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Use of microbiomics to study human impacts on complex microbial communities
Södertörn University, School of Life Sciences. Karolinska Institutet.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The study of bacterial communities in nature is currently a challenge. The majority of bacteria in clinical and environmental samples have not yet been cultured and therefore we cannot fully understand their roles in nature and how the ecological balance in a specific microbial ecosystem can be disrupted. For example, exposure to pollutants in soil and antibiotics in the human gut can have large consequences on microbial populations but the magnitude of these impacts is difficult to assess. In this thesis, a combination of molecular techniques, microbiomics, were used to assess complex microbial communities in soil and the human gut. One goal of this thesis was to study the impact of the toxic compound, 4-chlorophenol, on the soil microbiota. In addition, a specific 4-chlorophenol degrading bacterium, Arthrobacter chlorophenolicus, was monitored in soil. In order to monitor the cells they were chromosomally tagged with marker genes encoding either the green fluorescent protein (the gfp gene) or firefly luciferase (the luc gene). During degradation of high levels of 4-chlorophenol in soil, total cells counts of A. chlorophenolicus cells could be measured by flow cytometry (GFP protein) and the metabolic activity could be measured by lurninometry (luciferase activity). In addition, the relative abundance of A. chlorophenolicus in soil could be measured by terminal restriction fragment length polymorphism (T-RFLP) and a higher relative abundance was detected in soil contaminated with 4chlorophenol compared with non-treated soil. The impacts of 4-chlorophenol and A. chlorophenolicus on the dominant members of the soil microbiota were also assessed by T-RFLP. Another goal of this thesis was to study the impact of a short term antibiotic administration in a long term perspective, using either clindamycin, in a two year study or a triple therapy for eradication of Helicobacter pylori containing clarithromycin and metronidazole, in a four year study, on the human fecal microbiota. Both the total bacterial community and specific populations, i.e. Bacteroides spp. and Enterococcus spp., were monitored by T-RFLP. The Bacteroides populations never returned to their pre-treatment composition after clindamycin exposure during the two year study period. Selection and persistence of resistant Bacteroides clones up to two years after treatment was furthermore detected. In the four year study, Enterococcus populations increased as a response to the clarithromycin and metronidazole treatment. An increase in the levels of antibiotic resistance genes, specific erm genes, conferring resistance to macrolides and lincosamides were detected for up to 2 and 4 years after both types of antibiotic treatments in the respective studies. It was also possible to specifically monitor two probiotic Lactobacillus strains and their transient colonization by T-RFLP. In conclusion, the use of a polyphasic approach with complementary analytical tools made it possible to obtain a comprehensive picture of complex microbial communities. In addition, specific bacteria of interest in complex soil and fecal samples could be monitored using microbiomics approaches.

Place, publisher, year, edition, pages
Stockholm: Karolinska instiutet , 2006. , 37 p.
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:sh:diva-31990ISBN: 91-7140-960-2 (print)OAI: oai:DiVA.org:sh-31990DiVA: diva2:1072771
Public defence
2006-12-15, MB416, Alfred Nobels allé 7, Huddinge, 10:00 (English)
Opponent
Supervisors
Available from: 2017-02-08 Created: 2017-02-08 Last updated: 2017-02-08Bibliographically approved
List of papers
1. Use of green fluorescent protein and luciferase biomarkers to monitor survival and activity of Arthrobacter chlorophenolicus A6 cells during degradation of 4-chlorophenol in soil
Open this publication in new window or tab >>Use of green fluorescent protein and luciferase biomarkers to monitor survival and activity of Arthrobacter chlorophenolicus A6 cells during degradation of 4-chlorophenol in soil
2001 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 3, no 1, 32-42 p.Article in journal (Refereed) Published
Abstract [en]

The recently isolated novel species Arthrobacter chlorophenolicus A6 is capable of growth on and degradation of high concentrations of 4-chlorophenol (up to 350 mug ml(-1)) as the sole carbon and energy source, This strain shows promise for bioremediation of environmental sites contaminated with high levels of chlorophenols. In this study, green fluorescent protein (gfp) or luciferase (luc) genes were used as biomarkers for monitoring cell number and activity, respectively, during degradation of 4-chlorophenol by A. chlorophenolicus cells. The individual marked strains, Arthrobacter chlorophenolicus A6L (luc-tagged) and Arthrobacter chlorophenolicus A6G (gfp-tagged), were monitored during degradation of 250 mug ml(-1) 4-chlorophenol in pure culture and 175 mug g(-1) 4-chlorophenol in soil microcosms. Both gene-tagged strains were capable of cleaning up the contaminated soil during 9 d incubation. During the bioremediation experiments, the luc-tagged cells were monitored using luminometry and the gfp tagged cells using flow cytometry, in addition to selective plate counting for both strains. The cells remained at high population levels in the soil (evidenced by GFP-fluorescent cell counts) and the A. chlorophenolicus A6L population was metabolically active (evidenced by luciferase activity measurements). These results demonstrate that the Arthrobacter chlorophenolicus A6 inoculum is effective for cleaning-up soil containing high concentrations of 4-chlorophenol.

