Pyrosequencing Revealed SAR116 Clade as Dominant dddP-Containing Bacteria in Oligotrophic NW Pacific Ocean

Dimethyl sulfide (DMS) is a climatically active gas released into the atmosphere from oceans. It is produced mainly by bacterial enzymatic cleavage of dimethylsulfoniopropionate (DMSP), and six DMSP lyases have been identified to date. To determine the biogeographical distribution of bacteria relevant to DMS production, we investigated the diversity of dddP—the most abundant DMS-producing gene—in the northwestern Pacific Ocean using newly developed primers and the pyrosequencing method. Consistent with previous studies, the major dddP-containing bacteria in coastal areas were those belonging to the Roseobacter clade. However, genotypes closely related to the SAR116 group were found to represent a large portion of dddP-containing bacteria in the surface waters of the oligotrophic ocean. The addition of DMSP to a culture of the SAR116 strain Candidatus Puniceispirillum marinum IMCC1322 resulted in the production of DMS and upregulated expression of the dddP gene. Considering the large area of oligotrophic water and the wide distribution of the SAR116 group in oceans worldwide, we propose that these bacteria may play an important role in oceanic DMS production and biogeochemical sulfur cycles, especially via bacteria-mediated DMSP degradation.     

Vertical Profiles of Bacteria in the Tropical and Subarctic Oceans Revealed by Pyrosequencing

Community composition of Bacteria in the surface and deep water layers were examined at three oceanic sites in the Pacific Ocean separated by great distance, i.e., the South China Sea (SCS) in the western tropical Pacific, the Costa Rica Dome (CRD) in the eastern tropical Pacific and the western subarctic North Pacific (SNP), using high throughput DNA pyrosequencing of the 16S rRNA gene. Bioinformatic analysis rendered a total of 143600 high quality sequences with an average 11967 sequences per sample and mean read length of 449 bp. Phylogenetic analysis showed that Proteobacteria dominated in all shallow and deep waters, with Alphaproteobacteria and Gammaproteobacteria the two most abundant components, and SAR11 the most abundant group at family level in all regions. Cyanobacteria occurred mainly in the surface euphotic layer, and the majority of them in the tropical waters belonged to the GpIIa family including Prochlorococcus and Synechococcus, whilst those associated with Cryptophytes and diatoms were common in the subarctic waters. In general, species richness (Chao1) and diversity (Shannon index H′) were higher for the bacterial communities in the intermediate water layers than for those in surface and deep waters. Both NMDS plot and UPGMA clustering demonstrated that bacterial community composition in the deep waters (500 m ∼2000 m) of the three oceanic regions shared a high similarity and were distinct from those in the upper waters (5 m ∼100 m). Our study indicates that bacterial community composition in the DOC-poor deep water in both tropical and subarctic regions were rather stable, contrasting to those in the surface water layers, which could be strongly affected by the fluctuations of environmental factors.     

