The clinical impact of bacterial biofilms

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria （and fungi） are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.

Strategies for combating bacterial biofilm infections

Formation of biofilm is a survival strategy for bacteria and fungi to adapt to their living environment, especially in the hostile environment. Under the protection of biofilm, microbial cells in biofilm become tolerant and resistant to antibiotics and the immune responses, which increases the difficulties for the clinical treatment of biofilm infections. Clinical and laboratory investigations demonstrated a perspicuous correlation between biofilm infection and medical foreign bodies or indwelling devices. Clinical observations and experimental studies indicated clearly that antibiotic treatment alone is in most cases insufficient to eradicate biofilm infections. Therefore, to effectively treat biofilm infections with currently available antibiotics and evaluate the outcomes become important and urgent for clinicians. The review summarizes the latest progress in treatment of clinical biofilm infections and scientific investigations, discusses the diagnosis and treatment of different biofilm infections and introduces the promising laboratory progress, which may contribute to prevention or cure of biofilm infections. We conclude that, an efficient treatment of biofilm infections needs a well-established multidisciplinary collaboration, which includes removal of the infected foreign bodies, selection of biofilm-active, sensitive and well-penetrating antibiotics, systemic or topical antibiotic administration in high dosage and combinations, and administration of anti-quorum sensing or biofilm dispersal agents.

Phylogenetic diversity of bacterial biofilms covering the settlement substrates of nona-porous abalones (Haliotis diversicolor supertexta)

The settlement substrates of nona-porous abalones （Haliotis diversicolor supertexta） are covered with biofilms in which several types of microorganisms coexist and interact. These microorganisms are usually important causes of juvenile abalone disease as well as organisms useful in promoting abalones’ adhesion. The bacterial community structure of the biofilms remains unclear. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Total DNA of bacteria in biofilms was extracted, and 16S rRNA gene clone library was constructed using the primers specific for the domain bacteria. Subsequently, 30 randomly selected positive clones were screened by PCR-restriction fragment length polymorphism （PCR-RFLP） analysis, and resulted in 15 different RFLP patterns. Sequences analysis of representatives from each unique RFLP type revealed high genetic diversity in the bacterial populations. These sequences fell into nine major lineages of the bacterial domains： α-, β-, γ-and δ-subdivisions of the Proteobacteria; Planctomycete, Actinobacteria, Firmicutes, V errucomicrobium spp., and CytophagaFlexibacter-Bacteroides spp. Phylogenetic analysis indicated that the dominant phylotypes were most closely related to environmental and clinical Burkholderia cepacia of the β-Proteobacteria, and Roseobacteria of the α-Proteobacteria.

Bacterial Biofilm Degradation Using Extracellular Enzymes Produced by <i>Penicillium janthinellum</i>EU2D-21 under Submerged Fermentation

Bacterial biofilms are the bacterial aggregates that are embedded in the self-produced matrix of extracellular polymeric substances (EPS) that cause persistent bacterial infections posing significant medical challenges. They are recalcitrant to antibiotics and host defenses which make the treatments difficult and costly. Penicillium janthinellum mutant EU2D-21 was found to produce extracellular enzyme complex (amylase, cellulase, protease) under submerged fermentation. Maximum specific enzyme activities were found to be 3.04 IU/mg, 2.61 IU/mg and 3.39 IU/mg for alpha-amylase, cellulase and protease respectively, after 8 days of incubation at 30?C. We evaluated the enzyme complex for its ability to target and degrade the biofilms of different bacteria. We found that it degraded biofilms of Escherichia coli (85.5%), Salmonella enterica (79.72%), Pseudomonas aeruginosa (88.76%) and Staphyloccus aureus (87.42%) within 1 h of incubation at 50?C. The scanning electron microscopy (SEM), quantitation of biofilm removal assay and Crystal violet assay demonstrated that the enzyme complex detached the biofilm exo-polysaccharide matrix and bacteria from the cell surface. These results illustrate the feasibility and benefits of using this enzyme complex as anti-biofilm therapeutics to eradicate biofilms. This can also be used as a promising strategy to improve treatment of multidrug resistant bacterial infections.

