Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis: = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
2000
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2000 |
| Beschreibung: | IV, 148 Bl. Ill., graph. Darst. |
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| 100 | 1 | |a Rachid, Shwan |d 1968- |e Verfasser |0 (DE-588)122380886 |4 aut | |
| 245 | 1 | 0 | |a Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis |b = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis |c von Shwan Rachid |
| 246 | 1 | 1 | |a Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis |
| 264 | 1 | |c 2000 | |
| 300 | |a IV, 148 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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| 338 | |b nc |2 rdacarrier | ||
| 500 | |a Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2000 | ||
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| 650 | 0 | 7 | |a Biofilm |0 (DE-588)4232790-8 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Umweltfaktor |0 (DE-588)4124348-1 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Staphylococcus epidermidis |0 (DE-588)4318476-5 |2 gnd |9 rswk-swf |
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Datensatz im Suchindex
| _version_ | 1812460946751225856 |
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| adam_text |
TABLE OF CONTENTS
TABLE OF CONTENTS
LA. SUMMARY.1
LB. ZUSAMMENFASSUNG.3
II. INTRODUCTION.5
1.
STAPHYLOCOCCUS AUREUS
AS A HUMAN PATHOGEN.5
2. NOSOCOMIAL INFECTIONS BY COAGULASE NEGATIVE
STAPHYLOCOCCUS
(CNS).9
2.1 BIOFILM PRODUCTION IN 5.
EPIDERMIDIS
.10
2.1.1 REGULATION OF THE VIRULENCE FACTORS IN
S. EPIDERMIDIS
BY THE AGR SYSTEM.13
3. ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCI.15
III. MATERIALS.18
1. BACTERIAL STRAINS.18
2. PLASMIDS AND GENE PROBES.19
3. OLIGONUCLEOTIDES.20
4. CHEMICALS.21
5. EQUIPMENTS.22
6. MEDIA AND SUPPLEMENTS.23
7. ANTIBIOTICS.28
8. BUFFER AND SOLUTIONS.29
*
8.1. ELECTROPHORESIS RUNNING BUFFERS.29
IV. METHODS.30
1. ISOLATION OF PLASMID DNA.30
1.1 SMALL-SCALE PREPERATION OF PLASMID DNA.30
1.2 LARGE-SCALE PREPARATION OF PLASMID DNA.31
2. PHENOL TREATMENT OF DNA PERPERATION.32
3. ETHANOL PRECIPITATION OF DNA.32
4. DNA AND RNA QUANTITATION.32
5. DIGESTION OF PLASMID -DNA WITH RESTRICTION ENZYMES.33
6. AGAROSE GEL ELECTROPHORESIS FOR SEPARATION OF DNA FRAGMENTS.33
6.1 DNA-SIZE MARKER (MBI).34
7. ELUTION OF DNA FRAGMENTS FROM AGAROSE GELS (GENE CLEAN-KIT).35
8. DEPHOSPHORYLATION OF DNA FRAGMENTS. 35
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/959882278
