Influence of allochthonous organic matter on bacterioplankton biomass and activity in a eutrophic, sub-tropical estuary

Heterotrophic bacterial and phytoplankton biomass, production, specific growth rates, and growth efficiencies were studied in the Northern region of the Cananeia-Iguape estuarine system, which has recently experienced an intense eutrophication due to anthropogenic causes. Two surveys were carried out during spring and neap tide periods of the dry season of 2005 and the rainy season of 2006. This region receives large freshwater inputs with organic seston and phosphate concentrations that reach as high as 1.0 mg l(-1) and 20.0 mu M, respectively. Strong decreasing gradients of seston and dissolved inorganic nutrients were observed from the river/estuary boundary to the estuary/coastal interface. Gradients were also observed in phytoplankton and bacterial production rates. The production rates of phytoplankton were 5.6-fold higher (mean 8.5 mu g Cl(-1) h(-1)) during the dry season. Primary production rates (PP) positively correlated with salinity and euphoric depth, indicating that phytoplankton productivity was light-limited. On the other hand, bacterial biomass (BB) and production rates (BP) were 1.9- and 3.7-fold higher, respectively, during the rainy season, with mean values of up to 40.4 mu g Cl(-1) and 7.9 mu g Cl(-1) h-1, respectively. Despite such a high BP, bacterial abundance remained <2 x 106 cells ml(-1), indicating that bacterial production and removal were coupled. Mean specific growth rates ranged between 0.9 and 5.5 d(-1). BP was inversely correlated with salinity and positively correlated with temperature, organic matter, exopolymer particles, and particulate-attached bacteria; this last accounted for as much as 89.6% of the total abundance. During the rainy season, BP was generally much higher than PP, and values of BP/PP > 20 were registered during high freshwater input, suggesting that under these conditions, bacterial activity was predominantly supported by allochthonous inputs of organic carbon. In addition, BB probably represented the main pathway for the synthesis of high-quality (low C:N) biomass that may have been available to the heterotrophic components of the plankton food web, particularly nanoheterotrophs. (C) 2008 Elsevier Ltd. All rights reserved.

Bacterial and phytoplankton dynamics in a sub-tropical estuary

Heterotrophic bacterial and biomass, production, specific growth rates and growth efficiencies were studied in July 2001 and January 2002 during both spring and neap tides, along a tidal cycle, at three sites in a subtropical estuary. Major freshwater inputs located in the Northern region led to differences in both and bacterioplankton biomass and activity along the estuary. While in the Northern region is light-limited, with mean production (PP) between 1.1 and 1.9 mu g C l(-1) h(-1) and mean specific growth rates (PSG) between 0.14 and 0.16 d(-1), the Southern region registered values as high as 24.7 mu g C l(-1) h(-1) for PP and 2.45 d(-1) (mean PP between 3.4 and 7.3 mu g C l(-1) h(-1); mean PSG between 0.28 and 0.57 d(-1)). On the other hand, maximum bacterial production (BP: 63.8 mu g C l(-1) h(-1)) and specific growth rate (BSG: 32.26 d(-1)) were observed in the Northern region (mean BP between 3.4 and 12.8 mu g C l(-1) h(-1); mean BSG between 1.98 and 6.67 day(-1)). These bacterial activity rates are among the highest recorded rates in estuarine and coastal waters, indicating that this system can be highly heterotrophic, due to high loads of allochthonous carbon (mainly derived from mangrove forest). Our results also showed that, despite that BP rates usually exceeded PP, in the Southern region BP may be partially supported (similar to 45%) by PP, since a significant regression was observed between BP and PP (r = 0.455, P < 0.001).

Antimicrobial activity and enterococcus faecalis biofilm formation on chlorhexidine varnishes

Objective: To evaluate, in vitro, the antimicrobial activity and biofilm formation of three chlorhexidine varnishes in four Enterococcus faecalis strains: E. faecalis ATCC 29212, E. faecalis EF-D1 (from failed endodontic treatment), E. faecalis 072 (cheese) and E. faecalis U-1765 (nosocomial infection), and one Enterococcus durans strain (failed endodontic treatment). Study Design: The direct contact test was used to study the antimicrobial activity. Bacterial suspensions were exposed for one hour to EC40, Cervitec (CE) and Cervitec Plus (CEP) varnishes. "Eradication" was defined as 100% bacterial kill. The formation of enterococci biofilms was tested on the surface of the varnishes after 24 hours of incubation and expressed as percentage of biofilm reduction. Results: EC40 eradicated all strains except E. faecalis ATCC 29212, where 98.78% kill was achieved. CE and CEP showed antimicrobial activity against all the strains, but most clearly against E. durans and E. faecalis 072. EC40 completely inhibited the formation of biofilm of E. faecalis ATCC 29212, E. faecalis 072 and E. durans. CE and CEP led to over 92% of biofilm reduction, except in the case of E. faecalis U-1765 on CEP (76.42%). Conclusion: The three varnishes studied were seen to be effective in killing the tested strains of enterococci and in inhibiting the formation of biofilm, the best results being observed with EC40.

