Gram-positive Genera Research Articles

Microbial ecology of two hot springs of Sikkim: Predominate population and geochemistry

Northeastern regions of India are known for their floral and faunal biodiversity. Especially the state of Sikkim lies in the eastern Himalayan ecological hotspot region. The state harbors many sulfur rich hot springs which have therapeutic and spiritual values. However, these hot springs are yet to be explored for their microbial ecology. The development of neo generation techniques such as metagenomics has provided an opportunity for inclusive study of microbial community of different environment. The present study describes the microbial diversity in two hot springs of Sikkim that is Polok and Borong with the assist of culture dependent and culture independent approaches. The culture independent techniques used in this study were next generation sequencing (NGS) and Phospholipid Fatty Acid Analysis (PLFA). Having relatively distinct geochemistry both the hot springs are thermophilic environments with the temperature range of 50–77 °C and pH range of 5–8. Metagenomic data revealed the dominance of bacteria over archaea. The most abundant phyla were Proteobacteria and Bacteroidetes although other phyla were also present such as Acidobacteria, Nitrospirae, Firmicutes, Proteobacteria, Parcubacteria and Spirochaetes. The PLFA studies have shown the abundance of Gram Positive bacteria followed by Gram negative bacteria. The culture dependent technique was correlative with PLFA studies. Most abundant bacteria as isolated and identified were Gram-positive genus Geobacillus and Anoxybacillus. The genus Geobacillus has been reported for the first time in North-Eastern states of India. The Geobacillus species obtained from the concerned hot springs were Geobacillus toebii, Geobacillus lituanicus, Geobacillus Kaustophillus and the Anoxybacillus species includes Anoxybacillus gonensis and Anoxybacillus Caldiproteolyticus. The distribution of major genera and their statistical correlation analyses with the geochemistry of the springs predicted that the temperature, pH, alkalinity, Ca2+, Mg2+, Cl2+, and sulfur were main environmental variables influencing the microbial community composition and diversity. Also the piper diagram suggested that the water of both the hot springs are Ca-HCO3– type and can be predicted as shallow fresh ground waters. This study has provided an insight into the ecological interaction of the diverse microbial communities and associated physicochemical parameters, which will help in determining the future studies on different biogeochemical pathways in these hot springs.

The Genus Macrococcus: An Insight Into Its Biology, Evolution, and Relationship With Staphylococcus.

The Gram-positive genus Macrococcus is composed of eight species that are evolutionarily closely related to species of the Staphylococcus genus. In contrast to Staphylococcus species, species of Macrococcus are generally regarded to be avirulent in their animal hosts. Recent reports on Macrococcus have focused on the presence of novel methicillin resistance genes in Macrococcus caseolyticus and Macrococcus canis, with the discovery of the first plasmid-encoded methicillin resistance gene in clinical Staphylococcus aureus of probable macrococcal origin generating further interest in these organisms. Furthermore, M. caseolyticus has been associated with flavor development in certain fermented foods and its potential as a food bio-preservative has been documented. The potential application of these organisms in food seems at odds with the emerging information regarding antibiotic resistance and is prompting further examination of the potential safety issues associated with such strains, given the European Food Safety Authority framework for the safety evaluation of microorganisms in the food chain. A comprehensive understanding of the genus would also contribute to understanding the evolution of staphylococci in terms of its acquisition of antibiotic resistance and pathogenic potential. In this review, we discuss the current knowledge on Macrococcus with regard to their phenotypic capabilities, genetic diversity, and evolutionary history with Staphylococcus. Comparative genomics of the sequenced Macrococcus species will be discussed, providing insight into their unique metabolic features and the genetic structures carrying methicillin resistance. An in-depth understanding of these antibiotic resistance determinants can open the possibilities for devising better preventative strategies for an unpredictable future.