Comparison of MIC with MBEC Assay for in Vitro Antimicrobial Susceptibility Testing in Biofilm Forming Clinical Bacterial Isolates

Context: MIC results can be misleading for treatment of biofilm associated. The Minimum Biofilm Eradication Concentration (MBEC) measures the determination to be made for a biofilm susceptibility to antibiotics. Aims: Assessment of biofilm production and comparison of the MIC and MBEC assays evaluate differences in the antibiotic sensitivity patterns of different clinical bacterial isolates from patients implanted with medical devices. Settings and Design: Random sampling with experimental study at tertiary care institute. Methods and Material: The study was carried out during January 2014 to March 2014 on 50 positive bacteriological cultures of medical devices which were inserted in hospitalized patients. Biofilm forming strains were identified by tissue culture plate method & tube method. Biofilm-producing and non-biofilm forming reference strains were used as controls. Assay has been developed for the use with flat bottom, 96-well microtiter plates. Sterile autoclaved PCR tubes were used as pegs which provided surface for the biofilm formation. Amikacin, ciprofloxacin, trimethoprim-sulfamethoxazole, vancomycin, cefoperazone/ sulbactam, gentamycin were tested for MIC and MBEC assay. Statistical Analysis Used: Results will be discussed in the form of percentages. Results: Colonization by Klebsiella pneumoniae, Acinetobacter baumanni and Pseudomonas aeruginosa was prevalent bacterial isolates in medical devices. MBEC was higher for all the antibiotics as compared to MIC except amikacin MBEC for Pseudomonas was the same as MIC. Conclusions: Device associated bacterial biofilms are the major source of infections in patients of critical care setup. MIC misleads physician for organism’s drug susceptibility testing, which results in therapeutic failure. MBEC can guide regarding choice and proper dosing of antibiotics to be given. That’s why major studies for similar testing should be done with clinical evaluation.

In vitro anti-biofilm and anti-bacterial activity of Junceella juncea for its biomedical application

Objective:To investigate the anti-biofilm and anti-bacterial activity of Junceella juncea(J.juncea)against biofilm forming pathogenic strains.Methods:Gorgonians were extraeted with methanol and analysed with fourier transform infrared spectroscopy.Biofilm forming pathogens were identified by Congo red agar supplemented with sucrose.A quantitative spectrophotometric method was used to monitor in vitro biofilm reduction by microtitre plate assay.Anti-bacterial activity of methanolic gorgonian extract(MGE)was carried out by disc diffusion method followed by calculating the percentage of increase with crude methanol(CM).Results:The presence of active functional group was exemplified by FT-IR spectroscopy.Dry,black,crystalline colonies confirm the production of extracellular polymeric substances responsible for biofilm formation in Congo red agar.MGE exhibited potential anti-biofilm activity against all tested bacterial strains.The anti-bacterial activity of methanolic extract was comparably higher in Salmonella typhii followed by Escherichia colt,Vibrio cholerae and Shigella flexneri.The overall percentage of increase was higher by 50.2%to CM.Conclusions:To conclude,anti-biofilm and anti-bacterial efficacy of J.juncea is impressive over biofilm producing pathogens and are good source for novel anti-bacterial compounds.

