Christopher L. Kitts

Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities

Microbial populations in complex environmental samples are difficult to characterize; current techniques are incomplete and time consuming. We investigated a polymerase chain reaction (PCR)-based method for rapidly comparing bacterial communities independent of culture or cloning. Community 16S rRNA genes were amplified and fluorescently labeled by PCR. The labeled products were digested by a restriction enzyme and the labeled, terminal restriction fragments (TRFs) were separated by electrophoresis and detected by laser-induced fluorescence on an automated gene sequencer. PCR parameters were optimized using an in vitro model community of known organisms. Community comparisons were made between deer fecal pellets, petroleum hydrocarbon-contaminated sands and pristine sand. Principal components analysis (PCA) was used to compare the resulting TRF patterns (TRFPs). Patterns derived from a single enzyme digest did not result in accurate community characterizations. Accurate characterizations reflecting the expected bacterial community biology were only achieved by combining TRFP data derived from different enzyme digestions. Suggestions are offered for future use of this technique.

Bacterial Succession in a Petroleum Land Treatment Unit

ABSTRACT Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C10 to C32 range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.

Removal of PCR Inhibitors From Soil DNA by Chemical Flocculation

Extracting high-purity DNA directly from soil has become essential for the study of microorganisms in environmental samples. However, many soils contain compounds that inhibit enzymes involved in manipulating DNA. In this study, chemical flocculation using multivalent cations was investigated as a potential method for eliminating soil-based inhibitors during the extraction process. The addition of AlNH(4)(SO(4))(2) during extraction significantly reduced the co-purification of PCR inhibitors with minimal loss of DNA yield.

Variation Between Observed and True Terminal Restriction Fragment Length is Dependent on True TRF Length and Purine Content

Terminal Restriction Fragment (TRF) pattern analysis has become a widely used and informative tool for studying microbial communities. Variation between sequence-determined or true TRF length and observed TRF length (TRF drift) has been previously reported and can significantly affect identification of bacterial species using TRF lengths predicted from sequence databases. In this study TRF drift was determined for 21 bacterial species using an ABI 310 Genetic Analyzer. TRF drift was positively correlated with true TRF length and negatively correlated with TRF purine content. This implies that subtle differences in molecular weight, whether from purine content or dye label, can significantly affect the observed TRF length.

Assessment of fungal diversity using terminal restriction fragment (TRF) pattern analysis: comparison of 18S and ITS ribosomal regions

Assessment of fungal diversity in environmental samples is currently a challenge. Several recently developed molecular methods offer new avenues for determining the presence and diversity of fungi in complex microbial communities. Terminal restriction fragment (TRF) pattern analysis was tested as a method for assessing the fungal molecular diversity of a terrestrial microbial community. Community DNA was isolated from sand samples taken from a pilot-scale petroleum-contaminated land treatment unit. PCR amplification was carried out using primers, one of which was fluorescently labeled, designed to hybridize to conserved sequences in the fungal ribosomal small subunit (18S) or the internal transcribed spacer ITS1-5.8S-ITS2 (ITS) ribosomal region. Amplicons were then digested separately with HpaII or HaeIII; fluorescently labeled TRFs were detected by capillary gel electrophoresis. ITS region TRF patterns were predicted and observed to generate a greater richness than 18S TRF patterns. Unique TRF patterns were also observed for each community examined. Finally, the ITS region showed a higher degree of specificity in matching observed TRF profiles to those generated from GenBank sequence data for species identification. These data suggest that ITS rDNA TRF pattern analysis has great potential as a rapid and specific method for fungal community analysis and species identification.

16S ribosomal DNA terminal restriction fragment pattern analysis of bacterial communities in feces of rats fed Lactobacillus acidophilus NCFM.

ABSTRACT 16S ribosomal DNA terminal restriction fragment patterns from rat fecal samples were analyzed to track the dynamics ofLactobacillus acidophilus NCFM and discern bacterial populations that changed during feeding with NCFM. Lactobacillus johnsonii and Ruminococcus flavefaciens were tentatively identified as such bacterial populations. The presence ofL. johnsonii was confirmed by isolation from feces.

Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.

