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«(Über die Bedeutung der bakteriellen Genomplastizität für die Adaptation und Evolution asymptomatischer Bakteriurie (ABU) Escherichia coli ...»

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One feature that was seen, is that fitness gains are initially rapid but tend to decelerate over time (Cooper and Lenski, 2000; de Visser and Lenski, 2002; Lenski et al., 1991). In a 10,000 generations experiment with 12 Escherichia coli populations, the morphology (cell size) and average fitness (measured in competition with the ancestor) evolved rapidly for the first 2,000 generations and were nearly static for the last 5,000 generations (Lenski and Travisano, 1994). Such dynamics indicate that populations, after being placed in a new environment, are evolving from a region of low fitness towards an adaptive peak or plateau. Evolutionary adaptation in experimental microbial populations typically occurs through the substitution of relatively few mutations that confer large benefits, as opposed to countless mutations with small benefits (Rozen et al., 2002). 12 replicate bacterial populations, although founded by the same clone, and evolving in identical environments, diverged from one another in their relative fitness, morphological features and performance in other environments (Korona et al., 1994; Lenski et al., 1991).

31   Introduction 

These experimental conditions, however, are very distinct to those in the urinary tract. The inflow of urine into the bladder (Coolsaet et al., 1980) rather resembles a continuous than a batch bacterial culture. Moreover, intra-individual differences of patients are very prominent (Rezzi et al., 2007). Similar to a ‘natural experiment’ that involves the colonization of several neighbouring islands by the same ancestor species demonstrated that subtle environmental differences promoted divergence (Darwin, 1872), the urinary tract of individual patients might be considered as the “Galapagos archipelago” for the evolution of ABU E. coli strains.

2.8. Aims of this work 

The role of multiple virulence-associated factors of uropathogenic E. coli involved in the development of symptomatic and chronic UTI has been elucidated so far, but only little information was available on characteristics of ABU isolates. Therefore one aim of this study was the detailed characterisation of clinical ABU isolates. The geno- and phenotypic diversity and relatedness of ABU isolates should be assessed using comparative genome hybridisation, pulsed-field gel electrophoresis and multi locus sequence typing. Moreover, virulence and fitness factors of this group of organisms should be further characterised and compared to those of UPEC and non-pathogenic E. coli strains.

Another aim was the assessment of bacterial adaptation and microevolution in the urinary tract using ABU strain 83972 as a model. Consecutive re-isolates of this strain derived from a deliberate human colonisation study and in vitro continuous culture should be analysed with regard to phenotypic and genomic alterations. In vitro transcriptome and proteome analysis should be performed to assess bacterial adaptation upon prolonged growth of strain 83972 in the urinary bladder.

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3.3. Oligonucleotides All oligonucleotides used for PCR, RT-PCR, gene disruption using the λ Red-based method (Datsenko and Wanner, 2000) were purchased from Sigma-Genosys (Steinheim, Germany).

The sequences and the application of all oligonucleotides are listed in (Table 4).

  Table 4: Oligonucleotides used in this study.

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3.4. Chemicals and enzymes  

All chemicals and enzymes used in this study were purchased from the following companies:

New England Biolabs (Frankfurt am Main), Invitrogen (Karlsruhe), MBI Fermentas (St.

Leon-Roth), Roche Diagnostics (Mannheim), Gibco BRL (Eggenstein), Dianova (Hamburg), Difco (Augsburg), Merck (Darmstadt), Oxoid (Wesel), GE Healthcare/Amersham Biosciences (Freiburg), Roth (Karlsruhe), Serva and Sigma-Aldrich (Taufkirchen), Axxora (Lörrach). Radionucleotides were purchased from GE Healthcare/Amersham Biosciences (Freiburg).

The following commercial kits were used:

- Plasmid Mini and Midi kit, QIAGEN (Hilden)

- PCR purification kit, QIAGEN (Hilden)

- Gel extraction kit, QIAGEN (Hilden)

- RNeasy kit, QIAGEN (Hilden)

- ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit, Applied Biosystems (Foster City, USA)

- ECLTM Direct Acid Labeling and Detection System, and ECLTM advance system, GE Healthcare/Amersham Biosciences (Freiburg)

- Roti-Nanoquant, Roth (Karlsruhe)

- OpArray Hybridization Buffer Kit, Operon (Cologne)

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3.5. Media, agar plates and antibiotics All media were autoclaved for 20 min at 120 °C, if not stated otherwise. Supplements for media and plates were sterile filtered through a 0.22 μm pore filter and added after cooling down the media to 50 °C.