TABLE OF CONTENTS
9. PRODUCTION OF BLUNT END DNA FRAGMENTS.36
10. DNA LIGATION.36
11. PREPARATION OF COMPETENT BACTERIAL CELLS.36
11.1 PREPARATION OF COMPETENT BACTERIAL CELLS FOR TRANSFORMATION BY THE
CACU PROCEDURE.36
11.2 PREPARATION OF COMPETENT BACTERIAL CELLS FOR TRANSFORMATION BY
ELECTROPORATION.37
11.3 PREPARATION OF
STAPHYLOCOCCUS COMPETENT CELLS.37
11.4 PREPARATION OF
BACILLUS SUHTILIS COMPETENT CELLS.37
12. TRANSFORMATION OF COMPETENT CELLS.38
12.1 TRANSFORMATION BY THE CACL; PROCEDURE.38
12.2 TRANSFORMATION BY THE ELECTROPORATION METHOD.38
12.3 TRANSFORMATION OF
BACILLUS SUBTILIS COMPETENT CELLS.39
13. DNA SEQUENCING.39
14. POLYMERASE CHAIN REACTION (PCR).40
15. CLONING OF THE PCR-FRAGMENTS.40
15.1 "SURECLONE
1" LIGATION KIT.40
15.2 PGEM-T EASY VECTOR SYSTEM.41
16. ISOLATION OF
STAPHYLOCOCCUS CHROMOSOMAL DNA.42
17. SOUTHERN-HYBRIDIZATION.42
18. LABELLING OF THE GENE PROBE.43
18.1 RADIOACTIVE LABELLING METHOD.43
18.2 NON RADIOACTIVE LABELLING METHOD "ECL-KIT".44
19. ISOLATION OF TOTAL RNA FROM
STAPHYLOCOCCUS CELLS.45
19.1 RNA ISOLATION BY "RNEASY
YY
"-KIT.45
19.2 RNA ISOLATION BY "FASTRNA"-KIT.45
20. NORTHERN HYBRIDIZATION ANALYSIS.46
20.1 RNA GEL ELECTROPHORESIS.46
20.2 RNA-BLOTTING.47
21. P-GALACTOSIDASE-TEST "GALACTO-LIGHT
YY
"-KIT.48
22. DETERMINATION OF MINIMAL INHIBITORY CONCENTRATION OF ANTIBIOTICS
(MIC).49
23. QUANTITATIVE ADHERANCE ASSAY.49
24. STATISTICS.50
V. RESULTS.51
1. CONSTRUCTION OF A P
ICA-LACZ FUSION AND INTEGRATION IN THE CHROMOSOME OF
S. EPIDERMIDIS
220.51
1.1 CLONING OF THE PROMOTOR REGION OF
ICA GENE AND FUSION WITH P-GALACTOSIDASE
GENE FROM E. COLI.51
II
TABLE OF CONTENTS
1.2 INTEGRATION OF THE P
LCA-LACZ
FUSION IN THE CHROMOSOME OF 5.
EPIDERMIDIS
220.53
1.3 SOUTHERN HYBRIDIZATION TO DETERMINE THE CORRECT INTEGRATION.54
1.4 SEQUENCE ANALYSIS OF P
1(RFL AND
LACZ
IN THE RECOMBINANT STRAIN 220-1.55
2. EFFECT OF ENVIRONMENTAL FACTORS ON THE
ICA
EXPRESSION.56
2.1 EXPRESSION OF THE P
,CA-/ACZ DURING THE GROWTH PHASE IN
S. EPIDERMIDIS
220-1.57
2.2 EFFECT OF THE INCUBATION TEMPERATURE ON THE
?KI-LACZ
EXPRESSION IN
5.
EPIDERMIDIS
220-1.57
2.3 EFFECT OF PH ON THE P
KI-LACZ
EXPRESSION IN 5.
EPIDERMIDIS
220-1.58
2.4 EFFECT OF THE OSMOLARITY ON THE
ICA
EXPRESSION IN S.
EPIDERMIDIS
.59
2.4.1 EFFECT OF THE OSMOLARITY ON THE
?K -LACZ
EXPRESSION IN
S. EPIDERMIDIS
220-1.59
2.4.2 EFFECT OF THE OSMOLARITY ON THE BIOFILM PRODUCTION IN
S. EPIDERMIDIS
220 WILD TYPE STRAIN.61
2.4.3 EFFECT OF THE OSMOLARITY ON THE
ICA
TRANSCRIPTION IN S.
