Bacillus borbori sp. Nov., Isolated From an Electrochemically Active Biofilm

A Gram-positive, facultative anaerobic, motile, endospore-forming rod strain, designated DX-4^T, was isolated from an electrochemically active biofilm. Growth occurred at 30–65 °C (optimum 55 °C), at pH 6.0–8.5 (optimum pH 7.0–7.5) and with <6 % (w/v) NaCl. Cells were catalase- and oxidase-positive. The main respiratory quinone was MK-7, the predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol mannoside, and unidentified aminophospholipid, the DNA G+C content was 38.6 mol% and the major fatty acids (>5 %) were iso-C_15:0 (38.9 %), iso-C_17:0 (30.5 %), iso-C_16:0 (5.6 %), and anteiso-C_17:0 (5.2 %). The phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain DX-4^T is a member of the genus Bacillus. The results of phenotypic, chemotaxonomic, and genotypic analyses clearly indicated that strain DX-4^T represents a novel species, for which the name Bacillus borbori sp. nov. is proposed. The type strain is DX-4^T (= CCTCC AB2012196^T = KCTC 33103^T).     

Presence of a Polymicrobial Endometrial Biofilm in Patients with Bacterial Vaginosis

Objective

To assess whether the bacterial vaginosis biofilm extends into the upper female genital tract.

Study Design

Endometrial samples obtained during curettage and fallopian tube samples obtained during salpingectomy were collected. Endometrial and fallopian tube samples were analyzed for the presence of bacteria with fluorescence-in-situ-hybridisation (FISH) analysis with probes targeting bacterial vaginosis-associated and other bacteria.

Results

A structured polymicrobial Gardnerella vaginalis biofilm could be detected in part of the endometrial and fallopian tube specimens. Women with bacterial vaginosis had a 50.0% (95% CI 24.0–76.0) risk of presenting with an endometrial Gardnerella vaginalis biofilm. Pregnancy (AOR  = 41.5, 95% CI 5.0–341.9, p<0.001) and the presence of bacterial vaginosis (AOR  = 23.2, 95% CI 2.6–205.9, p<0.001) were highly predictive of the presence of uterine or fallopian bacterial colonisation when compared to non-pregnant women without bacterial vaginosis.

Conclusion

Bacterial vaginosis is frequently associated with the presence of a structured polymicrobial Gardnerella vaginalis biofilm attached to the endometrium. This may have major implications for our understanding of the pathogenesis of adverse pregnancy outcome in association with bacterial vaginosis.     

细菌生物被膜的耐药机制及控制策略

在特定的条件下,细菌可以形成生物被膜,包被有生物被膜的细菌称为被膜菌.被膜菌无论其形态结构、生理生化特性、致病性还是对环境因子的敏感性等都与浮游细菌有显著的不同,尤其对抗生素和宿主免疫系统具有很强的抵抗力,从而导致严重的临床问题,引起许多慢性和难治性感染疾病的反复发作.细菌生物被膜粘附在各种医疗器械及导管上极难清除,以至引发大量的医源性感染.近年来,随着人们对细菌致病机制认识的逐步深入,控制细菌生物被膜的方法已有较大发展.本文拟探讨被膜菌的耐药机制并着重综述细菌生物被膜控制方法的最新研究进展.     

The Use of New Methylene Blue in Pseudomonas aeruginosa Biofilm Destruction

A Pseudomonas aeruginosa biofilm was produced in a model system using the bacterial strain NCIMB 8295, grown on silicone tubing (bore size 0.75 cm). Destruction of the biofilm was attempted using either ampicillin or a combination of white light (light dose=7.2 J cm^{m 2}) and the phenothiazinium photosensitiser new methylene blue, and damage, both to extra-cellular polymeric substance (EPS) and to the organism, was monitored. It was found that although little damage to the EPS occurred with ampicillin, NMB caused both cell death and breakdown of the EPS, suggesting the use of photodynamic antimicrobial chemotherapy (PACT) in the disinfection of pathogenic biofilms, e.g. at external catheter surfaces.     

