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.     

Characterization of a biofilm-like extracellular matrix in FLO1-expressing Saccharomyces cerevisiae cells

Like bacteria, fungi growing in biofilms are often embedded in a so-called extracellular matrix (ECM), a complex and species-specific mixture of compounds secreted by cells in the biofilm. The precise physiological role of this ECM and its importance for the stress and drug resistance that is so characteristic of biofilms remain vague. Here, we describe the discovery of an ECM produced by flocculating Saccharomyces cerevisiae cells. Although S. cerevisiae has long been believed not to produce an ECM, our results indicate that flocculating cells secrete a mixture of glucose and mannose polysaccharides that surrounds flocculating cells. This matrix impedes the penetration of large molecules into the floc, but does not seem to play a role in the resistance of flocculating cultures to drugs and ethanol. Together, our results provide a new model system to study the formation and biological role of microbial extracellular matrices.     

Purification and culture characteristics of 36 benthic marine diatoms isolated from the Solthörn tidal flat (Southern North Sea)

Marine benthic diatoms growing in biofilms on sediment surfaces generally occur associated with heterotrophic bacteria, whereas modern molecular techniques and analyses of species‐specific physiology create a demand for axenic cultures. Numerous benthic diatoms were isolated from surface sediments during a monitoring of the Solthörn tidal flat (southern North Sea, Germany) from May 2008 to May 2009. Of these, around 50% could be purified from the accompanying heterotrophic bacteria using different antibiotics combined with physical separation methods (vortexing, ultrasound). Overall, seven different antibiotics were tested at different concentrations, and a best working protocol was developed. The axenic strains were stable on average for only around 15 months, indicating a symbiotic interaction between the benthic diatoms and the associated bacteria. While most short‐term effects during the purification process were restricted to differences in growth rates among xenic and axenic diatom strains, long‐term cultivation led to distinct changes in cell volumes and growth characteristics of the axenic strains.     

Combinatorial approaches with selected phytochemicals to increase antibiotic efficacy against Staphylococcus aureus biofilms

Combinations of selected phytochemicals (reserpine, pyrrolidine, quinine, morin and quercetin) with antibiotics (ciprofloxacin, tetracycline and erythromycin) were tested on the prevention and control of Staphylococcus aureus biofilms. The phytochemicals were also studied for their ability to avoid antibiotic adaptation and to inhibit antibiotic efflux pumps. Morin, pyrrolidine and quercetin at subinhibitory concentrations had significant effects in biofilm prevention and/or control when applied alone and combined with antibiotics. Synergism between antibiotics and phytochemicals was found especially against biofilms of NorA overexpressing strain S. aureus SA1199B. This strain when growing with subinhibitory concentrations of ciprofloxacin developed increased tolerance to this antibiotic. However, this was successfully reversed by quinine and morin. In addition, reserpine and quercetin showed significant efflux pump inhibition. The overall results demonstrate the role of phytochemicals in co-therapies to promote more efficient treatments and decrease antimicrobial resistance to antibiotics, with substantial effects against S. aureus in both planktonic and biofilm states.     

Interrelationships between colonies, biofilms, and planktonic cells of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic human pathogen that causes chronic infections in immunocompromised individuals. These infections are hard to treat, partly due to the high intrinsic resistance of the bacterium to clinically used antibiotics and partly due to the formation of antibiotic-tolerant biofilms. The three most common ways of growing bacteria in vitro are as planktonic cultures, colonies on agar plates, and biofilms in continuous-flow systems. Biofilms are known to express genes different from those of planktonic cells, and biofilm cells are generally believed to closely resemble planktonic cells in stationary phase. However, few, if any, studies have examined global gene expression in colonies. We used a proteomic approach to investigate the interrelationships between planktonic cells, colonies, and biofilms under comparable conditions. Our results show that protein profiles in colonies resemble those of planktonic cells. Furthermore, contrary to what has been reported previously, the protein profiles of biofilms were found to more closely resemble those of exponentially growing planktonic cells than those of planktonic cells in the stationary phase. These findings raise some intriguing questions about the true nature of biofilms.     

