Silver nanoparticles induced with aqueous black carpenter ant extract selectively inhibit the growth of Pseudomonas aeruginosa.

Aqueous black carpenter ant extract (ABCAE) was used to synthesize silver nanoparticles (AgNPs). The ABCAE was rich in water-soluble compounds such as hydrophilic polypeptides that behaved as both reducing and stabilizing agents for generating AgNPs from Ag+ ion precursors. The diameter of the observed AgNPs was mostly in the range of 20-60 nm. The AgNPs were tested as an antibacterial agent for the growth inhibition of two pathogenic bacteria (Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 27661) and one common bacteria (Escherichia coli K12 ATCC 10798). Disk diffusion test showed that the AgNPs selectively inhibited the growth of P. aeruginosa but not for the other two species, suggesting the potential application of the green-chemically synthesized AgNPs as a selective antibacterial agent without harming other beneficial bacteria.

Bacteria involved in the sulfur cycle in tarballs collected from the Alabama Gulf Coast.

Tarballs are formed from released or discharged crude oil containing sulfur compounds. A considerable amount and variety of sulfate-reducing bacteria (SRB) and sulfur-oxidizing bacteria (SOB) were identified in tarballs collected from the intertidal and supratidal zones of Alabama's Gulf beaches. Amplicon sequencing of the bacterial 16S rRNA gene showed that SRB were more abundantly distributed in the core than on the surface of tarballs, while no significant differences were observed in the distribution of SOB. To our best knowledge, this is the first report on the spatial distribution of diverse SRB and SOB in tarballs.

Kangiella spongicola sp. nov., a halophilic marine bacterium isolated from the sponge Chondrilla nucula.

A novel halophilic bacterium of the genus Kangiella was isolated from a marine sponge collected from the Florida Keys, USA. Strain A79(T), an aerobic, Gram-negative, non-motile, rod-shaped bacterium, grew in 2-15 % (w/v) NaCl, at a temperature of 10-49 °C and at pH 4.5-10. Phylogenetic analysis placed strain A79(T) in the family Alcanivoraceae in the class Gammaproteobacteria. Strain A79(T) showed 98.5 % 16S rRNA gene sequence similarity to Kangiella japonica KMM 3899(T), 96.6 % similarity to Kangiella koreensis DSM 16069(T) and 95.6 % similarity to Kangiella aquimarina DSM 16071(T). The major cellular fatty acids were iso-C(11 : 0), iso-C(11 : 0) 3-OH, iso-C(15 : 0), iso-C(17 : 0) and iso-C(17 : 1)ω9c and the G+C content of the genomic DNA was 44.9 mol%. On the basis of physiological, chemotaxonomic and phylogenetic comparisons, strain A79(T) represents a novel species in the genus Kangiella, for which the name Kangiella spongicola sp. nov. is proposed. The type strain is A79(T) ( = ATCC BAA-2076(T) = DSM 23219(T)).

The effect of co-substrate activation on indigenous and bioaugmented PCB dechlorinating bacterial communities in sediment microcosms.

Microbial reductive dechlorination by members of the phylum Chloroflexi, including the genus Dehalococcoides, may play an important role in natural detoxification of highly chlorinated environmental pollutants, such as polychlorinated biphenyls (PCBs). Previously, we showed the increase of an indigenous bacterial population belonging to the Pinellas subgroup of Dehalococcoides spp. in Anacostia River sediment (Washington DC, USA) microcosms treated with halogenated co-substrates ("haloprimers"), tetrachlorobenzene (TeCB), or pentachloronitrobenzene (PCNB). The PCNB-amended microcosms exhibited enhanced dechlorination of weathered PCBs, while TeCB-amended microcosms did not. We therefore developed and used different phylogenetic approaches to discriminate the effect of the two different haloprimers. We also developed complementary approaches to monitor the effects of haloprimer treatments on 12 putative reductive dehalogenase (rdh) genes common to Dehalococcoides ethenogenes strain 195 and Dehalococcoides sp. strain CBDB1. Our results indicate that 16S rRNA gene-based phylogenetic analyses have a limit in their ability to distinguish the effects of two haloprimer treatments and that two of rdh genes were present in high abundance when microcosms were amended with PCNB, but not TeCB. rdh gene-based phylogenetic analysis supports that these two rdh genes originated from the Pinellas subgroup of Dehalococcoides spp., which corresponds to the 16S rRNA gene-based phylogenetic analysis.

Nested PCR bias: a case study of Pseudomonas spp. in soil microcosms.