Ice nucleation active bacteria in precipitation are genetically diverse and nucleate ice by employing different mechanisms

A growing body of circumstantial evidence suggests that ice nucleation active (Ice+) bacteria contribute to the initiation of precipitation by heterologous freezing of super-cooled water in clouds. However, little is known about the concentration of Ice+ bacteria in precipitation, their genetic and phenotypic diversity, and their relationship to air mass trajectories and precipitation chemistry. In this study, 23 precipitation events were collected over 15 months in Virginia, USA. Air mass trajectories and water chemistry were determined and 33 134 isolates were screened for ice nucleation activity (INA) at -8 °C. Of 1144 isolates that tested positive during initial screening, 593 had confirmed INA at -8 °C in repeated tests. Concentrations of Ice+ strains in precipitation were found to range from 0 to 13 219 colony forming units per liter, with a mean of 384±147. Most Ice+ bacteria were identified as members of known and unknown Ice+ species in the Pseudomonadaceae, Enterobacteriaceae and Xanthomonadaceae families, which nucleate ice employing the well-characterized membrane-bound INA protein. Two Ice+ strains, however, were identified as Lysinibacillus, a Gram-positive genus not previously known to include Ice+ bacteria. INA of the Lysinibacillus strains is due to a nanometer-sized molecule that is heat resistant, lysozyme and proteinase resistant, and secreted. Ice+ bacteria and the INA mechanisms they employ are thus more diverse than expected. We discuss to what extent the concentration of culturable Ice+ bacteria in precipitation and the identification of a new heat-resistant biological INA mechanism support a role for Ice+ bacteria in the initiation of precipitation.

Identification of YfiH (PgeF) as a factor contributing to the maintenance of bacterial peptidoglycan composition

Peptidoglycan (PG) is an essential, envelope-fortifying macromolecule of eubacterial cell walls. It is a large polymer with multiple glycan strands interconnected by short peptide chains forming a sac-like structure around cytoplasmic membrane. In most bacteria, the composition of the peptide chain is well-conserved and distinctive; in E. coli, the peptide chain length varies from two to five amino acids with a tetrapeptide consisting of L-alanine - D-glutamic acid - meso-diaminopimelic acid - D-alanine. However, it is not known how bacteria conserve the composition and sequence of peptide chains of PG. Here, we find that a conserved open reading frame of unknown function, YfiH (renamed PgeF) contributes to the maintenance of peptide composition in E. coli. Using genetic, biochemical and mass spectrometrical analyses we demonstrate that absence of yfiH results in incorporation of non-canonical amino acids, L-serine or glycine in place of L-alanine in PG sacculi leading to β-lactam - sensitivity, lethality in mutants defective in PG remodelling or recycling pathways, altered cell morphology and reduced PG synthesis. yfiH orthologs from other Gram-positive genera were able to compensate the absence of yfiH in E. coli indicating a conserved pathway in bacterial kingdom. Our results suggest editing/quality control mechanisms exist to maintain composition and integrity of bacterial peptidoglycan.

An isolate of Haemophilus haemolyticus produces a bacteriocin-like substance that inhibits the growth of nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) frequently colonises the upper respiratory tract and is an important cause of respiratory infections. Resistance to antibiotics is an emerging trend in NTHi and alternative prevention or treatment strategies are required. Haemophilus haemolyticus is a common commensal occupying the same niche as NTHi and, if able to produce substances that inhibit NTHi growth, may have a role as a probiotic. In this study, ammonium sulphate extracts from broth culture of 100 H. haemolyticus isolates were tested for the presence of substances inhibitory to NTHi using a well diffusion assay. One isolate produced a substance that consistently inhibited the growth of NTHi. The substance was inactivated by protease enzymes and had a molecular size of ca. 30 kDa as determined by size exclusion chromatography. When the substance was tested against bacteria from eight Gram-negative and three Gram-positive genera, only Haemophilus spp. were inhibited. Quantitative PCR testing showed the substance to be different to ‘haemocin’, the previously described bacteriocin of H. influenzae type b. These molecular characteristics, together with narrow-spectrum activity, suggest the substance may be a novel bacteriocin, and there is potential for this H. haemolyticus isolate to function as a probiotic for reduction of colonisation and subsequent infection with NTHi.

AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus sp.