Evaluating the Growth Potential of Pathogenic Bacteria in Water

The degree to which a water sample can potentially support the growth of human pathogens was evaluated. For this purpose, a pathogen growth potential (PGP) bioassay was developed based on the principles of conventional assimilable organic carbon (AOC) determination, but using pure cultures of selected pathogenic bacteria (Escherichia coli O157, Vibrio cholerae, or Pseudomonas aeruginosa) as the inoculum. We evaluated 19 water samples collected after different treatment steps from two drinking water production plants and a wastewater treatment plant and from ozone-treated river water. Each pathogen was batch grown to stationary phase in sterile water samples, and the concentration of cells produced was measured using flow cytometry. In addition, the fraction of AOC consumed by each pathogen was estimated. Pathogen growth did not correlate with dissolved organic carbon (DOC) concentration and correlated only weakly with the concentration of AOC. Furthermore, the three pathogens never grew to the same final concentration in any water sample, and the relative ratio of the cultures to each other was unique in each sample. These results suggest that the extent of pathogen growth is affected not only by the concentration but also by the composition of AOC. Through this bioassay, PGP can be included as a parameter in water treatment system design, control, and operation. Additionally, a multilevel concept that integrates the results from the bioassay into the bigger framework of pathogen growth in water is discussed. The proposed approach provides a first step for including pathogen growth into microbial risk assessment.Pathogenic bacteria can survive and also grow in low-nutrient aquatic environments, such as surface waters or man-made water treatment systems (2, 17, 30). Studies on pathogen survival and/or die-off (including disinfection) in water are common, but little is known about the fundamental factors governing their growth in the environment (34, 35). Understanding the growth of pathogenic bacteria in aquatic ecosystems is essential for a holistic approach to microbial risk assessment as well as for improving drinking water treatment design and operation.A key factor governing growth of all organisms is nutrient availability. All human pathogens are heterotrophs, utilizing organic compounds as their carbon and energy source. Natural organic matter in water comprises a broad spectrum of many different compounds; it is usually determined as a bulk parameter, such as dissolved organic carbon (DOC). Only a fraction (0.1 to 44%) of this DOC pool is readily available for bacterial growth (18, 33). This bioavailable fraction is quantified using bioassays, such as the biodegradable dissolved organic carbon (BDOC) assay (27) or the assimilable organic carbon (AOC) assay (31). Typically, AOC represents small molecules readily available for growth, whereas BDOC can also include larger molecular compounds, which require predegradation before they can be taken up by microbial cells. Results from both of these assays are commonly used as indicators for bacterial growth potential and have previously been associated with regrowth and biofilm formation in drinking water distribution systems (7, 20, 32).Previous studies have pointed toward an apparent correlation between the concentration of AOC and the presence of enteric bacteria. For example, during two large surveys of drinking water treatment systems across North America, the occurrence (presence/absence) of coliform bacteria was found to be elevated above an AOC concentration of 100 μg liter⁻¹ (4, 21). Other studies also found that AOC concentrations were directly correlated to growth of pathogenic bacteria (30, 34, 35). However, AOC is a bulk parameter, which includes many different substrates (e.g., amino acids, sugars, and fatty acids) readily available for heterotrophic growth. Hence, its composition can differ distinctly, and it is assumed that every aquatic environment carries a complex and unique “fingerprint” of utilizable organic carbon compounds (22). Moreover, the spectrum of growth-supporting substrates (carbon compounds) of individual bacterial strains is specific—a fact also used for the classification of bacteria for taxonomic purposes. This principle has been integrated into conventional AOC assays, where the specific substrate spectrum of different pure cultures can be used to quantify different types of compounds present in water (26, 33). The term “pathogenic bacteria” is a collective term for many different bacterial species that can all cause disease in humans but their individual substrate spectra are unique for each species. Thus, we have hypothesized that the total concentration of AOC alone is not a sufficient parameter for describing the growth potential of pathogenic bacteria; the quality of the available carbon compounds has to be considered as well.There is no existing method that is capable of fractionating organic carbon in a way that allows for the quantification of individual compounds that support growth of specific pathogens. In this study, we have developed a pathogen growth potential (PGP) assay by combining the conventional AOC assay (31) with flow cytometric quantification of bacterial growth (11) and using pathogens as inocula. The PGP assay yields two main results, namely, (i) the extent of pathogen growth, and (ii) the relative fraction of AOC consumed by a pathogen. With this approach, we investigated the growth potential of three model pathogens from three different genera, namely, Escherichia coli O157, Vibrio cholerae O1, and Pseudomonas aeruginosa, in a broad range of water samples, differing considerably in their origin and quality.     

A Rare Y Chromosome Missense Mutation in Exon 25 of Human USP9Y Revealed by Pyrosequencing

Ubiquitin-specific protease 9, Y-linked (USP9Y), is a protein encoded by the Y chromosome. Its precise function in the cell is unknown, although a role in the regulation of protein turnover has been postulated. Nonetheless, mutations in this gene could result in the over- or under-abundance of proteins involved in the regulation of spermatogenesis. We have identified a novel mutation, SM1, located in exon 25 of USP9Y (c.3642G→A), which results in an amino acid substitution (p.V1214I). The mutation is in close linkage (four bases distant) from a silent mutation, referred to as M222 (p.E1212E, c.3636G→A). In our male population (n = 374), SM1 was found in one individual (0.3%) who belongs to the recently described haplogroup R1b3h, defined by the U152 SNP. This new mutation is expected to represent a new haplogroup, (R1b1c10a); therefore, within our population of individuals from haplogroup R1b3h (R1b110) (n = 16), it has a frequency of 6.3% (95% CI: 2.7–9.9%).     