Nested PCR methods combined with denaturing gradient gel electrophoresis (DGGE) are widely used for the detection of low copy number species or for the analysis of group-specific community profiles. With an appropriate number of PCR cycles during the first round of amplification, initial differences in the copy numbers of different DNA fragments that are targeted can be maintained during the second round without significant bias. However, if an excessive number of cycles in used in the first round, relative differences in the copy numbers of the targeted sequences can be obscured. Here we demonstrate the effect of "nested PCR bias" in a case study with PCR-DGGE of 16S rRNA gene sequences targeting Pseudomonas spp. following exposure of soil to naphthalene vapors. Our results demonstrate artifacts caused by nested PCR bias can be substantially minimized by calibrating the number of first round PCR cycles, thereby preserving the ability to obtain semiquantitative data for evaluating changes in gene copy numbers over time.

Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE.

The analysis of microbial communities in environmental samples requires accurate and reproducible methods for extraction of DNA from sample matrices that have different physical and chemical characteristics. Even with the same sample type, variations in laboratory methods can result in different DNA yields. To circumvent this problem, we have developed an easy and inexpensive way to normalize the quantities of DNA that involves the addition of an internal standard prepared from plasmid DNA. The method was evaluated by comparing DNA yields using different DNA extraction procedures, after which the DNA was used for microbial community analysis by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S ribosomal RNA (rRNA) and for quantification of 16S rRNA gene copy numbers in environmental samples by real-time PCR. Our results show that use of the internal standard allows normalization of the resulting data and more accurate quantification of gene copy numbers in soil samples. These methods should also have broad application for various other types of environmental samples.

Synergistic effect of crude oil plus dispersant on bacterial community in a louisiana salt marsh sediment.

A combined effect of crude oil plus dispersant (Corexit 9500A) significantly altered indigenous bacterial communities in a Louisiana salt marsh sediment after 30 days of incubation; the crude oil and/or Corexit 9500A treatments triggered shifts in bacterial communities and the shifted bacterial structure by crude oil plus Corexit 9500A was considerably different from those by either crude oil or Corexit 9500A. However, the synergistic effect of crude oil plus Corexit 9500A was not observed after 7 days of incubation; the bacterial community was slightly shifted by Corexit 9500A and the crude oil did not trigger any bacterial community shift after 7 days of incubation. DNA sequencing data indicated that Chromobacterium species was enriched in the Corexit 9500A microcosms after 7 days of incubation, while Pseudomonas, Advenella, Acidocella and Dyella spp. were enriched after 30 days of incubation. Parvibaculum was a dominant species in the crude oil microcosms after 30 days of incubation. Rhodanobacter, Dyella and Frateuria spp. were dominant in crude oil plus Corexit 9500A microcosms after 30 days of incubation. Our data show that the effect of crude oil plus Corexit 9500A on bacterial community is synergistic, and thus the dispersant effect should be considered with the spilled oil to correctly evaluate the environmental impact.

Enriching for microbial reductive dechlorination of polychlorinated dibenzo-p-dioxins and dibenzofurans.

Anaerobic enrichment cultures derived from contaminated Kymijoki River sediments dechlorinated 1,2,3,4-tetrachlorodibenzofuran (1,2,3,4-tetra-CDF), octachlorodibenzofuran (octa-CDF) and 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-tetra-CDD). 1,2,3,4-tetra-CDF was dechlorinated via 1,2,3-, 2,3,4-, and 1,3,4/1,2,4-tri-CDFs to 1,3-, 2,3-, and 2,4-di-CDFs and finally to 4-mono-CDF. The dechlorination rate of 1,2,3,4-tetra-CDF was generally slower than that of 1,2,3,4-tetra-CDD. The rate and extent of 1,2,3,4-tetra-CDD dechlorination was enhanced by addition of pentachloronitrobenzene (PCNB) as a co-substrate. Dechlorination of spiked octa-CDF was observed with the production of hepta-, hexa-, penta- and tetra-CDFs over 6 months. Two major phylotypes of the Chloroflexi community showed an increase, one of which was identical to the Dehalococcoides mccartyi Pinellas subgroup. A set of twelve putative reductive dehalogenase (rdh) genes increased in abundance with addition of 1,2,3,4-tetra-CDF, 1,2,3,4-tetra-CDD and/or PCNB. This information will aid in understanding how indigenous microbial communities impact the fate of PCDFs and in developing strategies for bioremediation of PCDD/F contaminated sediments.

Microbially mediated reductive dechlorination of weathered polychlorinated dibenzofurans in Kymijoki sediment mesocosms.