Planococcus is a Gram-positive halotolerant bacterial genus in the phylum Firmicutes, commonly found in various habitats in Antarctica. Quorum quenching (QQ) is the disruption of bacterial cell-to-cell communication (known as quorum sensing), which has previously been described in mesophilic bacteria. This study demonstrated the QQ activity of a psychrotolerant strain, Planococcus versutus strain L10.15T, isolated from a soil sample obtained near an elephant seal wallow in Antarctica. Whole genome analysis of this bacterial strain revealed the presence of an N-acyl homoserine lactonase, an enzyme that hydrolyzes the ester bond of the homoserine lactone of N-acyl homoserine lactone (AHLs). Heterologous gene expression in E. coli confirmed its functions for hydrolysis of AHLs, and the gene was designated as aidP (autoinducer degrading gene from Planococcus sp.). The low temperature activity of this enzyme suggested that it is a novel and uncharacterized class of AHL lactonase. This study is the first report on QQ activity of bacteria isolated from the polar regions.

Chronic periprosthetic hip infection: micro-organisms responsible for infection and re-infection.

This study aimed to delineate the infecting micro-organisms identified at the first-time revision for infected THA, analyze pre-operative versus intra-operative findings, as well as intra-operative ones against re-infection micro-organisms. Microbiological laboratory findings were studied in 73 patients (mean age, 51.93 ± 10.9years) with chronic periprosthetic hip infection pre-operatively and intra-operatively. Forty-three patients had a two-stage revision THA while 30 patients were treated with a modified resection arthroplasty using the Ilizarov apparatus. Re-infection developed in 29 cases. Its microbial species were identified. Pre-operative findings on micro-organisms coincided 50.7% with the intra-operative ones. Bacterial growth in the intra-operative tests was detected in 72 (98.5%) cases. Gram-positive single genus infection was identified in 35 patients (48%); microbe associations were present in 33 patients (45%). Staphylococcus species prevailed. Gram-negative infection was detected in 5.5% of cases. One case (1.5%) did not have any microbe growth. Re-infection happened in 10 cases (23.2%) in a two-stage revision THA. In the resection arthroplasty group, early re-infection was observed in 63.3% of cases. Among a total of 29 re-infection cases, staphylococcus species were identified in 19 cases, present either in associations or as single germs. Intra-operative microbiological tests at the first-time revision for infected THR detect a reliable spectrum of micro-organisms to assess microbial resistance to antibiotics, develop treatment protocols, and for prognostic purposes. Preventive measures at primary THR and strategies to fight periprosthetic infection and reinfection should be targeted on staphylococci.

Comparative genomic analysis of the flagellin glycosylation island of the Gram-positive thermophile Geobacillus.

BackgroundProtein glycosylation involves the post-translational attachment of sugar chains to target proteins and has been observed in all three domains of life. Post-translational glycosylation of flagellin, the main structural protein of the flagellum, is a common characteristic among many Gram-negative bacteria and Archaea. Several distinct functions have been ascribed to flagellin glycosylation, including stabilisation and maintenance of the flagellar filament, motility, surface recognition, adhesion, and virulence. However, little is known about this trait among Gram-positive bacteria.ResultsUsing comparative genomic approaches the flagellin glycosylation loci of multiple strains of the Gram-positive thermophilic genus Geobacillus were identified and characterized. Eighteen of thirty-six compared strains of the genus carry these loci, which show evidence of horizontal acquisition. The Geobacillus flagellin glycosylation islands (FGIs) can be clustered into five distinct types, which are predicted to encode highly variable glycans decorated with distinct and heavily modified sugars.ConclusionsOur comparative genomic analyses showed that, while not universal, flagellin glycosylation islands are relatively common among members of the genus Geobacillus and that the encoded flagellin glycans are highly variable. This suggests that flagellin glycosylation plays an important role in the lifestyles of members of this thermophilic genus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3273-2) contains supplementary material, which is available to authorized users.

Chassis organism from Corynebacterium glutamicum: the way towards biotechnological domestication of Corynebacteria.

Chassis organism from Corynebacterium glutamicum: the way towards biotechnological domestication of Corynebacteria.

Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalis generates a new host for the analysis of biomass deconstruction.

BackgroundMembers of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria.ResultsTo develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrF locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host.ConclusionsThe use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0132-8) contains supplementary material, which is available to authorized users.

Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalis generates a new host for the analysis of biomass deconstruction

BackgroundMembers of the thermophilic, anaerobic Gram-positive bacterial genus Caldicellulosiruptor grow optimally at 65 to 78°C and degrade lignocellulosic biomass without conventional pretreatment. Decomposition of complex cell wall polysaccharides is a major bottleneck in the conversion of plant biomass to biofuels and chemicals, and conventional biomass pretreatment includes exposure to high temperatures, acids, or bases as well as enzymatic digestion. Members of this genus contain a variety of glycosyl hydrolases, pectinases, and xylanases, but the contribution of these individual enzymes to biomass deconstruction is largely unknown. C. hydrothermalis is of special interest because it is the least cellulolytic of all the Caldicellulosiruptor species so far characterized, making it an ideal naïve system to study key cellulolytic enzymes from these bacteria.ResultsTo develop methods for genetic manipulation of C. hydrothermalis, we selected a spontaneous deletion of pyrF, a gene in the pyrimidine biosynthetic pathway, resulting in a strain that was a uracil auxotroph resistant to 5-fluoroorotic acid (5-FOA). This strain allowed the selection of prototrophic transformants with either replicating or non-replicating plasmids containing the wild-type pyrF gene. Counter-selection of the pyrF wild-type allele on non-replicating vectors allowed the construction of chromosomal deletions. To eliminate integration of the non-replicating plasmid at the pyrF locus in the C. hydrothermalis chromosome, we used the non-homologous Clostridium thermocellum wild-type pyrF allele to complement the C. hydrothermalis pyrF deletion. The autonomously replicating shuttle vector was maintained at 25 to 115 copies per chromosome. Deletion of the ChyI restriction enzyme in C. hydrothermalis increased the transformation efficiency by an order of magnitude and demonstrated the ability to construct deletions and insertions in the genome of this new host.ConclusionsThe use of C. hydrothermalis as a host for homologous and heterologous expression of enzymes important for biomass deconstruction will enable the identification of enzymes that contribute to the special ability of these bacteria to degrade complex lignocellulosic substrates as well as facilitate the construction of strains to improve and extend their substrate utilization capabilities.

Extended tracking of the microbial community structure and dynamics in an industrial synthetic metalworking fluid system

Understanding of the occupational exposure risk scenario and disease etiology associated with industrial metalworking fluids (MWFs) requires knowledge of the development and composition of their microbial diversity in relation to the underlying fluid management factors. In this study, a managed synthetic MWF operation freshly recharged following the dumping, cleaning, and recharge (DCR) process was tracked in real time for microbial community changes over a period of 1.25 years (65 weeks). The recharged fluid developed very high bacterial counts (viable and nonviable) fairly quickly after the DCR process, indicating its inadequacy. Genus-/group-specific real-time qPCR confirmed the prevalence of six potentially pathogenic/immunogenic microbial genera/groups, viz. pseudomonads, enterics, mycobacteria, legionellae, actinomycetes, and fungi. Selective culturing revealed Acinetobacter and Bacillus as the most frequently isolated Gram-negative and Gram-positive genera, respectively, in addition to the presence of fungi and actinomycetes. Endotoxin perturbations (< 1000 to > 100000 EU mL⁻¹) coincided with temporal increases in Gram-negative bacteria and/or periodic biocide additions. PCR-DGGE-sequencing revealed an expanded estimated bacterial richness (up to 23 bands per sample). Of the 16 dominant bacterial phylotypes identified, the majority were detected for the first time in MWF. Interestingly, the study revealed a crucial role for MWF brand, among other fluid factors, in modulating the community structure and dynamics.

Dihydroanthracenone atropodiastereomers from the endophytic fungus Talaromyces wortmanii with activity against multi-resistant Staphylococcus aureus

