Identification of Different Agrobacterium Strains Isolated from the Same Forest Nursery

Several Agrobacterium strains isolated from the same forest nursery from 1982 to 1988 were compared by serological, biochemical, and DNA-DNA hybridization methods. Similarities among strains belonging to biovar 2 were observed by indirect immunofluorescence, whereas biovar 1 strains showed serological heterogeneity. Electrophoretic analysis of bacterial envelope-associated proteins showed that few bands appeared in the strains belonging to biovar 1, whereas many proteins appeared in the case of biovar 2 strains. Chromosomal DNA was analyzed with six random C58 chromosomal fragments. None of the six probes hybridized to the DNA of the two biovar 2 strains. One of the probes gave the same hybridization pattern with all biovar 1 strains, whereas the other probes yielded different patterns. The vir regions were closely related in the different pathogenic strains. The T-DNA and replication regions were less conserved and showed some variations among the strains.     

Bacterial Strains Isolated from Different Niches Can Exhibit Different Patterns of Adhesion to Substrata

Various mechanisms have been demonstrated to be operative in bacterial adhesion to surfaces, but whether bacterial adhesion to surfaces can ever be captured in one generally valid mechanism is open to question. Although many papers in the literature make an attempt to generalize their conclusions, the majority of studies of bacterial adhesion comprise only two or fewer strains. Here we demonstrate that three strains isolated from a medical environment have a decreasing affinity for substrata with increasing surface free energy, whereas three strains from a marine environment have an increasing affinity for substrata with increasing surface free energy. Furthermore, adhesion of the marine strains related positively with substratum elasticity, but such a relation was absent in the strains from the medical environment. This study makes it clear that strains isolated from a given niche, whether medical or marine, utilize different mechanisms in adherence, which hampers the development of a generalized theory for bacterial adhesion to surfaces.     

Carbonate Precipitation of Bacterial Strains Isolated from Sediments and Seawater: Formation Mechanisms

This article presents a research study on carbonate formation in solid and liquid media by Thalassospira sp., Halomonas sp., Bacillus pumilus, and Pseudomonas grimontii, four bacterial strains isolated from sediments and deep seawater. As part of this study, we analyzed carbonic anhydrase activity, pH, adsorption of calcium and magnesium ions, and total organic and inorganic carbon. The geochemical program PHREEQC was also used to calculate the mineral saturation indexes in all the cultures. The minerals formed were studied with X-ray diffraction, X-ray dispersive energy microanalysis, and scanning electron microscopy. In addition, all four bacterial strains were found to induce carbonate precipitation and to have carbonic anhydrase activity. Sterile control experiments did not precipitate carbonate. In solid M1 and B4 media, all of the strains precipitated magnesium calcite, whereas in the liquid media, they precipitated different percentages of magnesium calcite, aragonite, and monohydrocalcite. In both cases, small amounts of amorphous precipitates were also produced. This article discusses carbonate formation and the possible role played by metabolic activity, bacterial surfaces and carbonic anhydrase in this process. Finally, the results obtained lead to a hypothesis regarding the importance of carbonate precipitation for the survival of bacteria populations in certain habitats.     

雪貂葡萄球菌的分离与鉴定

目的对雪貂分离菌株进行鉴定。方法通过菌落、菌体形态、生化特征等细菌学检测方法和药敏实验对所分离菌株进行鉴定。结果根据生物学特性和药敏实验确定所分离菌株为金黄色葡萄球菌和中间葡萄球菌。结论分离菌株为金黄色葡萄球菌和中间葡萄球菌。     

Photoacclimation of Prochlorococcus sp. (Prochlorophyta) Strains Isolated from the North Atlantic and the Mediterranean Sea

Two Atlantic (SARG and NATL1) strains and one Mediterranean (MED) strain of Prochlorococcus sp., a recently discovered marine, free-living prochlorophyte, were grown over a range of "white" irradiances (lg) and under low blue light to examine their photoacclimation capacity. All three strains contained divinyl (DV) chlorophylls (Chl) a and b, both distinguishable from "normal" Chls by their red-shifted blue absorption maximum, a Chl c-like pigment at low concentration, zeaxanthin, and [alpha]-carotene. The presence of two phaeophytin b peaks in acidified extracts from both Atlantic strains grown at high lg suggests that these strains also had a normal Chl b-like pigment. In these strains, the total Chl b to DV-Chl a molar ratio decreased from about 1 at 7.5 [mu]mol quanta m-2 s-1 to 0.4 to 0.5 at 133 [mu]mol quanta m-2 s-1. In contrast, the MED strain always had a low DV-Chl b to DV-Chl a molar ratio, ranging between 0.13 at low lg and 0.08 at high lg. The discrepancies between the Atlantic and MED strains could result from differences either in the number of light-harvesting complexes (LHC) II per photosystem II or in the Chl b-binding capacity of the apoproteins constituting LHC II. Photosynthesis was saturated at approximately 5 fg C(fg Chl)-1 h-1 or 6 fg C cell-1 h-1, and growth was saturated at approximately 0.45 d-1 for both MED and SARG strains at 18[deg]C, but saturating irradiances differed between strains. Atlantic strains exhibited increased light-saturated rates and quantum yield for carbon fixation under blue light.     