微生物生物膜研究的新进展

微生物在生长过程中为适应生存环境而形成了生物膜,Dr.Costerton JW在生物膜方面的研究为我们开拓了微生物学的新领域。微生物生物膜是由微生物群体及其包被的细胞外多聚物和基质网组成,它们彼此黏附或者黏附到组织或物体的表面。微生物生物膜与微生物的耐药性形成、基因的转移以及引起机体的持续性感染等都密切相关。目前对生物膜的研究重点已经深入到微生物相互间的信号传递、致病基因的转移以及如何干预微生物生物膜的形成等方面。此外,在治理污水和环境保护工程、生物材料工程和食品工业等方面,微生物生物膜技术已经得到了应用。     

The extracellular matrix protects Pseudomonas aeruginosa biofilms by limiting the penetration of tobramycin

Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non‐mucoid Pseudomonas aeruginosa biofilms. To our surprise, we found that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose‐dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration.     

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.     

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

Bacterial biofilms as a natural form of existence of bacteria in the environment and host organism

Advances in microscopic analysis and molecular genetics research methods promoted the acquisition of evidence that natural bacteria populations exist predominately as substrate attached biofilms. Bacteria in biofilms are able to exchange signals and display coordinated activity that is inherent to multicellular organisms. Formation of biofilm communities turned out to be one of the main survival strategies of bacteria in their ecological niche. Bacteria in attached condition in biofilm are protected from the environmental damaging factors and effects of antibacterial substances in the environment and host organism during infection. According to contemporary conception, biofilm is a continuous layer of bacterial cells that are attached to a surface and each other, and contained in a biopolymer matrix. Such bacterial communities may be composed of bacteria of one or several species, and composed of actively functioning cells as well as latent and uncultured forms. Particular attention has recently been paid to the role of biofilms in the environment and host organism. Microorganisms form biofilm on any biotic and abiotic surfaces which creates serious problems in medicine and various areas of economic activity. Currently, it is established that biofilms are one of the pathogenetic factors of chronic inflection process formation. The review presents data on ubiquity of bacteria existence as biofilms, contemporary methods of microbial community analysis, structural-functional features of bacterial biofilms. Particular attention is paid to the role of biofilm in chronic infection process formation, heightened resistance to antibiotics of bacteria in biofilms and possible mechanisms of resistance. Screening approaches for agents against biofilms in chronic infections are discussed.     

噬菌体及其裂解酶对细菌生物被膜作用的研究进展

细菌形成的生物被膜,可保护细菌不易被抗生素杀死,这给临床上相应疾病的治疗及医疗器械的消毒带来极大困难。研究表明,噬菌体及其裂解酶对生物被膜有降解作用。噬菌体能清除细菌在有生物活性或无生物活性的介质表面形成的生物被膜。此外,噬菌体裂解酶比如LySMP、肽酶CHAPk、细胞壁溶解酶CWHs等能清除特定的生物被膜,这可能与裂解酶直接溶菌和裂解细菌细胞外基质有关。同时,与抗生素、钴离子、氯等物质联合使用时,噬菌体对生物被膜的清除作用会更强。本文从噬菌体、噬菌体编码的裂解酶、以及它们联合其他物质对细菌生物被膜的作用进行综述,并对其实际应用做了展望。     

Adhesion to medical device materials and biofilm formation capability of some species of enterococci in different physiological states

Enterococci may survive in adverse environments including the human body where bacteriocins, antibiotics, iron-limitation and immune response represent stressing conditions for bacteria that cause division block. In those conditions, bacteria present in the human body would hardly be in an exponentially growing phase but would mostly be in physiological states such as starvation or the viable but nonculturable (VBNC) state. The possibility that the starved and VBNC bacteria can maintain their ability to adhere to living and inanimate substrates is the first mandatory step for them potentially to cause an infection process. In this study it is shown that starved and stationary enterococcal cells are able to form biofilms on plastic material albeit with reduced efficiency as compared to growing cells. Moreover, although VBNC enterococcal forms are not capable of forming biofilms, Enterococcus faecalis and other enterococcal species of medical interest maintain their ability to synthesize the polymeric matrix for a limited period of time under adverse environmental conditions. The data presented, together with those regarding the maintenance of the division recovery potential already proved in nonculturable bacteria, further support the possibility for the VBNC and other nondividing bacterial forms to have a role as infectious agents and to constitute a risk to human health.     