EPIDERMIDIS
220.62
2.5 EFFECT OF DIFFERENT CARBON SOURCES ON THE
ICA
EXPRESSION IN
S. EPIDERMIDIS
.63
2.5.1 EFFECT OF CARBON SOURCES ON THE
?KI-LACZ
EXPRESSION.63
2.5.2 EFFECT OF GLUCOSE ON THE BIOFILM PRODUCTION IN
S. EPIDERMIDIS
220.65
2.5.3 INFLUENCE OF GLUCOSE ON THE
ICA
TRANSCRIPTION IN
S. EPIDERMIDIS
220.66
2.6
EFFECT OF ETHANOL, HJO:, SDS AND UREA ON THE BIOFILM PRODUCTION IN
S. EPIDERMIDIS 2
15.66
2.7 IDENTIFICATION OF THE
SIGB
GENE IN
S. EPIDERMIDIS
AND ITS EXPRESSION IN
RESPONSE TO THE
BIOFILM INDUCING CONDITIONS.69
3. INFLUENCE OF SUBINHIBITORY CONCENTRATIONS OF ANTIBIOTICS ON THE
ICA
EXPRESSION.71
3.1 INFLUENCE OF PRISTINAMYCIN (QUINUPRISTIN/DALFOPRISTIN),TETRACYCLINE
AND
VIRIGINIAMYCIN ON THE BIOFILM PRODUCTION.72
3.1.1 INFLUENCE OF PRISTINAMYCIN AND TETRACYCLINE ON THE P
KT-LACZ
EXPRESSION.72
3.1.2 INFLUENCE OF PRISTINAMYCIN AND TETRACYCLINE ON THE BIOFILM
PRODUCTION IN
S. EPIDERMIDIS
220.75
3.1.3 INFLUENCE OF PRISTINAMYCIN AND TETRACYCLINE ON THE
ICA
TRANSCRIPTION.77
3.1.4 POST-EXPOSURE EFFECTS OF PRISTINAMYCIN AND TETRACYCLINE ON THE P
LCA-LACZ
EXPRESSION IN S. EPIDERMIDIS
220-1.77
3.1.5 POST-EXPOSURE EFFECTS OF PRISTINAMYCIN, TETRACYCLINE, AND
DALFOPRISTIN AND
QUINUPRISTIN SEPARATELY, ON THE BIOFILM PRODUCTION IN
S. EPIDERMIDIS
220.78
3.1.6. INFLUENCE OF SUBINHIBITORY CONCENTRAIONS OF VIRIGINIAMYCIN ON THE
ICA
EXPRESSION IN 5.
EPIDERMIDIS
220.79
3.2 INFLUENCE OF OTHER PROTEIN INHIBITORS ON THE P
,CL-LACZ
EXPRESSION IN
S. EPIDERMIDIS
220-1 .81
3.3 INFLUENCE OF CELL WALL AND DNA-GYRASE INHIBITOR ANTIBIOTICS ON THE
ICA
EXPRESSION.82
3.3.1 INFLUENCE OF CELL WALL AND DNA-GYRASE INHIBITOR ANTIBIOTICS ON THE
P
,YY-LACZ
EXPRESSION
IN 5.