Bacterial Biofilm Formation on Resorbing Magnesium Implants

Background: Implant-associated infections are a result of bacterial adhesion to an implant surface and subsequent biofilm formation at the implantation site. This study compares different magnesium materials based on their ability to resist bacterial adhesion as well as further biofilm formation. Material and Methods: The surfaces of four magnesium-based materials (Mg2Ag, Mg10Gd, WE43 and 99.99% pure Mg) were characterized using atomic force microscope. In addition, the samples were tested for their ability to resist biofilm formation. Planktonic bacteria of either S. epidermidis or E. faecalis were allowed to adhere to the magnesium surfaces for two hour followed by rinsing and, for S. epidermidis, further incubation of 24, 72 and 168 h was carried out. Results: E. faecalis had a significantly stronger adhesion to all magnesium surfaces compared to S. epidermidis (p = 0.001). Biofilm growth of S. epidermidis was different on various magnesium materials: the amount of bacteria increased up to 72 h but interestingly a significant decrease was seen at 168 h on Mg2Ag and WE43 surfaces. For pure Mg and Mg10Gd the biofilm formation reached plateau at 72 h. Surface characteristics of resorbable magnesium materials were changing over time, and the surface was generally less rough at 168 h compared to earlier time points. No correlation was found between the surface topology and the amount of adherent bacteria. Conclusion: In early stages of biofilm adhesion, no differences between magnesium materials were observed. However, after 72 h Mg2Ag and WE43 had the best ability to suppress S. epidermidis’ biofilm formation. Also, bacterial adhesion to magnesium materials was not dependent on samples’ surface topology.

Detection of bacterial biofilm in uterine of repeat breeder dairy cows

Objective:To determine the possibility of presence of bacterial biofilm in the uterus of repeat breeder cows and to evaluate the effect of mucolytic agent in cleanup of uterus from biofilm. Methods: Twenty repeat breeder cows were selected from a large commercial dairy farm near Shiraz, Fars province, southern Iran. Uterine secretion samples were collected before and after uterine lavage with dimethyl sulfoxide (DMSO) 10% solution and periodic acid Schiff (PAS) staining was used to detect bacterial biofilm in uterine samples. After sampling, all cows were treated with two doses of PGF2 and intrauterine infusion of Cefquinome sulphate. Artificial insemination (AI) was performed after that.Results: Bacterial biofilms were found in 12 out of 20 animals (60%) in the first sampling with sterile saline lavage (before DMSO) and in 7 cows (35%) after DMSO lavage. Fourteen cows (70%) became pregnant after AI. This evidence showed the presence of bacterial biofilm in the uterus of dairy cows for the first time. Although non-significant, decrease in biofilm detection after DMSO lavage may suggest the potential ability of mucolytic agent for cleaning the uterus from bacterial biofilm. Also, high pregnancy rate after antibiotic treatment in the present study might be attributed to improved effect of antibiotic following lavage of uterine by DMSO.Conclusions:These findings should be investigated in future researches with more sample size.

Current Concepts of <i>Gardnerella vaginalis</i>Biofilm: Significance in Bacterial Vaginosis

Gardnerella vaginalis (GV) has been implicated in BV development. Further, biofilm is accepted as one, if not the principle reason, for recurrent or recalcitrant BV. GV has defined virulence factors that contribute to biofilm, though more may be discovered within genomic information. Key players in genital tract microecology include GV, other species of the microbiome, and the epithelial base on which microbial interactions occur. The epithelium is influenced by various forces such as douching, smoking, diet, and estrogen: other potential factors are yet unidentified. All of these factors may contribute to bacterial vaginosis. Further, biofilms usually contain microbial species in addition to GV, and the mechanisms for supporting roles of these other species provide an opportunity for elucidation. Gaps in knowledge still exist in effective therapeutics aimed at biofilm, and better understanding of the process of bacterial quiescence, persistence, and biofilm formation is a key step in future research. Purpose: This review examines current literature for information about biofilm significance in relation to GV and bacterial vaginosis. Methods: Structured literature review.

