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.     

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.     

Identification of Bacterial Strains Isolated from the Mediterranean Sea Exhibiting Different Abilities of Biofilm Formation

The Mediterranean Sea has rarely been investigated for the characterization of marine bacteria as compared to other marine environments such as the Atlantic or Pacific Ocean. Bacteria recovered from inert surfaces are poorly studied in these environments, when it has been shown that the community structure of attached bacteria can be dissimilar from that of planktonic bacteria present in the water column. The objectives of this study were to identify and characterize marine bacteria isolated from biofilms developed on inert surfaces immersed in the Mediterranean Sea and to evaluate their capacity to form a biofilm in vitro. Here, 13 marine bacterial strains have been isolated from different supports immersed in seawater in the Bay of Toulon (France). Phylogenetic analysis and different biological and physico-chemical properties have been investigated. Among the 13 strains recovered, 8 different genera and 12 different species were identified including 2 isolates of a novel bacterial species that we named Persicivirga mediterranea and whose genus had never been isolated from the Mediterranean Sea. Shewanella sp. and Pseudoalteromonas sp. were the most preponderant genera recovered in our conditions. The phenotypical characterization revealed that one isolate belonging to the Polaribacter genus differed from all the other ones by its hydrophobic properties and poor ability to form biofilms in vitro. Identifying and characterizing species isolated from seawater including from Mediterranean ecosystems could be helpful for example, to understand some aspects of bacterial biodiversity and to further study the mechanisms of biofilm (and biofouling) development in conditions approaching those of the marine environment.     

Diversity of Microorganisms Isolated from Amber

Abstract Claims that organisms can be cultured from amber, if substantiated, would be significant contributions to our understanding of the evolution, tenacity, and potential spread of life. Three reports on the isolation of organisms from amber have been published. Cano and Borucki recently reported the isolation of Bacillus sphaericus and Lambert et al. have described a new species designated Staphylococcus succinus from 25–40 million year old Dominican amber. These characterized organisms were phylogenetically distant from extant relatives and the Staphylococcus sp. sufficiently far removed from other extant staphylococci to be considered a new species. Here we report the culture of bacteria from Dominican and previously untested 120 million year old Israeli (Lebanese lode) amber. Twenty-seven isolates from the amber matrix have been characterized by fatty-acid profiles (FAME) and/or 16S rRNA sequencing. We also performed a terminal restriction fragment pattern (TRF) analysis of the original amber before prolonged culture by consensus primer amplification of the 16S rRNA followed by restriction enzyme digestion of the amplicons. Sample TRFs were consistent with a sparse bacterial assemblage and included at least five of the isolated organisms. Finally, we microscopically mapped the internal topography of an amber slice. Received: 14 December 1998; Accepted: 16 March 1999     

Bacterial Biofilm in Seawater: Cell Surface Properties of Early-attached Marine Bacteria

The development of antifouling strategies in seawater requires knowledge of the physico-chemical properties of the cell surfaces of early adherent bacteria. The hydrophilic, electrostatic and the Lewis acid-base cell surface properties of eleven marine bacteria were characterized. Although these bacteria adhered to a hydrophilic support immersed for 3 and 6?h, they presented various physico-chemical properties. Eleven strains possessed a hydrophilic surface and five a hydrophobic surface. Although the majority of the bacteria presented an electron-donating character, some could not generate Lewis acid-base interactions with the support. On the other hand, all strains possessed an isoelectric point ranging from 2.2 to 3.4 and were negatively charged at the pH of seawater. Hydrophilicity was a preponderant property among these bacteria, but other properties should not be ignored. The development of new antifouling paints must take account all the possible interaction levels used by the bacteria to adhere to an immersed surface.     

Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.     

Proteolytic Activity of Microorganisms Isolated from Freshwater Fish

A raw fish-juice was prepared and sterilized through the use of (60)Co gamma-irradiation. It was evaluated for suitability in an agar medium for testing the proteolytic activity of bacteria isolated from fish. Microorganism proteolytic activity was also detected by conventional methods with skim milk-agar. We tested 1,145 isolates from fresh and spoiling irradiated (0.0, 0.3, and 0.6 Mrad) yellow perch fillets for proteolytic activity, by the use of both media. Most isolates that showed proteolytic activity exhibited this activity in both media. A few isolates showed proteolytic activity only in one medium or the other. Proteolysis was found mainly among bacteria isolated from nonirradiated perch fillets. Nonproteolytic organisms were slightly more abundant than were proteolytic ones throughout refrigerated storage (6 days); the latter constituted 48% of the total organisms. Irradiation eliminated essentially all proteolytic bacteria when the fillets were stored at 1 C. However, some proteolytic bacteria survived for a few days after irradiation when the fillets were stored at 5 C.     