National Category
Microbiology
Identifiers
urn:nbn:se:sh:diva-17374 (URN)10.1046/j.1462-2920.2001.00156.x (DOI)000167056100004 ()11225721 (PubMedID)2-s2.0-0035202047 (ScopusID)
Available from: 2012-11-22 Created: 2012-11-19 Last updated: 2017-02-15Bibliographically approved
2. Impact of 4-chlorophenol contamination and/or inoculation with the 4-chlorophenol-degrading strain, Arthrobacter chlorophenolicus A6L, on soil bacterial community structure
Open this publication in new window or tab >>Impact of 4-chlorophenol contamination and/or inoculation with the 4-chlorophenol-degrading strain, Arthrobacter chlorophenolicus A6L, on soil bacterial community structure
2002 (English)In: FEMS Microbiology Ecology, ISSN 0168-6496, E-ISSN 1574-6941, Vol. 42, no 3, 387-97 p.Article in journal (Refereed) Published
Abstract [en]

The 4-chlorophenol-degrading strain, Arthrobacter chlorophenolicus A6L (chromosomally tagged with the firefly luciferase gene, luc) was inoculated into 4-chlorophenol-contaminated soil to assess the impact of bioaugmentation with a biodegrading strain on the indigenous microbiota. Simultaneously, the impact of 4-chlorophenol alone, or inoculation with A. chlorophenolicus into non-contaminated soil, was addressed. Using terminal restriction fragment length polymorphism (T-RFLP) several significant changes were detected in community fingerprint patterns obtained from soil microcosms treated under the different conditions. The relative abundances of some populations, as judged by the relative intensity of terminal restriction fragments, were significantly impacted by either 4-chlorophenol, A. chlorophenolicus inoculation, or by a combination of both inoculation and 4-chlorophenol contamination. Some populations were significantly stimulated and others were significantly repressed when compared to control soil with no additions. For several peaks, the positive or negative impact imposed by the treatments increased over the 13-day incubation period. Some members of the bacterial community were specifically sensitive to A. chlorophenolicus inoculation or to 4-chlorophenol contamination, whereas other populations remained relatively unaffected by any of the treatments. The A. chlorophenolicus inoculum was also monitored by T-RFLP and was found to have a significantly higher relative abundance in soil contaminated with 4-chlorophenol. These results were substantiated by a high correlation to luciferase activity measurements and the number of colony forming units of the inoculum. Therefore, the A. chlorophenolicus A6L population was positively stimulated by the presence of the 4-chlorophenol substrate (180 microg g(-1) soil) that it catabolized during the first 8 days of the incubation period as a carbon and energy source. Together, these results demonstrate that specific populations in the soil bacterial community rapidly fluctuated in response to specific disturbances and the resulting shifts in the community may therefore represent an adjustment in community structure favoring those populations best capable of responding to novel stress scenarios.

National Category
Microbiology
Identifiers
urn:nbn:se:sh:diva-17639 (URN)10.1111/j.1574-6941.2002.tb01028.x (DOI)000179545100007 ()19709298 (PubMedID)2-s2.0-0036890005 (ScopusID)
Available from: 2012-12-14 Created: 2012-12-14 Last updated: 2017-02-08Bibliographically approved
3. Monitoring of antibiotic-induced alterations in the human intestinal microflora and detection of probiotic strains by use of terminal restriction fragment length polymorphism
Open this publication in new window or tab >>Monitoring of antibiotic-induced alterations in the human intestinal microflora and detection of probiotic strains by use of terminal restriction fragment length polymorphism
2005 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 71, no 1, 501-506 p.Article in journal (Refereed) Published
Abstract [en]

Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.