Little is known about the potential for indigenous microorganisms to reductively dechlorinate weathered polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) in contaminated sediments. The sediments of River Kymijoki, Finland are heavily contaminated with PCDFs originating from manufacture of the chlorophenol-based fungicide Ky-5. Reductive dechlorination of weathered PCDFs was monitored to examine strategies for stimulating such activities. Amendments with electron donors, a halogenated co-substrate (tetrachlorobenzene, TeCBz), and bioaugmentation with a mixed culture containing Dehalococcoides mccartyi strain 195 were used to stimulate dechlorination activity in 30 L River Kymijoki sediment mesocosms incubated from 18 °C to 21 °C. An initial onset of dechlorination of octa-, hepta- and hexa-CDFs was observed in all mesocosms in the first 2 years of incubation. During this initial 2-year period, the decrease in the mol% contribution of these PCDFs was coupled with an increase in the mol% contribution of tetra- and penta-CDFs. The ratio of 1,2,3,4,6,7,8- to 1,2,3,4,6,8,9-hepta-CDF increased significantly. Subtle differences were observed between amended and unamended mesocosms. For penta-CDFs, a decreasing mol% ratio of peri vs. total chlorines and increasing mol% ratio of lateral vs. total chlorines was observed in mesocosms amended with TeCBz, suggesting that the amendments may affect pathways of dechlorination. Analysis of congener patterns using principal components analysis supported the observation that dechlorination was most pronounced during the first 2 years. Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA genes revealed a diverse Chloroflexi community. This study showed evidence for dechlorination of weathered PCDFs in Kymijoki sediment mesocosms mediated by indigenous microorganisms.

Chronic laminitis is associated with potential bacterial pathogens in the laminae.

A common sequella of chronic laminitis in horses is repeated abscesses with variable lameness and drainage. It is unclear whether the exudate represents the debridement phase of a non-septic inflammatory process involving clearance of laminar tissue damaged during the acute episode of laminitis, or a response to a microbial infection developed by ascent of microbes from the environment to the tissue via the white line. The objective of this study was to evaluate the possibility that an undiagnosed microbial infection in laminar tissue is present in laminar tissue collected from chronically laminitic horses without an active hoof abscess. Methods to collect laminar tissue, aseptically, from control (non-laminitic) horses and those with chronic/recurrent laminitis are described. Laminae homogenates were evaluated for the presence of bacteria. Bacteria were identified using biochemical tests and sequencing of 16S rRNA and virulence genes. Laminae from chronically laminitic horses revealed 100-fold higher levels (P=0.002) of bacteria compared to control, non-laminitic horses. Although environmental organisms were identified, potential pathogens were identified. Included were Gram positive bacteria, Brevibacterium luteolum, coagulase-negative Staphylococcus spp. as well as Gram negative bacteria, enterohemorrhagic Escherichia coli and Alcaligenes faecalis. Further research is warranted to evaluate the role of bacteria in equine chronic laminitis.

Intestinal bacterial overgrowth includes potential pathogens in the carbohydrate overload models of equine acute laminitis.

Carbohydrate overload models of equine acute laminitis are used to study the development of lameness. It is hypothesized that a diet-induced shift in cecal bacterial communities contributes to the development of the pro-inflammatory state that progresses to laminar failure. It is proposed that vasoactive amines, protease activators and endotoxin, all bacterial derived bioactive metabolites, play a role in disease development. Questions regarding the oral bioavailability of many of the bacterial derived bioactive metabolites remain. This study evaluates the possibility that a carbohydrate-induced overgrowth of potentially pathogenic cecal bacteria occurs and that bacterial translocation contributes toward the development of the pro-inflammatory state. Two groups of mixed-breed horses were used, those with laminitis induced by cornstarch (n=6) or oligofructan (n=6) and non-laminitic controls (n=8). Cecal fluid and tissue homogenates of extra-intestinal sites including the laminae were used to enumerate Gram-negative and -positive bacteria. Horses that developed Obel grade2 lameness, revealed a significant overgrowth of potentially pathogenic Gram-positive and Gram-negative intestinal bacteria within the cecal fluid. Although colonization of extra-intestinal sites with potentially pathogenic bacteria was not detected, results of this study indicate that cecal/colonic lymphadenopathy and eosinophilia develop in horses progressing to lameness. It is hypothesized that the pro-inflammatory state in carbohydrate overload models of equine acute laminitis is driven by an immune response to the rapid overgrowth of Gram-positive and Gram-negative cecal bacterial communities in the gut. Further equine research is indicated to study the immunological response, involving the lymphatic system that develops in the model.