Chemical investigation of the endophytic fungal strain Talaromyces wortmannii, isolated from the medicinal plant Aloe vera (Xanthorrhoeaceae), yielded three atropodiastereomeric pairs, including two symmetrical bisdihydroanthracenones, flavomannin A and its new atropisomer flavomannin B, two new asymmetrical bisdihydroanthracenones, and two new dihydroanthracenone/anthraquinone dimers. The planar structures of the new derivatives were unequivocally identified by spectroscopic and mass spectrometric analyses. The axial chirality of the biaryls was deduced from TDDFT ECD and VCD calculations, which however failed to establish the central chirality elements of flavomannin A. All six compounds exhibited antibacterial activity, predominately directed against Staphylococcus aureus, including even high-level (multi)drug-resistant isolates, with MIC values from 4 to 8 µg/mL for the most active compounds. Further Gram-positive genera (Streptococcus, Enterococcus, Bacillus) were moderately affected (MIC 32 to 64 µg/mL). Reporter gene analyses in Bacillus subtilis indicated induction of SOS response for some derivatives, suggesting interference with DNA structure or metabolism. Accordingly, fluorescence microscopy demonstrated defective segregation of the bacterial chromosome and DNA degradation (J. Med. Chem. 2013, 56(8):3257 – 72). The compounds showed no activity when tested against eukaryotic THP-1 cells (leukemia cell line) and BALB cells (mouse embryonic fibroblasts) (> 32 µg/mL).

Screening and selection of novel animal probiotics isolated from bovine chyme

Probiotics, gut-colonizing microorganisms capable of conferring a number of health benefits to their hosts, are highly desirable as animal feed supplements. Members of the Gram-positive genus Bacillus are often utilized as probiotics, since endospores formed by those bacteria render them highly resistant to environmental extremes and therefore capable of surviving gastrointestinal tract conditions. In this study, 84 distinct bacterial colonies were obtained from bovine chyme and 29 isolates were determined as Bacillus species. These isolates were principally screened for their antimicrobial activity against a group of two Gram-positive and four Gram-negative bacteria, including known human and animal pathogens such as Salmonella enterica, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Seven strains displaying strong antimicrobial activity against the test cohort were further evaluated for other properties desirable from animal probiotics, including high spore-forming capacity and adhesiveness, resistance to pH extremes and ability to form biofilms. The isolates were found to resist simulated gastrointestinal conditions and most of the antibiotics tested. In addition, plasmid presence was checked and cytotoxicity tests were performed to evaluate the potential risks of antibiotic resistance transfer and unintended pathogenic effects on host, respectively. We propose that the bacterial isolates are suitable for use as animal probiotics.

Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram-Positive Genus Streptococcus

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans.

Methylation by a Unique α-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the Gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute) and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.

Streptomyces sp. JS520 produces exceptionally high quantities of undecylprodigiosin with antibacterial, antioxidative, and UV-protective properties

A Gram-positive, red-pigment-producing bacterial strain, designated JS520 was isolated from the pristine sediment from the cave on mountain Miroc in Serbia. Strain was confirmed to belong to Streptomyces genus based on phenotypic and genetic analysis. Streptomyces sp. JS520 has the ability to produce exceptionally high amounts of deep red pigment into both solid and liquid media. Liquid chromatography and mass spectroscopy of the purified pigments revealed the major component to be undecylprodigiosin (93%) with minor component being oxidatively cyclized derivative. The pigment production was affected by medium composition, temperature, pH, and the aeration rate. By medium optimization, yields of undecylprodigiosin of 138mgl(-1) were achieved, what is the highest level of undecylprodigiosin production reported for the members of Gram-positive Streptomyces genus. Purified pigment had antimicrobial properties against bacterial Bacillus and Micrococcus species (50μgml(-1)) and against Candida albicans species (100-200μgml(-1) range). The ability to affect auto-oxidation of the linoleic acid was demonstrated for the purified undecylprodigiosin, suggesting antioxidative properties of this pigment. Multiple ecophysiological roles of the pigment were revealed by comparing cultures grown under pigment-producing and pigment-nonproducing conditions. Cells grown under undecylprodigiosin-producing conditions could tolerate presence of hydrogen peroxide exhibiting three times smaller zones of inhibition at 100mM H(2)O(2). Undecylprodigiosin-producing cells were also less susceptible to tetracycline, kanamycin, chloramphenicol, and 8-hydroxyquinoline. While the growth of the cells not producing pigment was completely inhibited by 15min of exposure to ultraviolet light (254nm), cells producing undecylprodigiosin and cells supplied with purified pigment in vitro showed survival rates at 22 and 8%, respectively.

Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.