Combined action of antibiotics and deoxyribonuclease on bacteria]

The impact of DNase on formation of biofilms by grampositive and gramnegative bacteria was studied. The enzyme was shown to inhibit the biofilm formation and have no toxicity to bacteria. When applied to the formed associations, DNase potentiated the effect of antibiotics on the bacteria located in them. An increase in the antimicrobial effect of various nonrelated antibiotics was not connected with a change in the bacteria susceptibility but likely resulted from a break of the genetic cooperation of the cells' community, connected with functions of extracellular DNA of the biofilm's matrix.     

Covalently linked kanamycin – Ciprofloxacin hybrid antibiotics as a tool to fight bacterial resistance

To address the growing problem of antibiotic resistance, a set of 12 hybrid compounds that covalently link fluoroquinolone (ciprofloxacin) and aminoglycoside (kanamycin A) antibiotics were synthesized, and their activity was determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The hybrids were antagonistic relative to the ciprofloxacin, but were substantially more potent than the parent kanamycin against Gram-negative bacteria, and overcame most dominant resistance mechanisms to aminoglycosides. Selected hybrids were 42–640 fold poorer inhibitors of bacterial protein synthesis than the parent kanamycin, while they displayed similar inhibitory activity to that of ciprofloxacin against DNA gyrase and topoisomerase IV enzymes. The hybrids showed significant delay of resistance development in both E. coli and B. subtilis in comparison to that of component drugs alone or their 1:1 mixture. More generally, the data suggest that an antagonistic combination of aminoglycoside-fluoroquinolone hybrids can lead to new compounds that slowdown/prevent the emergence of resistance.     

The ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency

The penetration ability of 12 antimicrobial agents, including antibiotics and biocides, was determined against biofilms of B. cereus and P. fluorescens using a colony biofilm assay. The surfactants benzalkonium chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB), and the antibiotics ciprofloxacin and streptomycin were of interest due to their distinct activities. Erythromycin and CTAB were retarded by the presence of biofilms, whereas ciprofloxacin and BAC were not. The removal and killing efficacies of these four agents was additionally evaluated against biofilms formed in microtiter plates. The most efficient biocide was CTAB for both bacterial biofilms. Ciprofloxacin was the best antibiotic although none of the selected antimicrobial agents led to total biofilm removal and/or killing. Comparative analysis of the results obtained with colony biofilms and microtiter plate biofilms show that although extracellular polymeric substances and the biofilm structure are considered a determining factor in biofilm resistance, the ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency. Also, the results reinforce the role of an appropriate antimicrobial selection as a key step in the design of disinfection processes for biofilm control.     

The activity of a small lytic peptide PTP-7 on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilms

One of the most important features of bacterial biofilms is their resistance to antibiotics and to the host immune system. In this study, we have found that a small lytic peptide, PTP-7, is very potent to Gram-positive bacteria and is able to kill antibiotic sensitive and resistant Staphylococcus aureus indiscriminately. Further studies have revealed that despite being a cationic peptide, the antibacterial activity of PTP-7 was not affected by the negatively charged extracellular polymeric substance (EPS) of biofilms. PTP-7 could diffuse into the deep layer of S. aureus biofilms to kill bacteria inside biofilms efficiently and effectively. Neither the high concentrations of metal ions nor the acidic pH in biofilms affected the activity of peptide PTP-7. It seems that the unique sequence/structure together with the resistant bacteria killing ability of peptide PTP-7 confers its anti-biofilm activity. This study sheds new light on the treatment of bacterial biofilms, especially various biofilm related infections.     