Molecular analysis of spatial variation of iron-reducing bacteria in riverine alluvial aquifers of the Mankyeong River.

Alluvial aquifers are one of the mainwater resources in many countries. Iron reduction in alluvial aquifers is often a major anaerobic process involved in bioremediation or causing problems, including the release of As trapped in Fe(III) oxide. We investigated the distribution of potential iron-reducing bacteria (IRB) in riverine alluvial aquifers (B1, B3, and B6 sites) at the Mankyeong River, Republic of Korea. Inactive iron reduction zones, the diversity and abundance of IRB can be examined using a clone library and quantitative PCR analysis of 16S rRNA genes. Geobacter spp. are potential IRB in the iron-reducing zone at the B6 (9 m) site, where high Fe(II) and arsenic (As) concentrations were observed. At the B3 (16 m) site, where low iron reduction activity was predicted, a dominant clone (10.6%) was 99% identical in 16S rRNA gene sequence with Rhodoferax ferrireducens. Although a major clone belonging to Clostridium spp. was found, possible IRB candidates could not be unambiguously determined at the B1 (18 m) site. Acanonical correspondence analysis demonstrated that, among potential IRB, only the Geobacteraceae were well correlated with Fe(II) and As concentrations. Our results indicate high environmental heterogeneity, and thus high spatial variability, in thedistribution of potential IRB in the riverine alluvial aquifersnear the Mankyeong River.

PCB dechlorination enhancement in Anacostia River sediment microcosms.

In situ treatment of PCB contaminated sediments via microbial dechlorination is a promising alternative to dredging, which may be reserved for only the most contaminated areas. Reductive dechlorination of low levels of weathered PCB mixtures typical of urban environments may occur at slow rates. Here, we report that biostimulation and bioaugmentation enhanced dechlorination of low concentration (2.1 mg PCBs/kg dry weight) historical PCBs in microcosms prepared with Anacostia River, Washington, DC, sediment. Treatments included electron donors butyrate, lactate, propionate and acetate (1 mM each); alternate halogenated electron acceptors (haloprimers) tetrachlorobenzene (TeCB, 25 microM), pentachloronitrobenzene (PCNB, 25 microM), or 2,3,4,5,6-PCB (PCB116, 2.0 microM); and/or bioaugmentation with a culture containing Dehalococcoides ethenogenes strain 195 (3 x 10(6)cells/mL). Dechlorination rates were enhanced in microcosms receiving bioaugmentation, PCNB and PCNB plus bioaugmentation, compared to other treatments. Microcosm subcultures generated after 415 days and spiked with PCB116 showed sustained capacity for dechlorination of PCB116 in PCNB, PCNB plus bioaugmentation, and TeCB treatments, relative to other treatments. Analysis of Chloroflexi 16S rRNA genes showed that TeCB and PCNB increased native Dehalococcoides spp. from the Pinellas subgroup; however this increase was correlated to enhanced dechlorination of low concentration weathered PCBs only in PCNB-amended microcosms. D. ethenogenes strain 195 was detected only in bioaugmented microcosms and decreased over 281 days. Bioaugmentation with D. ethenogenes strain 195 increased PCB dechlorination rates initially, but enhanced capacity for dechlorination of a model congener, PCB116, after 415 days occurred only in microcosms with enhanced native Dehalococcoides spp.

Dynamic changes in nahAc gene copy numbers during degradation of naphthalene in PAH-contaminated soils.

Many bacteria that degrade polycyclic aromatic hydrocarbons (PAHs) contain the nahAc gene that encodes a component of multimeric naphthalene dioxygenases. Because the nahAc gene is highly conserved, this gene serves as a potential biomarker for PAH degradation activity. The aim of this research was to examine the relationship between the rate of naphthalene degradation and the copy number of the nahAc gene in soils using conventional and real-time PCR. Four sets of degenerate primers for real-time PCR were designed based on the nahAc DNA sequences of 33 bacterial species. Before addition of naphthalene, copy numbers of the nahAc gene were below the detection limits of the assay at 5 x 10(3) copy numbers per gram of soil, but increased by over a thousand fold to 10(7) copies after 6 days of exposure to naphthalene vapors (approximately 30 ppm soil water concentration). Two unreported naphthalene dioxygenase homologs were found in the naphthalene-spiked soil by cloning and sequencing of the PCR products from the nahAc primers. Results of these experiments demonstrate the highly dynamic changes that occur in soil microbial communities after exposure to naphthalene and suggest that there is a direct relationship between gene copy numbers and degradation rates for naphthalene in PAH-contaminated soils.