3.5.1. Media   LB medium (Luria-Bertani): (Sambrook, 1989)

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Urine Human urine was collected at least from 10 healthy male and female volunteers, pooled and sterilized by filtration. Sterile urine was stored at 4 °C not longer than one week.

  3.5.2. Agar plates

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3.5.3. Antibiotics When appropriate, media and plates were supplemented with the antibiotics listed in the Table 5, in the indicated concentrations. Stock solutions were sterile filtered and stored at -20 °C until usage.

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3.5.4. DNA Markers   To determine the size of DNA fragments in agarose gels, the “GenerulerTM” 1-kb DNA ladder, purchased from MBI Fermentas, was used (Fig. 4A), whereas in PFGE the Lambda Ladder PFG Marker from New England BioLabs was used (Fig. 4B).

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Vaccum pump Univac Uniequipe Videoprinter Mitsubishi Hitachi, Cybertech Cb1 Vortex-Genie 2TM Scientific Industries Vortexer UV-Crosslinker BioRad Waterbath GFL 1083, Memmert                      

4. Methods

4.1. Working with DNA 4.1.1. Isolation of chromosomal DNA   Bacteria from 1 ml an overnight culture were harvested by centrifugation for 4 min at maximum speed in a table centrifuge. After washing with 1 ml TNE buffer, cells were centrifuged for 4 min and resuspended in 270 µl TNE-X buffer. 30 µl lysozyme (5 mg mlwere added and samples were incubated for 20 min at 37 °C. Afterwards, 15 µl proteinase K (20 mg ml-1) were added and further incubated up to 2 h at 65 °C until the solution became clear. The genomic DNA was precipitated by addition of 0.05 vol 5 M NaCl (15 µl) and 500 µl ice-cold ethanol. After short incubation on ice DNA was collected be centrifugation for 15 min. After washing two times with 1 ml 70% (v/v) ethanol, DNA pellets were air-dried and redissolved in 100 µl 100 dH2O (Clermont et al., 2000b).

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4.1.2. Precipitation of DNA with alcohol   DNA was either precipitated with ethanol or isopropanol. In the first case, 0.1 vol 3 M Na-acetate (pH 4.8) were added to the sample prior to the addition of 2.5 vol ice-cold 100 % (v/v) ethanol. For the precipitation with isopropanol, 0.7 vol were used. Samples were incubated at -80 °C before centrifugation (13,000 rpm, 4 °C, 20 min). The DNA pellet was washed with 70 % (v/v) ethanol, air-dried and resuspended in dH2O.

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4.1.3. Determination of nucleic acid concentration and quality control Nucleic acid concentrations were determined either using a standard spectrophotometer in quartz cuvettes with a diameter of 1 cm or using a NanoDrop® instrument where cuvettes  were not needed. In both cases, absorption at 260 nm of 1.0 corresponds to 50 µg ml-1 double stranded DNA or 40 µg ml-1 RNA. The purity of the preparations was determined by measurement of the absorption of the sample. DNA and RNA were considered sufficiently pure when the ratio A260 / A280 was higher than 1.8 or 2.0, respectively.

Additionally, RNA integrity was determined by capillary electrophoresis using an Agilent 2100 Bioanalyzer instrument.

4.1.4. Polymerase chain reaction (PCR)   This method allows the exponential amplification of DNA regions in vitro by using a heat stable DNA polymerase from Thermus aquaticus (Taq). This way, even small amounts of template DNA can be amplified to high copy numbers and easily visualized.

Standard PCR   For routine PCR amplification, Taq DNA polymerase kits of different suppliers (QIAGEN, Sigma, Invitrogen, Roche) were used. Usually the reaction was performed in a final volume of 20 µl.

Mix for one sample:

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The thermal cycling profile was designed according to the elongation temperature (depending on the supplier), annealing temperature of the individual primers and the

length of the expected amplification product:

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PCR with proof-reading polymerases   Either for site directed mutagenesis using PCR products or for sequencing, a different polymerase with 3’ → 5’ proof-reading activity was used in order to prevent misincorporations during extension. The composition of a typical PCR mix is given below.