Barcoded amplicon sequencing is rapidly becoming a standard method for profiling microbial communities, including the human respiratory microbiome. While this approach has less bias than standard cultivation, several steps can introduce variation including the type of DNA extraction method used. Here we assessed five different extraction methods on pediatric bronchoalveolar lavage (BAL) samples and a mock community comprised of nine bacterial genera to determine method reproducibility and detection limits for these typically low complexity communities. Additionally, using the mock community, we were able to evaluate contamination and select a relative abundance cut-off threshold based on the geometric distribution that optimizes the trade off between detecting bona fide operational taxonomic units and filtering out spurious ones. Using this threshold, the majority of genera in the mock community were predictably detected by all extraction methods including the hard-to-lyse Gram-positive genus Staphylococcus. Differences between extraction methods were significantly greater than between technical replicates for both the mock community and BAL samples emphasizing the importance of using a standardized methodology for microbiome studies. However, regardless of method used, individual patients retained unique diagnostic profiles. Furthermore, despite being stored as raw frozen samples for over five years, community profiles from BAL samples were consistent with historical culturing results. The culture-independent profiling of these samples also identified a number of anaerobic genera that are gaining acceptance as being part of the respiratory microbiome. This study should help guide researchers to formulate sampling, extraction and analysis strategies for respiratory and other human microbiome samples.

Thirteen novel deoxynivalenol-degrading bacteria are classified within two genera with distinct degradation mechanisms

The mycotoxin deoxynivalenol (DON), a secondary metabolite produced by species of the plant pathogen Fusarium, causes serious problems in cereal crop production because of its toxicity towards humans and livestock. A biological approach for the degradation of DON using a DON-degrading bacterium (DDB) appears to be promising, although information about DDBs is limited. We isolated 13 aerobic DDBs from a variety of environmental samples, including field soils and wheat leaves. Of these 13 strains, nine belonged to the Gram-positive genus Nocardioides and other four to the Gram-negative genus Devosia. The degradation phenotypes of the two Gram types were clearly different; all washed cells of the 13 strains degraded 100 μg mL(-1) DON to below the detection limit (0.5 μg mL(-1)), but the conditions inducing the DON-degrading activities differed between the two Gram types. The HPLC profiles of the DON metabolites were also distinct between the two genera, although all strains produced 3-epi-deoxynivalenol. The Gram-positive strains showed DON assimilation in media containing DON as a carbon source, whereas the Gram-negatives did not. Our results suggest that aerobic DDBs are distributed within at least two phylogenetically restricted genera, suggesting independent evolution of the DON-degradation mechanisms.

Size Distribution of Particulate and Associated Endotoxin and Bacteria in Traditional Swine Barn Rooms and Rooms Sprinkled With Oil

ABSTRACT The objective of this pilot study was to investigate if a once-a-day sprinkling of canola oil in a swine confinement facility alters the airborne concentration and distribution of particulate matter and associated compounds (endotoxin and culturable microbes). Particulate was collected using an eight-stage cascade impactor in four identical swine grower/finisher rooms of a swine barn. Particulate (mg/m3) and endotoxin (EU/m3 and EU/mg) distribution was determined. A six-stage viable cascade impactor was used to quantify total bacteria, enteric bacteria, and fungi. Microbes were characterized from subcultures prepared from the 10 most predominant colony types on each stage 3 (aerodynamic size 3.3–4.7μm) of the collection plates. Results indicated that oil sprinkling reduced total dust by 86% and total endotoxin concentration by 82.5%. However, the distribution patterns indicate that reduction is observed predominantly on large dust particles. In addition, the proportion of endotoxin associated with smaller particulate sizes (i.e., particles <4.7 μm) was higher in the oil-sprinkled rooms. Oil sprinkling does not markedly alter distribution of total bacteria, enteric bacteria, or fungi. The most frequently identified species were gram-positive genera. Oil sprinkling in swine confinement grower/finisher rooms can significantly reduce airborne total dust and endotoxin; however, smaller particles and associated endotoxin appear to remain in suspension, suggesting the overall improvement in air quality is uncertain. Further distribution studies and exposure outcome studies would need to be undertaken to determine the impacts of oil sprinkling.