Bacterial Biofilm in Water Bodies of Cherrapunjee: The Rainiest Place on Planet Earth

Bacterial attachment is influenced by the cell surface, attachment media and other environmental factors. Bacterial community composition involved in biofilm formation in extremely high rainfall areas like Cherrapunjee has not been reported. The present study was undertaken to characterize bacteria involved in biofilm formation on different substrata in water bodies of Cherrapunjee, the highest rainfall receiving place on planet earth and to assess if the continuous rainfall has an effect on nature and colonization of biofilm bacteria. We developed the biofilm bacteria on stainless steel and glass surfaces immersed in water bodies of the study sites. Isolation of biofilm bacteria were performed on different culture media followed by estimation of protein and carbohydrate content of bacterial exopolysaccharides. 16S rRNA gene sequences were amplified for molecular characterization. The results showed that the biofilm bacterial diversity in water bodies of Cherrapunjee was influenced by substratum and was observed more in stainless steel than glass surface. Scanning electron microscopy images revealed that biofilm microstructure may represent a key determinant of biofilm growth and physiology of associated bacteria. The overall protein content of the extracted EPS of all the isolates were relatively higher than the carbohydrate content. Diverse bacteria proliferated on the substrata regardless of each other's presence, with more diverse bacteria colonizing the substrata on 7th day compared to 15th day of incubation. The biofilm bacteria compositions in the highest rainfall receiving habitat were not distinctly different from reports available, hence not unique from other water bodies.

Medicinal plant products targeting quorum sensing for combating bacterial infections

Traditional treatment of infectious diseases is based on compounds that aim to kill or inhibit bacterial growth. The bacterial resistance against antibiotics is a serious issue for public health. Today, new therapeutic targets other than the bacterial wall were deciphered. Quorum sensing or bacterial pheromones are molecules called auto-inducer secreted by bacteria to regulate some functions such as antibiotic resistance and biofilms formation. This therapeutic target is well-studied worldwide, nevertheless the scientific data are not updated and only recent researches started to look into its potential as a target to fight against infectious diseases. A major concern with this approach is the frequently observed development of resistance to antimicrobial compounds. Therefore, this paper aims to provide a current overview of the quorum sensing system in bacteria by revealing their implication in biofilms formation and the development of antibiotic resistance, and an update on their importance as a potential target for natural substances.

Efficient degradation of lube oil by a mixed bacterial consortium

A laboratory study was performed to assess the biodegradation of lube oil in bio-reactor with 304# stainless steel as a biofilm carrier. Among 164 oil degrading bacterial cultures isolated from oil contaminated soil samples, Commaonas acidovorans Px1, Bacillus sp. Px2, Pseudomonas sp. Px3 were selected to prepare a mixed consortium for the study based on the efficiency of lube oil utilization. The percentage of oil degraded by the mixed bacterial consortium decreased slightly from 99% to 97.2% as the concentration of lube oil was increased from 2000 to 10,000 mg/L. The degradation of TDOC （total dissolved organic carbon） showed a similar tendency compared with lube oil removal, which indicated that the intermediates in degradation process hardly accumulated. Selected mixed bacterial consortium showed their edge compared to activated sludge. Scanning electron microscopy （SEM） photos showed that biofilms on stainless steel were robust and with a dimensional framework constructed by EPS （extracellular polymeric substances）, which could promote the biodegradation of hydrocarbons. The increase of biofilm followed first-order kinetics with rate of 0.216 μg glucose/（cm2·day） in logarithm phase. With analysis of Fourier transform infrared spectroscopy （FT-IR） and gas chromatography-mass spectrometry （GC-MS） combined with removal of lube oil and TDOC, mixed bacterial consortium could degrade benzene and its derivatives, aromatic ring organic matters with a percentage over 97%.

