Modified implant surfaces show different biofilm compositions under in vivo conditions.

OBJECTIVE: Plaque accumulation on implant surfaces can result in peri-implantitis with potential implant loss. The aim of the present study was to examine the influence of zirconium nitride (ZrN) as a potential implant surface on the biofilm composition and diversity in vivo. MATERIAL AND METHODS: ZrN- or titanium (Ti)-coated glass specimens and ZrN or roughened Ti discs were used as substrates. Pure glass and polished titanium served as controls. The specimens were mounted on removable intraoral splints in five adults. After 24 h of intraoral exposure, the biofilms were analyzed applying single-strand conformation polymorphism (SSCP analysis) of 16S rRNA genes. Sequence analysis of the dominant bands excised from the SSCP fingerprints allowed to taxonomically describe bacteria derived from biofilm samples. RESULTS: The highest number of bands was counted on pure glass and Ti 800. ZrN-coated glass and ZrN-coated titanium discs showed the lowest values for species richness. However, no significant differences were observed regarding the diversity of the identified bacterial species among all the surfaces examined. A total of 46 different bacteria were identified. The dominant bands within the fingerprints indicated bacteria belonging to the Streptococcus group as identified by their 16S rDNA sequence. CONCLUSION: A coating of glass surfaces with ZrN significantly reduced the species richness in early bacterial colonization but the diversity was not significantly changed. In consideration of the results obtained by this and former studies a ZrN coating appears to rather modify the quantity of early bacterial adherence than the quality of the microbial community structure.

Diversity of biphenyl degraders in a chlorobenzene polluted aquifer.

Biphenyl degrading bacteria (40 strains) have been isolated along a gradient of chlorobenzene pollution from an aquifer which did not contain any PCB to answer the question of how metabolic/catabolic abilities exist in ecosystems that have not been stressed with the relevant substrates is important for intrinsic bioremediations. Only few of the isolates were characterized by 16S rRNA gene sequence analyses as Pseudomonas species while the majority were Gram-positive, belonging to the order Actinomycetales and representing the genera Rhodococcus and Arthrobacter. The strains could grow on a variety of chlorobenzoates but no pattern of substrate usage and phylogeny or pollution gradient could be found. Strains which were able to grow on 2,5-dichlorobenzoate were often also able to use 3,4- and 3,5-dichloro- and 2,3,5-trichlorobenzoate or those using 2-chlorobenzoate could usually use 2,6-dichlorobenzoate as well. From that results, it is concluded that a highly diverse, basic metabolic activity for PCB degradation existed at this site despite the absence of PCB.

In vitro alterations of intestinal bacterial microbiota in fecal samples during storage.

The human gastrointestinal tract harbors an extremely diverse and complex microbial ecosystem. Most of the existent data about the enteric microflora have been generated using stool samples, but the collection and storage of fecal samples are often problematic. The influence of the storage of stool samples on the bacterial diversity and the degradation of bacterial DNA was analysed in this study. Stool samples from 5 healthy volunteers were exposed to different storage temperatures and durations. The bacterial diversity and the amount of intact bacterial DNA were analysed by single-stranded conformation polymorphism analysis (SSCP) and real-time polymerase chain reaction (PCR), both using a 16S rDNA approach. Additionally, biopsy specimens were taken from 3 of the 5 individuals to compare fecal and mucosal flora. The bacterial diversity of the fecal flora and the total number of bacteria were significantly reduced after 8 and 24 hours at both room temperature and 4 degrees C. The mucosa-associated bacterial microflora showed substantial differences compared with the fecal flora. The observed alterations of fecal flora during storage point to the difficulty of the molecular analysis of the bacterial diversity and the enumeration of bacterial cells in fecal samples.

Microbial indicator groups in acidic mining lakes.

The salt composition of 14 acidic lakes was analysed, and their microbial communities were compared by their 16S rDNA-based single-strand conformation polymorphism (SSCP) fingerprints. The lakes were grouped into three chemically distinct types, and operational taxonomic units (OTUs) were identified in the 16S rDNA SSCP fingerprints. Discriminant analysis between these groups using a multivariate approach showed that not a single organism, but rather a set of seven OTUs (Ferromicrobium acidophilum, Bacillus pumilus, Acidophilium organovorum, Paenibacillus chibensis, Acidocella sp. and two different alpha-Proteobacteria), was found to be indicative for a given lake type. We conclude that, for these lakes, not single indicator strains but indicator groups exist that are controlled by the ionic composition of the lakes.

Do different implant surfaces exposed in the oral cavity of humans show different biofilm compositions and activities?

