (TRM 46200, TRM 41337, and TRM 46814) showed a significant inhibition against S. epidermidis biofilm formation without affecting the growth of planktonic cells. The characteristics of three strains of supernatants suggested that hydrophilic compound possibly extracellular peptides or proteins from these three strains, confer the biofilm reduction in S. epidermidis. An attempt was made to assess their effects on biofilm components and cell surface hydrophobicities in order to disclose acting mechanisms. The crude proteins from spent media of three strains degraded not only exopolysaccharides but also extracellular DNA in S. epidermidis biofilm. The active substances in crude proteins caused S. epidermidis cells to become less hydrophobic. Given these results, the metabolites from Actinomycete strains should keep further attention as potential antibiofilm agents against biofilm formation of S. epidermidis, even biofilm infections of the other bacteria. Staphylococcus epidermidis infections are frequently associated with biofilms that are difficult to eradicate with conventional antibiotics. The new biofilm inhibitors from Actinomycete will have a great value in the prevention and treatment of dairy cow mastitis and other biofilm‐related infections.
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