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering

The aim of this study was to characterize the physicochemical properties of bacterial cellulose (BC) membranes functionalized with osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP[10-14], and to evaluate in vitro osteoinductive potential in early osteogenesis, besides, to evaluate cytotoxic, genotoxic and/or mutagenic effects. Peptide incorporation into the BC membranes did not change the morphology of BC nanofibers and BC crystallinity pattern. The characterization was complemented by Raman scattering, swelling ratio and mechanical tests. In vitro assays demonstrated no cytotoxic, genotoxic or mutagenic effects for any of the studied BC membranes. Culture with osteogenic cells revealed no difference in cell morphology among all the membranes tested. Cell viability/proliferation, total protein content, alkaline phosphatase activity and mineralization assays indicated that BC-OGP membranes enabled the highest development of the osteoblastic phenotype in vitro. In conclusion, the negative results of cytotoxicity, genotoxicity and mutagenicity indicated that all the membranes can be employed for medical supplies, mainly in bone tissue engineering/regeneration, due to their osteoinductive properties.

Antimicrobial activity of two techniques for arm skin disinfection of blood donors in Brazil

Objective: Evaluation of the antimicrobial effect of skin disinfection techniques is essential to avoid the transmission of infectious agents during blood transfusion. The aim of this study was to examine the effectiveness of two methods of arm skin disinfection used in blood donors at a Hemotherapy Center in Brazil that represents an important centre for distributing haemocomponents to many cities in the country. Methods: Two skin disinfection techniques in 50 blood donors were evaluated. For the first arm, 10% povidone-iodine/two-stage technique was used. On the opposite arm, 0.5% chlorhexidine digluconate alcohol solution/one-stage technique was used. The swabs were seeded on three culture media: blood agar, mannitol salt agar and Mac Conkey agar. Automated bacterial classification based on biochemical tests/specific substrates was performed. Donor characteristics were collected using the computerised system of the Hemotherapy Center. Results: We found that microbial reduction was significantly higher for 10% povidone-iodine technique (98.57-98.87%) when compared with 0.5% chlorhexidine technique (94.38-95.06%). The species Leuconostoc mesenteroides and Staphylococcus hominis showed resistance to both disinfection techniques. We did not find statistically significant relationships between donor characteristics and microbial reduction. Conclusions: Arm skin disinfection with 10% povidone-iodine produced better antimicrobial activity. We must acknowledge that 10% povidone-iodine technique has the limitation of being a two-stage method. However, prevention of adverse events due to bacterial contamination and transfusion reactions should be prioritised. Production of hypoallergenic and stronger antiseptics that allowed a safe one-stage disinfection technique should be encouraged in health systems, not only in Brazil but also around the world.

Comparative Studies of the (Anti) Mutagenicity of Baccharis dracunculifolia and Artepillin C by the Bacterial Reverse Mutation Test

Baccharis dracunculifolia is a plant native from Brazil, commonly known as 'Alecrim-do-campo' and 'Vassoura' and used in alternative medicine for the treatment of inflammation, hepatic disorders and stomach ulcers. Previous studies reported that artepillin C (ArtC, 3-{4-hydroxy-3,5-di(3-methyl-2-butenyl)phenyl}-2(E)-propenoic acid), is the main compound of interest in the leaves. This study was undertaken to assess the mutagenic effect of the ethyl acetate extract of B. dracunculifolia leaves (Bd-EAE: 11.4-182.8 mu g/plate) and ArtC (0.69-10.99 mu g/plate) by the Ames test using Salmonella typhimurium strains TA98, TA97a, TA100 and TA102, and to compare the protective effects of Bd-EAE and ArtC against the mutagenicity of a variety of direct and indirect acting mutagens such as 4-nitro-O-phenylenediamine, sodium azide, mitomycin C, benzo[a]pyrene, aflatoxin B1, 2-aminoanthracene and 2-aminofluorene. The mutagenicity test showed that Bd-EAE and ArtC did not induce an increase in the number of revertant colonies indicating absence of mutagenic activity. ArtC showed a similar antimutagenic effect to that of Bd-EAE in some strains of S. typhimurium, demonstrating that the antimutagenic activity of Bd-EAE can be partially attributed to ArtC. The present results showed that the protective effect of whole plant extracts is due to the combined and synergistic effects of a complex mixture of phytochemicals, the total activity of which may result in health benefits.

