Volker S. Brözel

DNA as an Adhesin: Bacillus cereus Requires Extracellular DNA To Form Biofilms†

ABSTRACT The soil saprophyte Bacillus cereus forms biofilms at solid-liquid interfaces. The composition of the extracellular polymeric matrix is not known, but biofilms of other bacteria are encased in polysaccharides, protein, and also extracellular DNA (eDNA). A Tn917 screen for strains impaired in biofilm formation at a solid-liquid interface yielded several mutants. Three mutants deficient in the purine biosynthesis genes purA, purC, and purL were biofilm impaired, but they grew planktonically like the wild type in Luria-Bertani broth. Biofilm populations had higher purA, purC, and purL transcript ratios than planktonic cultures, as measured by real-time PCR. Laser scanning confocal microscopy (LSCM) of BacLight-stained samples indicated that there were nucleic acids in the cell-associated matrix. This eDNA could be mobilized off the biofilm into an agarose gel matrix through electrophoresis, and it was a substrate for DNase. Glass surfaces exposed to exponentially growing populations acquired a DNA-containing conditioning film, as indicated by LSCM. Planktonic exponential-phase cells released DNA into an agarose gel matrix through electrophoresis, while stationary-phase populations did not do this. DNase treatment of planktonic exponential-phase populations rendered cells more susceptible than control populations to the DNA-interacting antibiotic actinomycin D. Exponential-phase purA cells did not contain detectable eDNA, nor did they convey a DNA-containing conditioning film to the glass surface. These results indicate that exponential-phase cells of B. cereus ATCC 14579 are decorated with eDNA and that biofilm formation requires DNA as part of the extracellular polymeric matrix.

Analysis of the Life Cycle of the Soil Saprophyte Bacillus cereus in Liquid Soil Extract and in Soil

ABSTRACT Bacillus is commonly isolated from soils, with organisms of Bacillus cereus sensu lato being prevalent. Knowledge of the ecology of B. cereus and other Bacillus species in soil is far from complete. While the older literature favors a model of growth on soil-associated organic matter, the current paradigm is that B. cereus sensu lato germinates and grows in association with animals or plants, resulting in either symbiotic or pathogenic interactions. An in terra approach to study soil-associated bacteria is described, using filter-sterilized soil-extracted soluble organic matter (SESOM) and artificial soil microcosms (ASM) saturated with SESOM. B. cereus ATCC 14579 displayed a life cycle, with the ability to germinate, grow, and subsequently sporulate in both the liquid SESOM extract and in ASM inserted into wells in agar medium. Cells grew in liquid SESOM without separating, forming multicellular structures that coalesced to form clumps and encasing the ensuing spores in an extracellular matrix. Bacillus was able to translocate from the point of inoculation through soil microcosms as shown by the emergence of outgrowths on the surrounding agar surface. Microscopic inspection revealed bundles of parallel chains inside the soil. The motility inhibitor l-ethionine failed to suppress outgrowth, ruling out translocation by a flagellar-mediated mechanism such as swimming or swarming. Bacillus subtilis subsp. subtilis Marburg and four Bacillus isolates taken at random from soils also displayed a life cycle in SESOM and ASM and were all able to translocate through ASM, even in presence of l-ethionine. These data indicate that B. cereus is a saprophytic bacterium that is able to grow in soil and furthermore that it is adapted to translocate by employing a multicellular mode of growth.

Alteration of the Ileal Microbiota of Weanling Piglets by the Growth-Promoting Antibiotic Chlortetracycline†‡

ABSTRACT Antibiotics such as chlortetracycline (CTC) have been used to promote growth of pigs for decades, but concerns over increased antibiotic-resistant infections in humans have prompted the development of alternative strategies. Developing alternatives to antibiotic growth promoters (AGPs) could be informed by information on the mechanisms of growth promotion, notably, how AGPs affect the microbial populations of the gastrointestinal tract. Pigs from three sows were aseptically delivered by cesarean section. Six piglets were distributed to each of two foster mothers until weaning, when piglets were fed a diet with or without 50 mg/kg CTC for 2 weeks. The ileal bacterial microbiota was characterized by using a cultivation-independent approach based on DNA extraction, PCR amplification, cloning, and sequencing of the 16S rRNA gene pool. The ileal and mucosal communities of these growing pigs were dominated by Lactobacillus bacteria, various members of the family Clostridiaceae, and members of the poorly known genus Turicibacter. Overall, CTC treatment resulted in three shifts: a decrease in Lactobacillusjohnsonii, an increase in L. amylovorus, and a decrease in Turicibacter phylotypes. The composition of the microbiota varied considerably between individual pigs, as revealed by shared operational taxonomic units (OTUs) and similarity (SONS) analysis (θYC values). While the observed variation between untreated pigs obscured the possible effect of CTC, ∫-LIBSHUFF and SONS analyses of pooled libraries indicated a significant shift due to CTC in both the lumen and the mucosa, with some OTUs unique to either treated or control ileum. DOTUR analysis revealed little overlap between control and treated communities at the 3% difference level, indicating unique ileal communities in the presence of CTC.

Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.

Chemical characterization of soil extract as growth media for the ecophysiological study of bacteria.