Evaluation of an Orally Administered Multistrain Probiotic Supplement in Reducing Recurrences Rate of Bacterial Vaginosis: A Clinical and Microbiological Study

Background: Bacterial vaginosis (BV) is the most common urogenital disease in women, affecting about 19% - 24% of them in reproductive ages annually and after treatment, a single recurrence or more may occur in up to 58% of women within 12 months. Objective: The aim was to evaluate the effectiveness of a new orally administered food supplement, containing different probiotic strains, on women of childbearing age after the antibiotic treatment when compared with no probiotic intake. Methods: A prospective study was undertaken on 62 patients with BV. All patients were cured with metronidazole vaginal formulations (5 g of 0.75% gel once daily for 5 days or 500 mg ovules once daily for 7 days), then after was offered the option of using a new an orally administered food supplement containing: Lactobacillus plantarum PBS067, Lactobacillus rhamnosus LRH020 and Bifidobacterium animalis lactis BL050, with a total viability of 3 × 109 CFU/capsule (Intimique&#174;Femme). Among these women, 50 accepted to use the new orally food supplement, while 25 patients decided to use only metronidazole (control group). Results: The recurrence rate of BV after treatment with Intimique&#174;Femme was about 16%, compared to 40% in the control group. The incidence of abnormal vaginal microbiota decreased in both groups, but it was significantly higher in the Intimique&#174;Femme group at the end of treatment. Conclusion: This study showed that, in case of BV diagnosis, the complementary treatment of a strain-specific probiotic complex after antibiotics prophylaxis, is mandatory to reduce potential recurrences and cyclic use of further antibiotics.

Bacterial Cellulose Nanofibers as Reinforcement for Preparation of Bamboo Pulp Based Composite Paper

Bamboo fibers(BFs),with features of renewability and biodegradability,have been widely used in paper-making products.In order to improve the mechanical properties and water absorption behaviors of the BF paper,bacterial cellulose nanofibers(BCNFs)as environmentally friendly nano-fibrillated cellulose(NFC)were combined with BFs.The structures and properties of the BF/BCNF composite paper were characterized by field emission scanning electron microscopy(FE-SEM),X-ray diffraction(XRD),Fourier transforms infrared(FTIR)spectroscopy,mechanical tests,pore size tests,and water absorption tests.The results indicated that the addition of BCNFs could significantly improve the water absorption capacity and mechanical properties.The water absorption ratio of the BF/BCNF composite paper with a BCNF mass fraction of 9%comes to 443%,about 1.33 times that of the pure BF paper.At the same BCNF content,the tensile strength of the BF/BCNF composite paper in dry and wet states was 12.37 MPa and 200.9 kPa,respectively,increasing by 98.24%and 136.91%as compared with that of the BF paper.

Inhibition of Bacterial Adherence on the Surface of Biliary Stent Materials Modified With Chitosan

Bacterial infection plays an important role in the initiation of biliary sludge formation. Bacterial adherence and biofilm formation on the surface of a material have been considered as one of the main factors of stent re-occlusion in clinic. This work reported preventing bacterial adherence and bacterial biofilm formation on the surface of biliary stent material using chitosan film. The chitosan film was deposited on 316 L stainless steel （SS） plate by electrophoresis method and was characterized by X-ray diffraction （XRD）, Fourier Transform infrared spectroscopy （FTIR） and atomic force microscopy （AFM）. The ability of inhibiting bacterial adherence was investigated by incubating in human fresh bile adding E. coli and Enterobacter at 37±1 ℃ . Scanning electron microscopy （SEM） and fluorescence staining were used for observing bacterial colonization and biofilm formation. The results show that chitosan film was uniformly deposited on material surface, and the composition of the film did not change through cross-linking, but the crystallinity of chitosan film become well. Comparing to un-modified sample, the E. coli and Enterococcus adhesion amount and colonization on the surface of modified sample were significantly decreased by fluorescence staining and SEM. It is suggested that chitosan could be applied to biliary stent in clinical because of its antimicrobial activities.