Inactivation of Efflux Pumps Abolishes Bacterial Biofilm Formation

Bacterial biofilms cause numerous problems in health care and industry; notably, biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Bacteria rely on efflux pumps to get rid of toxic substances. We discovered that efflux pumps are highly active in bacterial biofilms, thus making efflux pumps attractive targets for antibiofilm measures. A number of efflux pump inhibitors (EPIs) are known. EPIs were shown to reduce biofilm formation, and in combination they could abolish biofilm formation completely. Also, EPIs were able to block the antibiotic tolerance of biofilms. The results of this feasibility study might pave the way for new treatments for biofilm-related infections and may be exploited for prevention of biofilms in general.     

Study of the Response of a Biofilm Bacterial Community to UV Radiation

We have developed a bioluminescent whole-cell biosensor that can be incorporated into biofilm ecosystems. RM4440 is a Pseudomonas aeruginosa FRD1 derivative that carries a plasmid-based recA-luxCDABE fusion. We immobilized RM4440 in an alginate matrix to simulate a biofilm, and we studied its response to UV radiation damage. The biofilm showed a protective property by physical shielding against UV C, UV B, and UV A. Absorption of UV light by the alginate matrix translated into a higher survival rate than observed with planktonic cells at similar input fluences. UV A was shown to be effectively blocked by the biofilm matrix and to have no detectable effects on cells contained in the biofilm. However, in the presence of photosensitizers (i.e., psoralen), UV A was effective in inducing light production and cell death. RM4440 has proved to be a useful tool to study microbial communities in a noninvasive manner.     

Biosorption of Copper by a Bacterial Biofilm on a Flexible Polyvinyl Chloride Conduit

Inexpensive technologies with less-than-optimal efficiencies as a strategy for countering economic restraints to pollution control have been evaluated by using a laboratory-scale biotreatment process for copper-containing effluent. Economizing measures include the use of polyvinyl chloride (PVC) cylinders fashioned from commercially available flexible PVC conduit to support a biofilm that was cultured in an inexpensive medium prepared in wastewater. The biofilm was challenged by aqueous copper solution in a bioreactor and subsequently analyzed under a scanning electron microscope with energy-dispersive X-ray microanalysis.     

Spatially Resolved Characterization of Biogenic Manganese Oxide Production within a Bacterial Biofilm

Pseudomonas putida strain MnB1, a biofilm-forming bacterial culture, was used as a model for the study of bacterial Mn oxidation in freshwater and soil environments. The oxidation of aqueous Mn⁺² [Mn⁺²_(aq)] by P. putida was characterized by spatially and temporally resolving the oxidation state of Mn in the presence of a bacterial biofilm, using scanning transmission X-ray microscopy (STXM) combined with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the Mn L_2,3 absorption edges. Subsamples were collected from growth flasks containing 0.1 and 1 mM total Mn at 16, 24, 36, and 48 h after inoculation. Immediately after collection, the unprocessed hydrated subsamples were imaged at a 40-nm resolution. Manganese NEXAFS spectra were extracted from X-ray energy sequences of STXM images (stacks) and fit with linear combinations of well-characterized reference spectra to obtain quantitative relative abundances of Mn(II), Mn(III), and Mn(IV). Careful consideration was given to uncertainty in the normalization of the reference spectra, choice of reference compounds, and chemical changes due to radiation damage. The STXM results confirm that Mn⁺²_(aq) was removed from solution by P. putida and was concentrated as Mn(III) and Mn(IV) immediately adjacent to the bacterial cells. The Mn precipitates were completely enveloped by bacterial biofilm material. The distribution of Mn oxidation states was spatially heterogeneous within and between the clusters of bacterial cells. Scanning transmission X-ray microscopy is a promising tool for advancing the study of hydrated interfaces between minerals and bacteria, particularly in cases where the structure of bacterial biofilms needs to be maintained.     