Fate of Pathogenic Bacteria in Microcosms Mimicking Human Body Sites

During the infectious process, pathogens may reach anatomical sites where they are exposed to substances interfering with their growth. These substances can include molecules produced by the host, and his resident microbial population, as well as exogenous antibacterial drugs. Suboptimal concentrations of inhibitory molecules and stress conditions found in vivo (high or low temperatures, lack of oxygen, extreme pH) might induce in bacteria the activation of survival mechanisms blocking their division capability but allowing them to stay alive. These “dormant” bacteria can be reactivated in particular circumstances and would be able to express their virulence traits. In this study, it was evaluated the effect of some environmental conditions, such as optimal and suboptimal temperatures, direct light and antibiotic sub-inhibitory concentrations doses of antibiotic, on the human pathogens Escherichia coli and Enterococcus faecalis when incubated in fluids accumulated in the body of patients with different pathologies. It is shown that inoculation in a number of accumulated body fluids and the presence of gentamicin, reliable conditions encountered during pathological states, induce stress-responding strategies enabling bacteria to persist in microcosms mimicking the human body. Significant differences were detected in Gram-negative and Gram-positive species with E. faecalis surviving, as starved or viable but non-culturable forms, in any microcosm and condition tested and E. coli activating a viable but non-culturable state only in some clinical samples. The persistence of bacteria under these conditions, being non-culturable, might explain some recurrent infections without isolation of the causative agent after application of the standard microbiological methods.     

Spatial and Species Variations in Bacterial Communities Associated with Corals from the Red Sea as Revealed by Pyrosequencing

Microbial associations with corals are common and are most likely symbiotic, although their diversity and relationships with environmental factors and host species remain unclear. In this study, we adopted a 16S rRNA gene tag-pyrosequencing technique to investigate the bacterial communities associated with three stony Scleractinea and two soft Octocorallia corals from three locations in the Red Sea. Our results revealed highly diverse bacterial communities in the Red Sea corals, with more than 600 ribotypes detected and up to 1,000 species estimated from a single coral species. Altogether, 21 bacterial phyla were recovered from the corals, of which Gammaproteobacteria was the most dominant group, and Chloroflexi, Chlamydiae, and the candidate phylum WS3 were reported in corals for the first time. The associated bacterial communities varied greatly with location, where environmental conditions differed significantly. Corals from disturbed areas appeared to share more similar bacterial communities, but larger variations in community structures were observed between different coral species from pristine waters. Ordination methods identified salinity and depth as the most influential parameters affecting the abundance of Vibrio, Pseudoalteromonas, Serratia, Stenotrophomonas, Pseudomonas, and Achromobacter in the corals. On the other hand, bacteria such as Chloracidobacterium and Endozoicomonas were more sensitive to the coral species, suggesting that the host species type may be influential in the associated bacterial community, as well. The combined influences of the coral host and environmental factors on the associated microbial communities are discussed. This study represents the first comparative study using tag-pyrosequencing technology to investigate the bacterial communities in Red Sea corals.     

Antibacterial Potential of Actinomycetes from North-Western Himalayas against Pathogenic Bacteria

Biology Bulletin - Actinomycetes derived from unfamiliar surroundings are considered as a promising supply for novel bioactive compounds comprising a broad range of biological actions. The current...     

Bacterial Community Composition and Diversity in Methane Charged Sediments Revealed by Multitag Pyrosequencing

Bacterial diversity in sediments obtained along the Chilean margin from areas containing methane seeps, and a hydrate mound were explored by cloning and sequencing and multitag pyrosequencing (MTPS). These libraries were statistically compared to determine the robustness of taxonomic assignment derived from multiplexed pyrosequencing strategies targeting variable regions V1 and V2 of the small subunit rRNA gene for environmental studies. There was no statistical difference in the composition of the libraries, thus, MTPS was utilized to describe diversity in three geochemical zones in these environments. Unidentified Cyanobacteria isolates were abundant in the sulfate reduction zone (SRZ), Deltaproteobacteria were concentrated at the sulfate methane transition zone (SMTZ) and Chloroflexi/GNS dominated methanogenesis zone (MGZ). Although there was variation among specific groups, communities in the SRZ and MGZ did not differ significantly. However, the community dominated by Deltaproteobacteria differentiates the SMTZ from the other zones. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental file.     