Biofilm formation under high temperature causes the commensal bacteria Bacillus cereus WPySW2 to shift from friend to foe in Neoporphyra haitanensis in vitro model

Although biofilm formation may promote growth,biofilms are not always beneficial to their hosts.The biofilm formation characteristics of Bacillus cereus WPySW2 and its changes at different temperatures were studied.Results show that B.cereus WPySW2 promoted the growth of Neoporphyra haitanensis(an economically cultivated seaweed)at 20℃ but accelerated algal rot at 28℃.Thicker B.cereus WPySW2 biofilms covered the surface of N.haitanensis thalli at 28℃,which hindered material exchange between the algae and surrounding environment,inhibited algal photosynthesis and respiration,and accelerated algal decay.Compared with planktonic bacteria,mature biofilm cells had lower energy consumption and metabolic levels.The biofilm metabolic characteristics of B.cereus WPySW2 changed significantly with temperature.High temperature accelerated biofilm maturation,which made it thicker and more stable,allowing the bacteria to easily adapt to environmental changes and obtain greater benefits from their host.High temperature did not affect the production or increased the abundance of toxic metabolites,indicating that the negative effects of B.cereus WPySW2 on algae were not caused by toxins.This study shows that increased temperature can transform a harmless bacterium into a detrimental one,demonstrating that temperature may change the ecological function of phycospheric bacteria by affecting their morphology and metabolism.

An Impact of Different Silicone Breast Implants on the Bacterial Attachment and Growth

Bacterial biofilms have been implicated with breast implant complications including capsular contracture, double-capsule formation, and breast implant-associated anaplastic large cell lymphoma. However, the relationship between implant surface texture and microbial biofilm formation is insufficiently evaluated. In the present study, we examined the antimicrobial activities of different types of silicone breast implant. The growth of bacterial including Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa was compared using implants with various surface textures, including Hans Smooth, Hans SmoothFine, Allergan Smooth, Eurosilicone Smooth, Eurosilicone Texture, Sebbin Smooth, Sebbin Micro, Sebbin Texture, and Motiva Smooth. Microbial investigation revealed the increased growth of S. aureus on breast implants after 48 h, except Eurosilicone Smooth, Eurosilicone Texture, Hans SmoothFine and Sebbin Smooth material. At 48 hours, there was no major difference between the S. aureus attachment on smooth and textured implants. The results of S. epidermis attachment on the implant after 48 h showed that their growth decreased on surfaces of Motiva Smooth, Sebbin Smooth, and Eurosilicone Smooth. These results indicated that S. epidermis was unable to survive on these breast implants. Eventually, P. aeruginosa count had showed decrease of bacterial count after 48 hours compared to 24 hours in most of the implants except for Eurosilicone Texture, Sebbin Smooth and Sebbin Micro, where the count of P. aeruginosa slightly increased. This indicated that P. aeruginosa was unable to exist on the smooth surfaces. Our results show that the in vitro assay revealed no significant difference between smooth and textured surfaces and showed variable interactions and needed further molecular analysis to assess their adherence nature.

耐替加环素鲍曼不动杆菌中外排泵AdeB与Bfs过表达生物膜形成的关系

目的研究生物内被膜相关基因和向外排水泵基因之间在对耐替加环素鲍曼不动杆菌抗性上的相互作用。方法根据替加环素药敏敏感试验,将72株临床分离的鲍曼不动杆菌分为耐替加环素鲍曼不动杆菌组和替加环素敏感鲍曼不动杆菌组。采用结晶紫染色法观察两株菌的生物被膜形成情况。用RT-PCR方法分析生物被膜相关基因和外排泵基因。结果耐替加环素组生物被膜形成率为82.2%,替加环素敏感组生物被膜形成率为14.8%。转录水平上,耐药组生物被膜合成基因BfmS阳性率明显高于敏感组(P<0.01)。替加环素的最小抑菌浓度生物被膜组明显高于生物被膜缺失组(P<0.01)。外排泵AdeB基因在生物被膜形成组阳性率为95.2%,生物被膜缺失组为38.7%。结论BfS基因和AdeB外排泵基因的共表达可能与鲍曼不动杆菌生物被膜的形成有关。AdeB与BfS转录水平的提高促进了生物膜的形成,从而提高了鲍曼不动杆菌对替加环素的抗性。