National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-17365 (URN)10.1128/AEM.71.1.501-506.2005 (DOI)000226458800062 ()15640226 (PubMedID)2-s2.0-12244292700 (ScopusID)
Available from: 2012-11-22 Created: 2012-11-19 Last updated: 2017-02-08Bibliographically approved
4. Long-term ecological impacts of antibiotic administration on the human intestinal microbiota
Open this publication in new window or tab >>Long-term ecological impacts of antibiotic administration on the human intestinal microbiota
2007 (English)In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 1, no 1, 56-66 p.Article in journal (Refereed) Published
Abstract [en]

Antibiotic administration is known to cause short-term disturbances in the microbiota of the human gastrointestinal tract, but the potential long-term consequences have not been well studied. The aims of this study were to analyse the long-term impact of a 7-day clindamycin treatment on the faecal microbiota and to simultaneously monitor the ecological stability of the microbiota in a control group as a baseline for reference. Faecal samples from four clindamycin-exposed and four control subjects were collected at nine different time points over 2 years. Using a polyphasic approach, we observed highly significant disturbances in the bacterial community that persisted throughout the sampling period. In particular, a sharp decline in the clonal diversity of Bacteroides isolates, as assessed by repetitive sequence-based PCR (rep-PCR) and long-term persistence of highly resistant clones were found as a direct response to the antibiotic exposure. The Bacteroides community never returned to its original composition during the study period as assessed using the molecular fingerprinting technique, terminal restriction fragment length polymorphism (T-RFLP). Furthermore, using real-time PCR we found a dramatic and persistent increase in levels of specific resistance genes in DNA extracted from the faeces after clindamycin administration. The temporal variations in the microbiota of the control group were minor compared to the large and persistent shift seen in the exposed group. These results demonstrate that long after the selection pressure from a short antibiotic exposure has been removed, there are still persistent long term impacts on the human intestinal microbiota that remain for up to 2 years post-treatment.

Keyword
Bacteroides, clindamycin, rep-PCR, faeces, T-RFLP
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31968 (URN)10.1038/ismej.2007.3 (DOI)000249215800009 ()18043614 (PubMedID)2-s2.0-34248152283 (ScopusID)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Erratum: Jernberg, C., Löfmark, S., Edlund, C., & Jansson, J. K. (2013). Erratum: Long-term ecological impacts of antibiotic administration on the human intestinal microbiota (ISME journal (2007) 1 (56-66) DOI: 10.1038/ismej.2007.3). ISME Journal, 7(2), 456. doi:10.1038/ismej.2012.91

Available from: 2017-02-07 Created: 2017-02-06 Last updated: 2017-02-08Bibliographically approved
5. Clindamycin-induced enrichment and long-term persistence of resistant Bacteroides spp. and resistance genes
Open this publication in new window or tab >>Clindamycin-induced enrichment and long-term persistence of resistant Bacteroides spp. and resistance genes
2006 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 58, no 6, 1160-1167 p.Article in journal (Refereed) Published
Abstract [en]

Objectives: The aim was to study the long-term consequences of 1 week clindamycin administration regarding selection and persistence of resistance, resistance determinants and diversity of the Bacteroides spp. in the intestinal microflora. Methods: A total of 1306 Bacteroides isolates were collected from constitutively cultured faecal samples during a 2 year period from eight healthy volunteers. The strains were identified by biochemical and genotyping methods. MIC values were determined by the agar dilution method and presence of resistance genes was screened by real-time PCR. Results: Ecological changes in the intestinal microflora persisting up to 24 months were recorded after a 7 day clindamycin administration to four healthy volunteers. Compared to a control group, not exposed to clindamycin, an enrichment and stabilization of resistant Bacteroides strains and resistance determinants were discovered up to 2 years after clindamycin exposure. Conclusions: The results indicate that even a short-term antibiotic administration can cause long-term alterations in the commensal microbiota of individual subjects, detectable 2 years after dosing. The recorded selection and persistence of resistant strains and resistance genes, illustrates the importance of increasing our knowledge of the role of the abundant intestinal microbial community as a reservoir for spread of resistance.

National Category
Microbiology Pharmacology and Toxicology Pharmaceutical Sciences
Identifiers
urn:nbn:se:sh:diva-14266 (URN)10.1093/jac/dkl420 (DOI)000242716600009 ()17046967 (PubMedID)2-s2.0-33845349674 (ScopusID)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2011-12-21 Created: 2011-12-20 Last updated: 2017-02-08Bibliographically approved
6. Molecular analysis of ecological changes in the human normal microflora after treatment with clarithromycin and metronidazole
Open this publication in new window or tab >>Molecular analysis of ecological changes in the human normal microflora after treatment with clarithromycin and metronidazole
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-31989 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2017-02-08 Created: 2017-02-08 Last updated: 2017-02-08Bibliographically approved

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  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
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  • Other locale
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Output format
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