Biofilm Interactions between Distinct Bacterial Genera Isolated from Drinking Water

In the environment, multiple microorganisms coexist as communities, competing for resources and often associated as biofilms. In this study, single- and dual-species biofilm formation by, and specific activities of, six heterotrophic intergeneric bacteria were determined using 96-well polystyrene plates over a 72-h period. These bacteria were isolated from drinking water and identified by partial 16S rRNA gene sequencing. A series of planktonic studies was also performed, assessing the bacterial growth rate, motility, and production of quorum-sensing inhibitors (QSI). This constituted an attempt to identify key attributes allowing bacteria to effectively interact and coexist in a drinking-water environment. We observed that in both pure and dual cultures, all of the isolates formed stable biofilms within 72 h, with specific metabolic activity decreasing, in most cases, with an increase in biofilm mass. The largest single- and dual-biofilm amounts were found for Methylobacterium sp. and the combination of Methylobacterium sp. and Mycobacterium mucogenicum, respectively. Evidences of microbial interactions in dual-biofilm formation, associated with appreciable biomass variation in comparison with single biofilms, were found for the following cases: synergy/cooperation between Sphingomonas capsulata and Burkholderia cepacia, S. capsulata and Staphylococcus sp., and B. cepacia and Acinetobacter calcoaceticus and antagonism between S. capsulata and M. mucogenicum, S. capsulata and A. calcoaceticus, and M. mucogenicum and Staphylococcus sp. A neutral interaction was found for Methylobacterium sp.-M. mucogenicum, S. capsulata-Staphylococcus sp., M. mucogenicum-A. calcoaceticus, and Methylobacterium sp.-A. calcoaceticus biofilms, since the resultant dual biofilms had a mass and specific metabolic activity similar to the average for each single biofilm. B. cepacia had the highest growth rate and motility and produced QSI. Other bacteria producing QSI were Methylobacterium sp., S. capsulata, and Staphylococcus sp. However, only for S. capsulata-M. mucogenicum, S. capsulata-A. calcoaceticus, and M. mucogenicum-Staphylococcus sp., dual-biofilm formation seems to be regulated by the QSI produced by S. capsulata and Staphylococcus sp. and by the increased growth rate of S. capsulata. The parameters assessed by planktonic studies did not allow prediction and generalization of the exact mechanism regulating dual-species biofilm formation between the drinking-water bacteria.     

Voice Prosthetic Biofilm Formation and Candida Morphogenic Conversions in Absence and Presence of Different Bacterial Strains and Species on Silicone-Rubber

Morphogenic conversion of Candida from a yeast to hyphal morphology plays a pivotal role in the pathogenicity of Candida species. Both Candida albicans and Candida tropicalis, in combination with a variety of different bacterial strains and species, appear in biofilms on silicone-rubber voice prostheses used in laryngectomized patients. Here we study biofilm formation on silicone-rubber by C. albicans or C. tropicalis in combination with different commensal bacterial strains and lactobacillus strains. In addition, hyphal formation in C. albicans and C. tropicalis, as stimulated by Rothia dentocariosa and lactobacilli was evaluated, as clinical studies outlined that these bacterial strains have opposite results on the clinical life-time of silicone-rubber voice prostheses. Biofilms were grown during eight days in a silicone-rubber tube, while passing the biofilms through episodes of nutritional feast and famine. Biofilms consisting of combinations of C. albicans and a bacterial strain comprised significantly less viable organisms than combinations comprising C. tropicalis. High percentages of Candida were found in biofilms grown in combination with lactobacilli. Interestingly, L. casei, with demonstrated favorable effects on the clinical life-time of voice prostheses, reduced the percentage hyphal formation in Candida biofilms as compared with Candida biofilms grown in absence of bacteria or grown in combination with R. dentocariosa, a bacterial strain whose presence is associated with short clinical life-times of voice prostheses.     

Attachment and Biofilm Forming Capabilities of Staphylococcus epidermidis Strains Isolated from Preterm Infants

Staphylococcus epidermidis, a human commensal, is an important opportunistic, biofilm-forming pathogen and the main cause of late onset sepsis in preterm infants, worldwide. In this study we describe the characteristics of S. epidermidis strains causing late onset (>72 h) bloodstream infection in preterm infants and skin isolates from healthy newborns. Attachment and biofilm formation capability were analyzed in microtiter plates and with transmission electron microscopy (TEM). Clonal relationship among strains was studied with pulsed-field gel electrophoresis. Antimicrobial susceptibility testing was performed, as well as the detection of biofilm-associated genes and of the invasiveness marker IS256 with polymerase chain reaction. Blood and skin isolates had similar attachment and biofilm-forming capabilities and biofilm formation was not related to the presence of specific genes. Filament-like membrane structures were seen by TEM early in the attachment close to the device surface, both in blood and skin strains. Nine of the ten blood isolates contained the IS256 and were also resistant to methicillin and gentamicin in contrast to skin strains. S. epidermidis strains causing bloodstream infection in preterm infants exhibit higher antibiotic resistance and are provided with an invasive genetic equipment compared to skin commensal strains. Adhesion capability to a device surface seems to involve bacterial membrane filaments.     