We investigated the composition of soil-extracted solubilized organic and inorganic matter (SESOM) prepared from three different soils. Growth of various bacterial strains in these soil extracts was evaluated to find appropriate conditions for ecophysiological approaches. Analysis of SESOM by 1H-NMR and gas chromatography/mass spectrometry revealed a complex mixture of organic compounds. An oak forest SESOM supported the growth of several gram-positive and gram-negative soil-derived heterotrophic bacteria, whereas beech forest and grassland soil extracts did not. A metabolomic approach was performed by determining the extracellular metabolite profile of Bacillus licheniformis in SESOM. The results demonstrated that determination of the organic composition of SESOM during batch culturing is feasible. This makes SESOM amenable to studying the ecophysiology of a range of soil bacteria growing on soil-dissolved organic matter under more defined laboratory conditions. SESOM may also increase success in isolating previously uncultured or novel soil bacteria. Cell populations and the corresponding extracellular medium can be obtained readily and specific components extracted, paving the way for proteomic, transcriptomic, and metabolomic analyses. The synthetic carbon mixture based on SESOM, which mimics soil abilities, shows a positive impact on higher cell yields and longer cultivation time for biotechnological relevant bacteria.

GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles.

Fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene pools have become a popular tool for comparisons between microbial communities. The GC-clamp portion of primers for DGGE amplicon preparation provides a key component in resolving fragments of similar size but different sequence. We hypothesized that repeat syntheses of identical 40-base GC-clamp primers lead to different DGGE profiles. Three repeat syntheses of the same GC-clamp primer and two different GC-clamp primers directed at the V3-5 region of the 16S rRNA gene were compared. Genomic DNA of two separate soil bacterial communities and three bacterial species was amplified and resolved by DGGE. The DGGE profiles obtained with repeat-synthesized primers differed among each other as much as with alternate primers, for both soil DNA and pure single species. The GC-clamp portion of members of amplicon pools varied among each other, deviating from the design sequence, and was the likely cause for multiple bands derived from a single 16S rRNA gene sequence. We recommend procuring an oligonucleotide batch large enough to conduct an entire project. This should help to avoid any DGGE profile variations due to performance differences between repeat syntheses of GC-clamp oligonucleotide primers.

Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole‐genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm‐specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.

An Indigenous Gut Bacterium, Enterococcus faecalis (Lactobacillales: Enterococcaceae), Increases Seed Consumption By Harpalus pensylvanicus (Coleoptera: Carabidae)

Abstract Microbial symbioses likely drive the evolution of diet within animals, yet these symbiotic relationships remain poorly understood for many organisms. The bacterial endosymbiont Enterococcus faecalis is found in the intestinal tract of the beetle Harpalus pensylvanicus (DeGeer) (Coleoptera: Carabidae) and is thought to contribute to the digestion of the insects seed diet. We tested whether E. faecalis increases seed consumption by H. pensylvanicus. The feeding assay consisted of 4 dietary treatments fed: 1) antibiotics and E. faecalis; 2) antibiotics and no E. faecalis; 3) no antibiotics and E. faecalis; and 4) no antibiotics and no E. faecalis, in which seed consumption of the beetles was measured. Beetles administered antibiotics and then E. faecalis consumed greater weights of seeds and had both decreased efficiency of conversion of ingested material to biomass (E.C.I.) per beetle and decreased efficiency of conversion of digested material (E.C.D.) to biomass per beetle. These data provide further evidence that a gut microbiota dominated by E. faecalis facilitate seed consumption by H. pensylvanicus, possibly by contributing digestive enzymes to their host. Further research is needed on the evolutionary relationship between E. faecalis and granivorous insects, and on how these facultative symbioses could influence the trophic placement of animals within complex food webs.

Gut Bacterial Symbiont Diversity within Beneficial Insects Linked to Reductions in Local Biodiversity

ABSTRACT Understanding the factors that constrain or promote symbiotic microbial communities gives a clearer picture of the niches that can be occupied by a host organism. Many insects harbor symbiotic microbes that can alter various aspects of insect behavior and biology including digestion, sex determination, and pathogen defense. Habitat diversity has a major influence on insect and microbial diversity within an environment. In the current study, we assessed how habitat biodiversity affects the bacterial species richness within the gastrointestinal tract of insects. We measured species abundance of plants and insects present in three replicated habitats (prairie, pasture, and maize fields) that inherently represent a continuum of biological diversities. Gut bacterial symbiont diversity of the crickets Gryllus pennsylvanicus Burmeister and Allonemobius sp. (Orthoptera: Gryllidae) were described using terminal restriction fragment length polymorphism analysis of rRNA genes. The resulting data show that gut bacterial diversity of both cricket species is positively correlated with biodiversity according to habitat type. This demonstrates that microbial diversity within insect gastrointestinal tracts, and possibly their functions within these insects, is tied to the biodiversity within the habitats where insects live. These results have important implications as to how reductions in habitat biodiversity may affect the ecological functions and services that the remaining species can perform.

Assessment of Toll-like Receptors in the Ileum of Weanling Pigs- Responses to Feed Antibiotic Chlortetracycline and Gnotobiotic Conditions

It has been suggested that changes in diet at weaning in pigs induce intestinal inflammation which may be mediated through toll-like receptors (TLRs). We hypothesized that the use of antibiotics as growth promoters and subsequent changes in intestinal microbiota may mediate changes in the expression of TLRs in the intestine. Thus, this study was performed to assess the changes in intestinal TLRs in weanling pigs in response to the use of chlortetracycline as a growth promotant, and under gnotobiotic conditions. Eighteen cesarean-derived half-sib piglets were divided into three groups; antibiotic-fed, control (normal-fed) and gnotobiotic groups.TLR-2, -4, -5 and -9 gene expression and abundance of TLR-2 and -9 proteins in the ileum of pigs was assessed at 5 wk of age. No significant differences (p ?0.5) in TLR-2, -4, -5 and -9transcript levels and abundanceof TLR-2 and -9 proteins among three groups of pigswere observed.