Probiotics to minimize the disruption of faecal microbiota in healthy subjects undergoing antibiotic therapy

A novel combination of culturing and DNA-based terminal restriction fragment length polymorphism (TRFLP) analysis was used to investigate the effect of probiotics on antibiotic-induced gut microbiota alterations to determine if a probiotic preparation containing bifidobacteria and lactobacilli, taken during and after antibiotic therapy, can minimize antibiotic disturbance of faecal microbiota. Healthy subjects administered amoxicillin/clavulanate were randomized and concomitantly received a placebo or probiotic mixture. The primary end point was similarity of faecal microbiota as determined by culturing and TRFLP from subjects taking probiotics compared to those taking a placebo measured by comparing data from baseline to post-treatment for each subject. TRFLP analysis revealed a high subject to subject variation in the baseline faecal microbiota. The most common antibiotic-induced disturbance was a relative increase in Clostridium, Eubacterium, Bacteroides and Enterobacteraceae. The mean similarity to the baseline increased over time in both treatment groups, although the probiotic group was less disturbed according to both TRFLP and culture data. The culture method revealed that post-antibiotic faecal microbiota in probiotic-consuming subjects were more similar to the baseline microbiota than the control group (P=0.046). Changes in Enterobactereaceae (P=0.006) and Bifidobacterium (P=0.030) counts were significantly different between the groups. Analysis of TRFLP data reinforced the trend between groups but was not statistically significant (P=0.066). This study indicates this mixture of probiotics promotes a more rapid return to pre-antibiotic baseline faecal bacterial microbiota.

Diversity of Microorganisms Isolated from Amber

A bstractClaims that organisms can be cultured from amber, if substantiated, would be significant contributions to our understanding of the evolution, tenacity, and potential spread of life. Three reports on the isolation of organisms from amber have been published. Cano and Borucki recently reported the isolation of Bacillus sphaericus and Lambert et al. have described a new species designated Staphylococcus succinus from 25–40 million year old Dominican amber. These characterized organisms were phylogenetically distant from extant relatives and the Staphylococcus sp. sufficiently far removed from other extant staphylococci to be considered a new species. Here we report the culture of bacteria from Dominican and previously untested 120 million year old Israeli (Lebanese lode) amber. Twenty-seven isolates from the amber matrix have been characterized by fatty-acid profiles (FAME) and/or 16S rRNA sequencing. We also performed a terminal restriction fragment pattern (TRF) analysis of the original amber before prolonged culture by consensus primer amplification of the 16S rRNA followed by restriction enzyme digestion of the amplicons. Sample TRFs were consistent with a sparse bacterial assemblage and included at least five of the isolated organisms. Finally, we microscopically mapped the internal topography of an amber slice.

Distinct microbiome in pouchitis compared to healthy pouches in ulcerative colitis and familial adenomatous polyposis

Background: Pouchitis occurs in up to 50% of patients with ulcerative colitis (UC) undergoing ileal pouch anal anastomosis (IPAA). Pouchitis rarely occurs in patients with familial adenomatous polyposis (FAP) who undergo IPAA. Our aim was to compare mucosal and luminal flora in patients with UC‐associated pouchitis (UCP), healthy UC pouches (HUC), and healthy FAP pouches (FAP). Methods: Nineteen patients were enrolled in this cross‐sectional study (nine UCP, three HUC, seven FAP). Patients with active pouchitis were identified using the Pouchitis Disease Activity Index (PDAI). Ileal pouch mucosal biopsies and fecal samples were analyzed with a 16S rDNA‐based terminal restriction fragment length polymorphism (TRFLP) approach. Pooled fecal DNA from four UCP and four FAP pouches were sequenced for further speciation. Results: TRFLP data revealed statistically significant differences in the mucosal and fecal microbiota between each group of patients. UCP samples exhibited significantly more TRFLP peaks matching Clostridium and Eubacterium genera compared to HUC and FAP pouches and fewer peaks matching Lactobacillus and Streptococcus genera compared to FAP. DNA Sanger sequencing of a subset of luminal samples revealed UCP having more identifiable sequences of Firmicutes (51.2% versus 21.2%) and Verrucomicrobia (20.2% versus 3.2%), and fewer Bacteroidetes (17.9% versus 60.5%) and Proteobacteria (9.8% versus 14.7%) compared to FAP. Conclusions: The pouch microbial environment appears to be distinctly different in the settings of UC pouchitis, healthy UC, and FAP. These findings suggest that a dysbiosis may exist in pouchitis which may be central to understanding the disease. (Inflamm Bowel Dis 2010;)