EPIDERMIDIS
220-1.82
III
TABLE OF CONTENTS
3.3.2 INFLUENCE OF PENICILLIN ON THE BIOFTLM PRODUCTION IN
S. EPIDERMIDIS 220 WILD TYPE STRAIN.84
4. THE ROLE OF THE ACCESSORY GENE REGULATOR (
AGR) GENE IN THE REGULATION OF THE BIOFTLM
PRODUCTION IN 5. EPIDERMIDIS.86
4.1 EFFECT OF THE OSMOLARITY ON THE RNAIII TRANSCRIPTION.87
4.2 CONSTRUCTION OF AN AGR MUTANT IN 5.
EPIDERMIDIS 567.87
4.3 INACTIVATION OF THE
AGR GENE IN S. EPIDERMIDIS 567.89
4.4 SOUTHEN BLOT ANALYSIS TO DETERMINE THE
AGR GENE REPLACEMENT IN S. EPIDERMIDIS 567-1.90
4.5 SEQUENCE ANALYSIS OF THE
AGR.ERMB SITE IN THE RECOMBINANT STRAIN 567-1.90
4.6 BIOFTLM PRODUCTION IN THE
AGR MUTANT S. EPIDERMIDIS 567-1.91
5. THE ROLE OF THE ALTERNATIVE SIGMA FACTOR (
SIGB) IN THE REGULATION OF BIOFTLM PRODUCTION.92
5.1. CONSTRUCTION OF A
SIGB KNOCK OUT MUTANT.92
5.1.1 CONSTRUCTION OF A
SIGB KNOCK OUT MUTATOR PLASMID PSK8.94
5.1.2 ISOLATION OF THE
SIGB KNOCK OUT MUTANTS.7T.94
5.1.3 PCR ANALYSIS OF THE
SIGB KNOCK OUT MUTANT.95
5.1.4 SOUTHERN AND BLOT ANALYSIS OF THE
SIGB MUTANT.96
5.1.5 EVALUATION OF THE
SIGB DEPENDENT ASPLZ EXPRESSION.96
5.2 COMPLEMENTATION OF THE
SIGB MUTANT S. AUREUS MA12.2 WITH A FUNCTIONAL SIGB.97
5.3 INFLUENCE OF THE
SIGB INACTIVATION ON THE ICA EXPRESSION IN S. AUREUS MAI 2.2.98
VI. DISCUSSION.
1()-
1. BIOFILM EXPRESSION IS GROWTH DEPENDENT AND CONTROLLED BY
ENVIRONMENTAL FACTORS.103
2. ALTERATION OF THE
ICA FEXPRESSION BY SUBINHIBITORY CONCENTRATION OF ANTIBIOTICS.110
3. THE ROLE OF THE AGR-QUORUM SENSING SYSTEM IN THE REGULATION OF THE
ICA OPERON
EXPRESSION IN
S. EPIDERMIDIS.113
4. THE ROLE OF THE ALTERNATIVE TRANSCRIPTION FACTOR
SIGB IN THE S. AUREUS BIOFTLM PRODUCTION. 114
VII. REFERENCES.I*
7
VIII. APPENDIX.13
7
1. RESTRICTION MAP OF THE PLASMIDS USED IN THIS STUDY.137
2. CRONYMS AND ABBREVIATIONS.142
3. PUBLICATIONS AND PRESENTATIONS.145
4A. CURRICULUM VITAE.147
4B. LEBENSLAUF.148
IV |
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| id | DE-604.BV013221805 |
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| indexdate | 2024-10-09T18:10:52Z |
| institution | BVB |
| language | English |
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| physical | IV, 148 Bl. Ill., graph. Darst. |
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| spelling | Rachid, Shwan 1968- Verfasser (DE-588)122380886 aut Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis von Shwan Rachid Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis 2000 IV, 148 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Zsfassung in dt. Sprache. - Würzburg, Univ., Diss., 2000 Antibiotikum (DE-588)4002257-2 gnd rswk-swf Biofilm (DE-588)4232790-8 gnd rswk-swf Umweltfaktor (DE-588)4124348-1 gnd rswk-swf Staphylococcus epidermidis (DE-588)4318476-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Staphylococcus epidermidis (DE-588)4318476-5 s Biofilm (DE-588)4232790-8 s Umweltfaktor (DE-588)4124348-1 s DE-604 Antibiotikum (DE-588)4002257-2 s DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009009433&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rachid, Shwan 1968- Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis Antibiotikum (DE-588)4002257-2 gnd Biofilm (DE-588)4232790-8 gnd Umweltfaktor (DE-588)4124348-1 gnd Staphylococcus epidermidis (DE-588)4318476-5 gnd |
| subject_GND | (DE-588)4002257-2 (DE-588)4232790-8 (DE-588)4124348-1 (DE-588)4318476-5 (DE-588)4113937-9 |
| title | Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis |
| title_alt | Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis |
| title_auth | Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis |
| title_exact_search | Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis |
| title_full | Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis von Shwan Rachid |
| title_fullStr | Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis von Shwan Rachid |
| title_full_unstemmed | Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis von Shwan Rachid |
| title_short | Molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in Staphylococcus epidermidis |
| title_sort | molecular investigations of the influence of environmental factors and subinhibitory antibiotic concentrations on the biofilm formation in staphylococcus epidermidis molekulare untersuchungen zum einfluß von umweltfaktoren und subinhibitorischen antibiotikakonzentrationen auf die biofilmbildung in staphylococcus epidermidis |
| title_sub | = Molekulare Untersuchungen zum Einfluß von Umweltfaktoren und subinhibitorischen Antibiotikakonzentrationen auf die Biofilmbildung in Staphylococcus epidermidis |
| topic | Antibiotikum (DE-588)4002257-2 gnd Biofilm (DE-588)4232790-8 gnd Umweltfaktor (DE-588)4124348-1 gnd Staphylococcus epidermidis (DE-588)4318476-5 gnd |
| topic_facet | Antibiotikum Biofilm Umweltfaktor Staphylococcus epidermidis Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=009009433&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT rachidshwan molecularinvestigationsoftheinfluenceofenvironmentalfactorsandsubinhibitoryantibioticconcentrationsonthebiofilmformationinstaphylococcusepidermidismolekulareuntersuchungenzumeinflußvonumweltfaktorenundsubinhibitorischenantibiotikakonzentrationenaufdiebiof AT rachidshwan molekulareuntersuchungenzumeinflußvonumweltfaktorenundsubinhibitorischenantibiotikakonzentrationenaufdiebiofilmbildunginstaphylococcusepidermidis |