Residual Structure of Streptococcus mutans Biofilm following Complete Disinfection Favors Secondary Bacterial Adhesion and Biofilm Re-Development

Chemical disinfection of oral biofilms often leaves biofilm structures intact. This study aimed to examine whether the residual structure promotes secondary bacterial adhesion. Streptococcus mutans biofilms generated on resin-composite disks in a rotating disc reactor were disinfected completely with 70% isopropyl alcohol, and were again cultured in the same reactor after resupplying with the same bacterial solution. Specimens were subjected to fluorescence confocal laser scanning microscopy, viable cell counts and PCR-Invader assay in order to observe and quantify secondarily adhered cells. Fluorescence microscopic analysis, particularly after longitudinal cryosectioning, demonstrated stratified patterns of viable cells on the disinfected biofilm structure. Viable cell counts of test specimens were significantly higher than those of controls, and increased according to the amount of residual structure and culture period. Linear regression analysis exhibited a high correlation between viable and total cell counts. It was concluded that disinfected biofilm structures favored secondary bacterial adhesion.     

Dissociation of Tissue Destruction and Bacterial Expansion during Bubonic Plague

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host.     

Efficacy of a Marine Bacterial Nuclease against Biofilm Forming Microorganisms Isolated from Chronic Rhinosinusitis

Background

The persistent colonization of paranasal sinus mucosa by microbial biofilms is a major factor in the pathogenesis of chronic rhinosinusitis (CRS). Control of microorganisms within biofilms is hampered by the presence of viscous extracellular polymers of host or microbial origin, including nucleic acids. The aim of this study was to investigate the role of extracellular DNA in biofilm formation by bacteria associated with CRS.

Methods/Principal Findings

Obstructive mucin was collected from patients during functional endoscopic sinus surgery. Examination of the mucous by transmission electron microscopy revealed an acellular matrix punctuated occasionally with host cells in varying states of degradation. Bacteria were observed in biofilms on mucosal biopsies, and between two and six different species were isolated from each of 20 different patient samples. In total, 16 different bacterial genera were isolated, of which the most commonly identified organisms were coagulase-negative staphylococci, Staphylococcus aureus and α-haemolytic streptococci. Twenty-four fresh clinical isolates were selected for investigation of biofilm formation in vitro using a microplate model system. Biofilms formed by 14 strains, including all 9 extracellular nuclease-producing bacteria, were significantly disrupted by treatment with a novel bacterial deoxyribonuclease, NucB, isolated from a marine strain of Bacillus licheniformis. Extracellular biofilm matrix was observed in untreated samples but not in those treated with NucB and extracellular DNA was purified from in vitro biofilms.

Conclusion/Significance

Our data demonstrate that bacteria associated with CRS form robust biofilms which can be reduced by treatment with matrix-degrading enzymes such as NucB. The dispersal of bacterial biofilms with NucB may offer an additional therapeutic target for CRS sufferers.     

Effect of Bacterial Interference on Biofilm Development by Legionella pneumophila

In the ecology of Legionella pneumophila a crucial role may be played by its relationship with the natural flora; thus we investigated the interactions between Legionella and other aquatic bacteria, particularly within biofilms. Among 80 aquatic bacteria screened for the production of bacteriocin-like substances (BLSs), 66.2% of them were active against L. pneumophila. The possible effect of some of these aquatic bacteria on the development and stability of L. pneumophila biofilms was studied. Pseudomonas fluorescens, the best BLS producer, showed the greatest negative effect on biofilm formation and strongly enhanced the detachment of Legionella. Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Aeromonas hydrophila, and Stenotrophomonas maltophilia, although producing BLSs at different levels, were less active in the biofilm experiments. Acinetobacter lwoffii did not produce any antagonistic compound and was the only one able to strongly enhance L. pneumophila biofilm. Our results highlight that BLS production may contribute to determining the fate of L. pneumophila within ecological niches. The interactions observed in this study are important features of L. pneumophila ecology, which knowledge may lead to more effective measures to control the persistance of the germ in the environment.     