Biofilm-Forming Abilities of Listeria monocytogenes Serotypes Isolated from Different Sources

A total of 98 previously characterized and serotyped L. monocytogenes strains, comprising 32 of 1/2a; 20 of 1/2b and 46 of 4b serotype, from clinical and food sources were studied for their capability to form a biofilm. The microtiter plate assay revealed 62 (63.26%) strains as weak, 27 (27.55%) strains as moderate, and 9 (9.18%) strains as strong biofilm formers. Among the strong biofilm formers, 6 strains were of serotype 1/2a and 3 strains were of serotype 1/2b. None of the strain from 4b serotype exhibited strong biofilm formation. No firm correlation (p = 0.015) was noticed between any serotype and respective biofilm formation ability. Electron microscopic studies showed that strong biofilm forming isolates could synthesize a biofilm within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Cell enumeration of strong, moderate, and weak biofilm was performed to determine if the number of cells correlated with the biofilm-forming capabilities of the isolates. Strong, moderate, and weak biofilm showed 570±127× 10³ cells/cm², 33±26× 10³ cells/cm², 5±3× 10³ cells/cm², respectively, indicating that the number of cells was directly proportional to the strength of the biofilm. The hydrophobicity index (HI) analysis revealed higher hydrophobicity with an increased biofilm formation. Fatty acid methyl esterase analysis revealed the amount of certain fatty acids such as iso-C15:0, anteiso-C15:0, and anteiso-C17:0 fatty acids correlated with the biofilm-forming capability of L. monocytogenes. This study showed that different strains of L. monocytogenes form biofilm of different intensities which did not completely correlate with their serotype; however, it correlated with the number of cells, hydrophobicity, and amount of certain fatty acids.     

16S rRNA Targeted Probes for the Identification of Bacterial Strains Isolated from Cultures of the Toxic Dinoflagellate Alexandrium tamarense

Abstract Bacteria have been implicated in the production of paralytic shellfish poison (PSP) toxins, which are normally associated with bloom-forming algal species, specifically toxic dinoflagellate algae. To clarify the role that these bacteria may play in the production of PSP toxins, it is desirable to identify and localize the bacteria associated with the dinoflagellates. 16S rRNA-targeted probes offer the possibility for both, and thus, probes have been made to putatively toxigenic bacteria isolated from the PSP-related dinoflagellate Alexandrium tamarense and tested for their specificity in dot blot and in situ hybridization experiments. Received: 5 August 1999; Accepted: 19 January 2000; Online Publication: 5 May 2000;     

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.     

Variation in Biofilm Formation among Strains of Listeria monocytogenes

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.     

罗勒多糖对博莱霉素诱导小鼠肺纤维化的作用

目的：研究罗勒多糖对博莱霉素诱导肺纤维化小鼠肺组织病理的影响。方法：将40只C57BL/6J雄性小鼠随机分为假手术组,模型组,罗勒多糖高、中、低剂量组。模型组和罗勒多糖组小鼠,气管注射博来霉素（1.5mg/kg）,诱导其肺纤维化,假手术组注射等量生理盐水同法造模。罗勒多糖组小鼠每天用100、50、25mg/kg罗勒多糖,假手术组、模型组小鼠同法给药相应剂量的生理盐水,每天给药。造模28d后处死小鼠,肺组织病理切片进行HE和Masson染色,观察肺泡炎和肺纤维化程度,ELISA检测肺组织羟脯氨酸含量。结果：与模型组相比,罗勒多糖不同剂量组小鼠肺组织胶原染色明显减少,肺泡间隔增厚程度较轻,区域性实质性病变少见,炎症细胞浸润减少,纤维化程度均有所减轻,羟脯氨酸含量下降,中、高剂量组优于低剂量组。结论：罗勒多糖能减轻博莱霉素诱导肺纤维化小鼠肺组织炎症和肺纤维化程度,是一种潜在的可用于特发性肺纤维化（IPF）治疗的中药提取物。     

2株海绵细菌的分离与鉴定

利用R2A培养基对海绵中的细菌进行分离,获得89株菌落形态有差异的菌株。通过在R2A培养基和营养丰富的LB培养基上的长势比较,发现有13株菌在LB培养基上生长缓慢、长势较弱。对此13株菌进行16S rDNA序列测定,发现菌株HB09009与Bacillus carboniphilus JCM9731T(AB021182)同源性最高,为97.1%;菌株HB09012与Planctomyces maris DSM8797T(NR025327)同源性最高,为97.4%,在发育树上处于一个分支,但在生长条件、培养特征和生理生化等方面都存在较大的差异,初步鉴定HB09009可能是Bacillus属的一个新种,暂定名为Bacillus sp.HB09009;鉴定HB09012可能是Planctomyces属的一个新种,暂定名Plancto-myces sp.HB09012。     

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.     

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。