Antimicrobial susceptibility and sessile behaviour of bacteria isolated from a minimally processed vegetables plant

Abstract

In this study, 20 heterotrophic bacteria from a minimally processed vegetables (MPV) plant were tested for their susceptibilities to five antibiotics (tetracycline, erythromycin, ampicillin, levofloxacin and ciprofloxacin), their (co)aggregation abilities and their survival under gastric simulated conditions. Peracetic acid (PA) and sodium hypochlorite (SH), both at 50?ppm, were evaluated for their abilities to control biofilms of these bacteria. In general, the Gram-negative bacteria were found to be more resistant to the selected antibiotics. Two isolates, Rhanella aquatilis and Stenotrophomonas maltophilia, demonstrated multidrug resistance. Only Rhodococcus erythropolis presented aggregation potential, while no bacterium survived under the gastric conditions. The biofilm experiments showed PA as less efficient than SH in killing biofilms and neither of the disinfectants was able to fully eliminate the biofilms. Significant regrowth was observed for most of the biofilms. The results indicate that alternative and/or complementary disinfection strategies are required to guarantee food safety.     

Modality of bacterial growth presents unique targets: how do we treat biofilm-mediated infections?

It is well accepted that bacterial pathogens growing in a biofilm are recalcitrant to the action of most antibiotics and are resistant to the innate immune system. New treatment modalities are greatly warranted to effectively eradicate these infections. However, bacteria growing in a biofilm are metabolically unique in comparison to the bacteria growing in a planktonic state. Unfortunately, most antibiotics have been developed to inhibit the growth of bacteria in a planktonic mode of growth. This review focuses on the metabolism and physiology of biofilm growth with special emphasis on staphylococci. Future treatment options should include targeting unique metabolic niches found within bacterial biofilms in addition to the enzymes or compounds that inhibit biofilm accumulation molecules and/or interact with quorum sensing and intercellular bacterial communication.     

细菌显微追踪技术在生物被膜中的应用

细菌生物被膜是粘附于物体表面的由细菌细胞及其胞外物质组成的复杂膜样物聚集体,具有很强的耐药性和免疫逃逸能力。生物被膜内细菌的代谢活性、运动状态等与浮游细菌有明显区别。近年来,先进的显微成像技术结合新型图像处理方法,在研究细菌的运动、生理等方面发挥了重要作用。本文围绕生物被膜,概述了细菌显微追踪技术在其研究中的应用。主要从细菌的运动方式和生物被膜形成过程的调控两方面出发,介绍了在单细胞水平上利用该技术研究生物被膜的进展,包括细菌的游泳、蹭行、群集运动和多种信号通路调控下生物被膜的形成过程等,并展望了该技术在生物被膜其他相关研究领域的应用前景。     

The synergistic activity of triclosan and ciprofloxacin on biofilms of Salmonella Typhimurium

Triclosan is a biocide whose wide use has raised a debate about the potential benefits vs. hazards of the incorporation of antimicrobials in consumer products. The purpose of the present study was to determine whether exposure of biofilms of Salmonella enterica serovar Typhimurium to triclosan influences the tolerance of the bacteria towards antibiotics such as ciprofloxacin and vice versa. A synergistic antibiofilm activity was observed when the biofilms were treated with triclosan before or together with ciprofloxacin, and an additive activity was observed with planktonic cells. For example 500 μg mL⁻¹ triclosan and 500 μg mL⁻¹ ciprofloxacin reduced the number of viable cells in the biofilm by 1.6 and 0.5 log, respectively. However, the sequential treatment of 500 μg mL⁻¹ triclosan followed by ciprofloxacin resulted in 4.8 log reduction. Combination indexes (CI) for biofilms treated with triclosan followed by ciprofloxacin were 0.7, 0.32 and 0.25 for reduction of 90%, 99% and 99.9%, respectively, indicating a synergism. For planktonic cells, CIs were 1±0.1, indicating an additive effect. Therefore, it was suggested that triclosan weakens the ability of biofilm-associated cells to survive exposure to ciprofloxacin in the biofilm, probably by improving the permeability or the activity of ciprofloxacin.