海洋微生物活性物质研究进展

由于海洋环境的特殊性和多样性,海洋生物产生了与陆地生物不同的代谢途径和防御体系,分泌出多种结构新颖、活性特异的物质,如抗生素、抗肿瘤活性物质、酶等,这些活性物质在化工、医药、食品以及生命科学等领域有着广阔的应用前景。本文主要介绍了海洋微生物活性物质的主要类型及研究现状。     

Passive Electrical Properties of Microorganisms: III. Conductivity of Isolated Bacterial Cell Walls

The dielectric properties of isolated Micrococcus lysodeikticus cell walls have been studied to establish more firmly the view that wall-associated ions play a major role in the conduction of low frequency electric current by intact bacterial cells. The conductivity of isolated walls was found to be about 0.40 mho/m. If counterions associated with fixed, ionized groups in the wall have average mobilities equal to that of sodium ions in free solution, the fixed charge concentration required to account for the measured conductivity is between 75 and 95 meq/liter of wet wall volume. Estimates of the numbers of titratable amino and carboxyl groups in wall polymers indicate that conductivity is more closely related to net wall charge than to total wall charge. The measured wall conductivity was used to predict a value of 0.15 ± 0.03 mho/m for whole cell conductivity. This prediction is close to the measured value of 0.25 ± 0.05 mho/m and it is thought that much of the disparity in values is related to changes in wall structure and composition during the isolation procedures.     

Biodegradation of Free Phytol by Bacterial Communities Isolated from Marine Sediments under Aerobic and Denitrifying Conditions

Biodegradation of (E)-phytol [3,7,11,15-tetramethylhexadec-2(E)-en-1-ol] by two bacterial communities isolated from recent marine sediments under aerobic and denitrifying conditions was studied at 20°C. This isoprenoid alcohol is metabolized efficiently by these two bacterial communities via 6,10,14-trimethylpentadecan-2-one and (E)-phytenic acid. The first step in both aerobic and anaerobic bacterial degradation of (E)-phytol involves the transient production of (E)-phytenal, which in turn can be abiotically converted to 6,10,14-trimethylpentadecan-2-one. Most of the isoprenoid metabolites identified in vitro could be detected in a fresh sediment core collected at the same site as the sediments used for the incubations. Since (E)-phytenal is less sensitive to abiotic degradation at the temperature of the sediments (15°C), the major part of (E)-phytol appeared to be biodegraded in situ via (E)-phytenic acid. (Z)- and (E)-phytenic acids are present in particularly large quantities in the upper section of the core, and their concentrations quickly decrease with depth in the core. This degradation (which takes place without significant production of phytanic acid) is attributed to the involvement of alternating β-decarboxymethylation and β-oxidation reaction sequences induced by denitrifiers. Despite the low nitrate concentration of marine sediments, denitrifying bacteria seem to play a significant role in the mineralization of (E)-phytol.     

Microbiological Community Structure of the Biofilm of a Methanol-Fed,Marine Denitrification System,and Identification of the Methanol-Utilizing Microorganisms

We demonstrated in a previous study that the biofilm of the methanol-fed fluidized marine denitrification reactor at the Montreal Biodome was composed of at least 15 bacterial phylotypes. Among those were 16S ribosomal RNA (rDNA) gene sequences affiliated to Hyphomicrobium spp., and Methylophaga spp.; the latter made up 70% of a clone library. By using fluorescent in situ hybridization (FISH), we investigated the structure of the biofilm during the colonization process in the denitrification reactor by targeting most of the bacterial families that the 16S rDNA gene library suggested would occur in the biofilm. Our results revealed that gamma-Proteobacteria (mostly Methylophaga spp.) accounted for up to 79% of the bacterial population, confirming the abundance of Methylophaga spp. within the biofilm. alpha-Proteobacteria represented 27–57% of the population, which included Hyphomicrobium spp. that appeared after 20 days of colonization and represented 7–8% of the population. We noticed a great abundance and diversity of eukaryotic cells, which made up 20% of the biomass at the beginning of the colonization but decreased to 3–5% in the mature biofilm. We then used FISH combined with microautoradiography (MAR–FISH) to identify the methylotrophs in the biofilm. The results showed that alpha-Proteobacteria used ¹⁴C methanol in the presence of nitrate, suggesting their involvement in denitrification. Despite their abundance, Methylophaga spp. did not assimilate methanol under those conditions.     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Involvement of B2 Receptor in Bradykinin-Induced Proliferation and Proinflammatory Effects in Human Nasal Mucosa-Derived Fibroblasts Isolated from Chronic Rhinosinusitis Patients