Structural, spectral and potentiometric characterization, and antimicrobial activity studies of [Zn(phen)(2)(cnge)(H2O)](NO3)(2)∙H2O

An octahedral Zn complex with o-phenanthroline (o-phen) and cyanoguanidine (cnge) has been synthesized and characterized. The crystal structural data show the formation of a ZnN5O core where the metal coordinates to two mutually perpendicular o-phenanthrolines as bidentate ligands [Zn-N bond lengths in the 2.124(2)-2.193(2) angstrom range], the cyanide nitrogen of a cnge [d(Zn-N) = 2.092(2) angstrom, angle(Zn-N-C) = 161.1(2)degrees], and a water molecule [d(Zn-Ow) = 2.112(2) angstrom]. Spectral data (FT-IR, Raman, and fluorescence) and speciation studies are in agreement with the structure found in the solid state and the one proposed to exist in the solution. To evaluate the changes in the microbiological activity of Zn, antibacterial studies were carried out by observing the changes in minimum inhibitory concentration of the complex, the ligands, and the metal against five different bacterial strains. The antibacterial activity of Zn improved upon complexation in three of the tested strains.

Bacterial cellulose-collagen nanocomposite for bone tissue engineering

A nanocomposite based on bacterial cellulose (BC) and type I collagen (COL) was evaluated for in vitro bone regeneration. BC membranes were modified by glycine esterification followed by cross-linking of type I collagen employing 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. Collagen incorporation was studied by spectroscopy analysis. X-Ray diffraction showed changes in the BC crystallinity after collagen incorporation. The elastic modulus and tensile strength for BC-COL decreased, while the strain at failure showed a slight increase, even after sterilization, as compared to pristine BC. Swelling tests and contact angle measurements were also performed. Cell culture experiments performed with osteogenic cells were obtained by enzymatic digestion of newborn rat calvarium revealed similar features of cell morphology for cultures grown on both membranes. Cell viability/proliferation was not different between BC and BC-COL membranes at day 10 and 14. The high total protein content and ALP activity at day 17 in cells cultured on BC-COL indicate that this composite allowed the development of the osteoblastic phenotype in vitro. Thus, BC-COL should be considered as alternative biomaterial for bone tissue engineering.

Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range

Abstract Background Citrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain Aw12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime. Results To characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306. Conclusions Comparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Structure of the PilZ-FimXEAL-c-di-GMP complex responsible for the regulation of bacterial type IV pilus biogenesis

Signal transduction pathways mediated by cyclic-bis(3'→5')-dimeric GMP (c-di-GMP) control many important and complex behaviors in bacteria. C-di-GMP is synthesized through the action of GGDEF domains that possess diguanylate cyclase activity and is degraded by EAL or HD-GYP domains with phosphodiesterase activity. There is mounting evidence that some important c-di-GMP-mediated pathways require protein-protein interactions between members of the GGDEF, EAL, HD-GYP and PilZ protein domain families. For example, interactions have been observed between PilZ and the EAL domain from FimX of Xanthomonas citri (Xac). FimX and PilZ are involved in the regulation of type IV pilus biogenesis via interactions of the latter with the hexameric PilB ATPase associated with the bacterial inner membrane. Here, we present the crystal structure of the ternary complex made up of PilZ, the FimX EAL domain (FimXEAL) and c-di-GMP. PilZ interacts principally with the lobe region and the N-terminal linker helix of the FimXEAL. These interactions involve a hydrophobic surface made up of amino acids conserved in a non-canonical family of PilZ domains that lack intrinsic c-di-GMP binding ability and strand complementation that joins β-sheets from both proteins. Interestingly, the c-di-GMP binds to isolated FimXEAL and to the PilZ-FimXEAL complex in a novel conformation encountered in c-di-GMP-protein complexes in which one of the two glycosidic bonds is in a rare syn conformation while the other adopts the more common anti conformation. The structure points to a means by which c-di-GMP and PilZ binding could be coupled to FimX and PilB conformational states