A polymyxin B loaded hypoxia-responsive liposome with improved biosafety for efficient eradication of bacterial biofilms

Biofilm-associated bacterial infection brings serious threats to global public health owing to serious antibiotic resistance.It is urgently needed to develop innovative strategies to combat biofilm-associated bacterial infections.Polymyxins stand out as the last line of defense against Gram-negative bacteria.However,serious nephrotoxicity of polymyxins severely limits their clinical utility.Herein,a hypoxia-responsive liposome is designed as the nanocarrier of polymyxin B(PMB)to combat biofilms developed by Gram-negative bacteria.A metronidazole modified lipid(hypoxia-responsive lipid(HRLipid))is synthesized to fabricate hypoxia-responsive liposomes(HRLip).PMB loaded hypoxia-responsive liposomes(HRL-PMB)is then prepared to mitigate the nephrotoxicity of PMB while preserving its excellent bactericidal activity.HRL-PMB shows very low hemolysis and cytotoxicity due to liposomal encapsulation of PMB.PMB can be readily released from HRL-PMB in response to hypoxic biofilm microenvironment,exerting its bactericidal activity to realize biofilm eradication.The excellent in vivo antibiofilm ability of HRL-PMB is confirmed by a Pseudomonas aeruginosa infected zebrafish model and a P.aeruginosa pneumonia infection model.Meanwhile,HRL-PMB can greatly reduce the nephrotoxicity of PMB after intravenous injection.The hypoxia-sensitive liposomes held great promise to improve the biosafety of highly toxic antibiotics while preserving their intrinsic bactericidal ability,which may provide an innovative strategy for combating biofilm-associated infections.

Novel Cathelicidins with Potent Antimicrobial,Biofilm Inhibitory,and Anti-inflammatory Activities from the Frog Fejervarya multistriata

Antimicrobial peptides（AMPs）,a class of gene-encoded peptides,are the first line of immune system to defense microbial invasions in multicellular organisms.Cathelicidins are an important family of AMPs that have been identified exclusively in vertebrates.However,up to now,cathelicidins from amphibians are poorly understood.In the present study,we reported the identification and characterization of two novel cathelicidins（FM-CATH1 and FMCATH2） from the frog Fejervarya multistriata.The c DNA sequences encoding FM-CATHs were successfully cloned from the constructed lung c DNA library of F.multistriata.Both of the c DNA sequences encoding FM-CATHs are 447 bp in length,and the deduced mature peptides of FM-CATHs are composed of 34 residues.Structural analysis indicated that FM-CATH1 and FM-CATH2 mainly assume amphipathic alpha-helical conformations.Antimicrobial and bacterial killing kinetic analysis indicated that both FM-CATH1 and FM-CATH2 possess potent,broad-spectrum and rapid antimicrobial potency.And cytoplasmic membrane permeabilization analysis indicated that FM-CATH1 and FMCATH2 kill bacteria by inducing the permeabilization of bacterial membrane.Besides direct antimicrobial activities,FM-CATHs also exhibited significant inhibitory effect on the formation of bacterial biofilms at low concentrations below 1×MIC.Furthermore,FM-CATH1 and FM-CATH2 exhibited potent anti-inflammatory activities by inhibiting LPS-induced transcription and production of pro-inflammatory cytokines TNF-α,IL-1β,and IL-6 in mouse peritoneal macrophages.Meanwhile,FM-CATHs showed relatively low cytotoxic activity against mammalian normal and tumor cell lines,and low hemolytic activity against human erythrocytes.In summary,the identification of FM-CATHs provides novel clues for our understanding of the roles of cathelicidins in amphibian immune systems.The potent antimicrobial,biofilm inhibitory,anti-inflammatory activities,and low cytotoxicity of FM-CATHs imply their great potential in novel antibiotics development.