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinonasal mucosa either accompanied by polyp formation (CRSwNP) or without polyps (CRSsNP). CRSsNP accounts for the majority of CRS cases and is characterized by fibrosis and neutrophilic inflammation. However, the pathogenesis of CRS, especially CRSsNP, remains unclear. Immunohistochemistry of CRSsNP specimens in the present study showed that the submucosa, perivascular areas, and the mucous glands were abundant in fibroblasts. Therefore, we investigated the effects bradykinin (BK), an autacoid known to participate in inflammation, on human CRSsNP nasal mucosa-derived fibroblasts (NMDFs). BK increased CXCL1 and -8 secretion and mRNA expression with EC₅₀ ranging from 0.15~0.35 μM. Moreover, BK enhanced cell proliferation and upregulated the expressions of proinflammatory molecules, including cell adhesion molecules (CAMs) and cyclooxygenase (COX)-1 and -2. These functionally caused an increase in monocyte adhesion to fibroblast monolayer. Using pharmacological intervention and BKR siRNA knockdown, we demonstrated that the BK-induced CXCL chemokine release, cell proliferation and COX and CAM expressions were mainly through the B2 receptor (B2R). Accordingly, the B2R was preferentially expressed in the NMDFs than B1R. The B2R was highly expressed in the CRSsNP than the control specimens, while the B1R and kininogen (KNG)/BK expression slightly increased in the CRSsNP mucosa. Collectively, we report here for the first time that fibroblasts, KNG/BK, and BKRs are overexpressed in CRSsNP mucosa and BK upregulates chemokine expression, proliferation, and proinflammatory molecule expression in NMDFs via B2R activation, which lead to a functional increase in monocyte-fibroblast interaction. Our findings reveal a critical role of fibroblast, KNG/BK, and BKRs in the development of CRSsNP.     

Hydrolytic Enzymes from Marine Organisms as Inhibitors of Biofilm Formation

Russian Journal of Marine Biology - The effects of some hydrolytic enzymes from marine organisms on the formation and destruction of bacterial biofilms have been studied. As the results show, the...     

从铅锌矿渣中分离的微生物对重金属吸附特性的研究

从铅锌矿渣中分离到 16种菌 (包括 7株细菌和 9株真菌 ) ,并研究了它们对Zn2 + ,Pb2 + ,Cu2 + 的吸附特性。发现大多数菌株对Pb2 + 与Zn2 + 有不同程度的吸附 ,但对Cu2 + 的吸附能力较小。菌株对Zn2 + 的吸附率大于对Pb2 + 的吸附 ,能吸附Pb2 + 的菌株也能吸附Zn2 + 。pH 4～ 6是真菌吸附金属离子的较好范围 ,细菌仅在pH =5 .0条件下 ,对Pb2 + 与Zn2 + 有吸附。在测试的不同金属离子浓度范围内 (5 0mg/L 相似文献   

A Halophilic Vibrio Isolated from a Case of Chronic Cholecystitis

We found a halophilic vibrio in B bile from a 75-year-old female patient with chronic cholecystitis, and examined its biochemical characteristics. The organisms are gram-negative short rods or comma shaped, with some ring forms. They have a single polar flagellum, but no capsule. The strains can grow in peptone water with 1.0 to 4.0% NaCl, but not with no NaCl or 6.0% NaCl. The characteristics of the organisms are positive dextrose fermentation, catalase, oxidase, and ornithine decarboxylase, and negative lysine decarboxylase, arginine dihydrolase, and absence of gas from glucose. They are sensitive to 2,4-diamine-6,7-diisopropyl pteridine (0/129). These characteristics indicate that the isolated strain should be a halophilic vibrio. However, no growth on Salmonella-Shigella (SS), SS with added sucrose and bromcresol purple (SS-SB), MacConkey's or thiosulfate citrate bile salts (TCBS) agar plates was demonstrated. Nitrate reduction, Simmons' citrate agar, indole, o-nitrophenol-β-d-galactopyranoside (ONPG), motility and esculin hydrolysis were positive. Urease, gelatinase, Voges-Proskauer, phenylalanine deaminase and malonate reactions were negative. Acid was produced from amygdalin, arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannitol, salicin, starch, sucrose, trehalose, and xylose, but not from adonitol, dulcitol, inositol, mannose, melibiose, raffinose, rhamnose and sorbitol. From these characteristics the isolate is considered to be not identical with V. parahaemolyticus, V. cholerae, V. vulnificus or other vibrios. It can be presumed that this isolate represents another species of halophilic vibrio.     

Enumeration and Characterization of Acidophilic Microorganisms Isolated from a Pilot Plant Stirred-Tank Bioleaching Operation

Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.