Susceptibility of Human Pathogenic Bacteria to Antimicrobial Peptides from Sesame Kernels

Hospital infection caused by Gram-negative bacteria is a serious and common problem, especially in developing countries. Aiming to reduce these infections, this report focuses on the identification and characterization of novel antimicrobial peptides from sesame (Sesamum indicum) kernel meals. Thus, sesame flour was extracted and precipitated with ammonium sulfate (100%). After dialysis, a rich fraction was applied to affinity red-Sepharose CL-6B chromatography, followed by reversed-phase high-performance liquid chromatography. Mass spectrometry analysis indicated the presence of a major peptide with molecular mass of ∼5.8 kDa in both cultivars. The bactericidal activities of antimicrobial peptides were evaluated against several human pathogens that had been effective only against Klebsiella sp., a Gram-negative bacterium responsible for human urinary infection. These data indicate the biotechnological potential of sesame peptides as an alternative method for hospital infection control and also the decrease of bacterial resistance to synthetic antibiotics.     

Cellobiose Transport by Mixed Ruminal Bacteria from a Cow

The transport of cellobiose in mixed ruminal bacteria harvested from a holstein cow fed an Italian ryegrass hay was determined in the presence of nojirimycin-1-sulfate, which almost inhibited cellobiase activity. The kinetic parameters of cellobiose uptake were 14 μM for the K_m and 10 nmol/min/mg of protein for the V_max. Extracellular and cell-associated cellobiases were detected in the rumen, with both showing higher V_max values and lower affinities than those determined for cellobiose transport. The proportion of cellobiose that was directly transported before it was extracellularly degraded into glucose increased as the cellobiose concentration decreased, reaching more than 20% at the actually observed levels of cellobiose in the rumen, which were less than 0.02 mM. The inhibitor experiment showed that cellobiose was incorporated into the cells mainly by the phosphoenolpyruvate phosphotransferase system and partially by an ATP-dependent and proton-motive-force-independent active transport system. This finding was also supported by determinations of phosphoenolpyruvate phosphotransferase-dependent NADH oxidation with cellobiose and the effects of artificial potentials on cellobiose transport. Cellobiose uptake was sensitive to a decrease in pH (especially below 6.0), and it was weakly but significantly inhibited in the presence of glucose.     

质谱法检测食源性致病菌技术研究进展

食源性致病菌存在广泛,能够引起人类的疾病甚至死亡,研究发现超过一半的食品安全问题来源于食源性致病菌的污染。如何快速有效地检测出食源性致病菌是预防和控制食品安全问题的关键环节。系统地介绍了检测食源性致病菌的方法,包括传统培养法、代谢学法、分子生物学法、免疫学方法等传统方法以及新兴的质谱法。质谱法有检测效率高、操作简便、灵敏度高等优点,着重对质谱法的原理、应用以及未来的发展趋势进行了阐述,以期为该技术的研究开发和推广应用提供参考。     

In Vitro Fermentation of Oat Bran Obtained by Debranning with a Mixed Culture of Human Fecal Bacteria

The prebiotic potential of oat samples was investigated by in vitro shaker-flask anaerobic fermentations with human fecal cultures. The oat bran fraction was obtained by debranning and was compared with other carbon sources such as whole oat flour, glucose, and fructo-oligosaccharide. The oat bran fraction showed a decrease in culturable anaerobes and clostridia and an increase in bifidobacteria and lactobacilli populations. A similar pattern was observed in fructo-oligosaccharide. Butyrate production was higher in oat bran compared to glucose and similar to that in fructo-oligosaccharide. Production of propionate was higher in the two oat media than in fructo-oligosaccharide and glucose, which can be used as energy source by the liver. This study suggests that the oat bran fraction obtained by debranning is digested by the gut ecosystem and increases the population of beneficial bacteria in the indigenous gut microbiota. This medium also provides an energy source preferred by colonocytes when it is metabolized by the gut flora.     

Increased Complexity of the Species Composition of Lactic Acid Bacteria in Human Feces Revealed by Alternative Incubation Condition

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with primers specific for lactic acid bacteria (LAB) was applied to investigate various media and incubation conditions to recover LAB from human feces. Samples were plated on selective and nonselective media and incubated under standard condition (37 degrees C, anaerobiosis) for fecal LAB as well as alternative condition (30 degrees C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected more strongly by the incubation condition than by the used medium. It was observed that food-associated LAB, such as Lactobacillus sakei and Leuconostoc mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition showed that L. sakei is one of the predominant food-associated LAB species, reaching counts of up to 106 CFU/g feces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in feces.