Osseointegrated dental implants play an important role in restorative dentistry. However, plaque accumulation may cause inflammatory reactions around the implants, sometimes leading to implant failure. In this in vivo study the influence of two physical hard coatings on bacterial adhesion was examined in comparison with a pure titanium surface. Thin glass sheets coated with titanium nitride (TiN), zirconium nitride (ZrN) or pure titanium were mounted on removable intraoral splints in two adults. After 60 h of intraoral exposure, the biofilms were analyzed to determine the number of bacteria, the types of bacteria [by applying single-strand conformation polymorphism (SSCP analysis) of 16S rRNA genes], and whether or not the bacteria were active (by SSCP analysis of 16S rRNA). The results showed that bacterial cell counts were higher on the pure titanium-coated glass sheets than on the glass sheets coated with TiN or ZrN. The lowest number of bacterial cells was present on theZrN-coated glass. However, the metabolic activity (RNA fingerprints) of bacteria on TiN- and ZrN-coated glass sheets seemed to be lower than the activity of bacteria on the titanium-coated surfaces, whereas SSCP fingerprints based on 16S rDNA revealed that the major 16S bands are common to all of the fingerprints, independently of the surface coating.

Leitbakteria of microbial biofilm communities causing occlusion of biliary stents.

Biliary stents inserted to relieve obstructive jaundice caused by biliary or pancreatic malignancies inevitably become occluded by microbial growth in the form of diverse microbial community biofilms. The scarce information available on these communities is based on cultivation methods, but such methods usually provide distorted overviews of community composition, so commonalities and differences in biliary stent communities are uncertain. We extracted DNA and RNA from the microbial communities of 11 biliary stents explanted from nine patients in hospitals from two different countries, amplified 16S rRNA and rDNA sequences, analysed the amplicons by the single-strand conformation polymorphism (SSCP) method, and sequenced and deduced phylogenetic assignments of the major amplicons representing the major biofilm community members. We used a Modified Robbins Device (MRD) to study de novo development of a stent biofilm from a patient stent microbial community. Single-strand conformation polymorphism fingerprinting revealed the same six abundant bacterial species, here designated Leitbakteria, namely Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter aerogenes, and two unculturable bacteria distantly related to E. coli and Shigella sonnei, in all of the stent biofilm communities. In the experimental biliary stent system, a sequential colonization of the stent surface was observed, with P. aeruginosa being the pioneer colonizer, followed by K. pneumoniae and one of the unculturable Leitbakteria, followed by the remainder of the community. The overview of microbial biofilm communities of biliary stents gained by the use of culture-independent methods revealed new unculturable bacteria as major members of biliary stent biofilms, and the diversity of the abundant members of the stent biofilms is considerably lower than suggested from earlier studies based on cultivation methods, and that communities from different stents from different patients in different countries are remarkably similar and have similar major members, the stent Leitbakteria.

Chlorobenzene biodegradation under consecutive aerobic-anaerobic conditions.

The biodegradation of monochlorobenzene, the main contaminant in a quaternary aquifer at Bitterfeld, Central Germany, was studied in microcosm experiments employing either original groundwater or defined mineral media together with the indigenous microbial community from the polluted site. The impact of consecutive aerobic-anaerobic-aerobic incubations on monochlorobenzene biodegradation, microbial diversity, and pH development was examined. The related changes in microbial community composition were analyzed by 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of dominant bands and by quantitative analysis of bacterial respiratory chain quinones as biomarkers. Under aerobic conditions, the indigenous microbial community of the groundwater degraded monochlorobenzene mainly via the modified ortho-pathway. Respiratory chain quinones and SSCP analysis suggested dominance of the genera Acidovorax and Pseudomonas. A shift to anoxic conditions resulted in monochlorobenzene biotransformation but no dechlorination. The ability to degrade monochlorobenzene aerobically remained preserved throughout a fortnightly anoxic period at sufficiently high buffer capacity. Acidification, caused by monochlorobenzene biodegradation, was alkalinity-controlled. At low initial alkalinity a substantial decrease in pH, monochlorobenzene degradation, and total counts of live cells, accompanied by a change of the microbial community composition, was observed.

Simultaneous extraction from bacterioplankton of total RNA and DNA suitable for quantitative structure and function analyses.

The aim of this study was to develop a protocol for the simultaneous extraction from bacterioplankton of RNA and DNA suitable for quantitative molecular analysis. By using a combined mechanical and chemical extraction method, the highest RNA and DNA yield was obtained with sodium lauryl sarcosinate-phenol or DivoLab-phenol as the extraction mix. The efficiency of extraction of nucleic acids was comparatively high and varied only moderately in gram-negative bacterial isolates and bacterioplankton (RNA, 52 to 66%; DNA, 43 to 61%); significant amounts of nucleic acids were also obtained for a gram-positive bacterial isolate (RNA, 20 to 30%; DNA, 20 to 25%). Reverse transcription-PCR and PCR amplification products of fragments of 16S rRNA and its genes were obtained from all isolates and communities, indicating that the extracted nucleic acids were intact and pure enough for community structure analyses. By using single-strand conformation polymorphism of fragments of 16S rRNA and its gene, community fingerprints were obtained from pond bacterioplankton. mRNA transcripts encoding fragments of the enzyme nitrite reductase gene (nir gene) could be detected in a pond water sample, indicating that the extraction method is also suitable for studying gene expression. The extraction method presented yields nucleic acids that can be used to perform structural and functional studies of bacterioplankton communities from a single sample.