Mapping of Bacterial Biofilm Local Mechanics by Magnetic Microparticle Actuation

Most bacteria live in the form of adherent communities forming three-dimensional material anchored to artificial or biological surfaces, with profound impact on many human activities. Biofilms are recognized as complex systems but their physical properties have been mainly studied from a macroscopic perspective. To determine biofilm local mechanical properties, reveal their potential heterogeneity, and investigate their relation to molecular traits, we have developed a seemingly new microrheology approach based on magnetic particle infiltration in growing biofilms. Using magnetic tweezers, we achieved what was, to our knowledge, the first three-dimensional mapping of the viscoelastic parameters on biofilms formed by the bacterium Escherichia coli. We demonstrate that its mechanical profile may exhibit elastic compliance values spread over three orders of magnitude in a given biofilm. We also prove that heterogeneity strongly depends on external conditions such as growth shear stress. Using strains genetically engineered to produce well-characterized cell surface adhesins, we show that the mechanical profile of biofilm is exquisitely sensitive to the expression of different surface appendages such as F pilus or curli. These results provide a quantitative view of local mechanical properties within intact biofilms and open up an additional avenue for elucidating the emergence and fate of the different microenvironments within these living materials.     

厌氧污泥体系脱氢酶活性表征细菌数的研究

以硫化钠作还原剂,甲苯为提取溶剂,用氯化三苯基四氮唑(TTC)法测定污泥脱氢酶活性,探讨了脱氢酶活性与细菌数对数(lgABN)的相关性,并建立了脱氢酶活性表征细菌数的数学模型。结果表明:在lgABN为7.5～12范围内,脱氢酶表征的污泥活性与细菌数对数(lgABN)呈明显线性相关,从理论上分析了其惟一线性相关性;在细菌数的对数达到7.5～12的对数期、稳定期与衰亡期,脱氢酶活性换算成对应细菌数所绘制的曲线与细菌生长曲线有明显拟合;在有重金属离子存在的污泥体系中,脱氢酶活性可用来作为评价重金属离子的毒性指标;从而脱氢酶活性可取代细菌计数表征污泥活性。     

细菌生物膜的结构及形成机制研究进展

细菌生物膜是细菌在特定条件下形成的一种特殊细菌群体结构,菌体被包裹在其自身分泌的多聚物中。近年来,有关生物膜组成结构、形成机制、抗逆性机制及其应用防治等诸方面的研究工作进展迅速,本文主要针对细菌生物膜的结构及形成机制方面的研究进展进行了介绍。     

Problems in Measuring Bacterial Diversity and a Possible Solution

The indices of species diversity used by plant and animal ecologists are not appropriate for bacterial diversity because of the inherent difficulty of defining a bacterial species. Arbitrary cutoff points to define a species or biotype lead to severe statistical problems. We suggest in this paper that a mean dissimilarity-based index without any attempt to define a species provides a statistically sound measurement of bacterial diversity.     

In Vitro Model of Bacterial Biofilm Formation on Polyvinyl Chloride Biomaterial

The aim of the study was to establish an in vitro model of Staphylococcus epidermidis biofilms on polyvinyl chloride (PVC) material, and to investigate bacterial biofilm formation and its structure using the combined approach of confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM). Staphylococcus epidermidis bacteria (stain RP62A) were incubated with PVC pieces in Tris buffered saline to form biofilms. Biofilm formation was examined at 6, 12, 18, 24, 30, and 48 h. Thicknesses of these biofilms and the number, and percentage of viable cells in biofilms were measured. CT scan images of biofilms were obtained using CLSM and environmental SEM. The results of this study showed that Staphylococcus epidermidis biofilm is a highly organized multi-cellular structure. The biofilm is constituted of large number of viable and dead bacterial cells. Bacterial biofilm formation on the surface of PVC material was found to be a dynamic process with maximal thickness being attained at 12–18 h. These biofilms became mature by 24 h. There was significant difference in the percentage of viable cells along with interior, middle, and outer layers of biofilms (P < 0.05). Staphylococcus epidermidis biofilm is sophisticated in structure and the combination method involving CLSM and SEM was ideal for investigation of biofilms on PVC material.