Mix for one sample (DAP Goldstar polymerase; Eurogentec):

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Box PCR   The Box PCR is a PCR-based DNA fingerprinting technique for identification and discrimination of bacterial strains. Repetitive intergenic sequence elements are amplified and this results in a strain-specific DNA band pattern (fingerprint), what allows a direct strain to strain comparison. PCR products obtained by amplification wit Box A1 primer were separated by 1.5 % agarose gel electrophoresis for 2 h at 140 mA.

Mix for one sample:

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    Triplex PCR   The affiliation of E. coli isolates to the four main phylogenetic groups (ECOR A, B1, B2, and D) was based on triplex PCR (Clermont et al., 2000a). In a single PCR reaction two genes (chuA and yjaA) and an anonymous DNA fragment (TspE4.C2) were amplified (standard PCR assay) and seperated by 1 % agarose gel electrophoresis. ECOR group determination was done as shown in Fig. 5.

Fig. 5: A) Triplex PCR profiles specific for the four E. coli phylogenetic groups. Each combination of chuA and yjaA gene and DNA fragment TSPE4.C2 amplification allowed the determination of the phylogenetic group of a given strain; B) Lanes 1 and 2, ECOR group A; lane 3, ECOR group B1; lanes 4 and 5, ECOR group D; lanes 6 and 7, group ECOR B2. Lane M, DNA size marker.

Multiplex PCR 

In order to detect fitness- and virulence associated genes of extraintestinal pathogenic E.

coli, a multiplex PCR was used (Johnson and Stell, 2000). For this, 29 primer pairs according to their respective PCR product size were sorted in five pools (Fig. 6). After electrophoresis in a 2 % agarose gel, each of the PCR products was represented by a single DNA band.

Inverse PCR (IPCR) In order to amplify unknown DNA with the help of a small region of known DNA sequence, an inverse PCR was carried out. Amplification by IPCR of unknown DNA regions flanking a particular gene requires a previous digestion of genomic DNA and identification of the corresponding restriction fragment which contains a part of the known sequence as well as unknown flanking sequences by Southern hybridization. The size of the required fragment should not exceed 1 – 3 kb to facilitate amplification by PCR. Genomic DNA was then digested with the chosen restriction enzymes. The sample was diluted and the restriction fragments were ligated in order to obtain circular DNA fragments. PCR amplification was performed with primers complementary to the ones used for probe generation, using the circularized genomic DNA fragments as a template.

4.1.5. Sequence analysis   The nucleotide sequences of genomic DNA or plasmid constructs were determined using fluorescent dye terminators (ABI prism BigDye terminator kit, Applied Biosystems). The

sequencing-PCR mix for one sample was:

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The thermal cycling profile for the PCR reaction was: 40 cycles of denaturation at 96 °C for 30 s, annealing at 60 °C for 15 s, and extension at 60 °C for 4 min, followed by final extension at 60 °C for 2 min. Sequencing products were purified by ethanol precipitation and analyzed in a ABI prism sequencer (Perkin Elmer).

4.1.6. Multi locus sequence typing (MLST) seven house-keeping genes of being typed E coli strain were amplified. The PCR reaction contained 50 ng of chromosomal DNA, 20 pmol of each primer, 200 µmol (10 µl of a 2 mM solution) of the dNPTs, 10 µl of 10x PCR buffer, 5 units of proof-reading Taq polymerase and water to 100 µl. The thermal profile was as follows: 2 min at 95 °C, 30 cycles of 1 min at 95 °C, 1 min at annealing temp, 2 min at 72 °C followed by 5 min at 72 °C. Resulted PCR products were purified and subjected to sequencing.

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Sequences were analyzed using Vector NTI™ software. Sequence types (STs) were assigned using the E. coli MLST database hosted at the Max-Planck-Institute for Infection Biology (Berlin).

4.1.7. Isolation of plasmids   While using the QIAGEN Plasmid Midi and Mini Kit, bacteria were collected from 100 ml over night cultures by centrifugation (6,000 rpm, 4 °C, 15 min) and resuspended in 4 ml buffer P1, according to the manufacturer’s recommendations. After 5 min incubation at room temperature, 4 ml buffer P2 was added for lysis of the cells. After clearing of the suspension, 4 ml neutralization buffer P3 was added and samples were incubated for 10 min on ice. Cell debris and genomic DNA was removed by centrifugation (11,000 rpm, 4 °C, 30 min). Plasmid DNA containing supernatant was loaded on equilibrated columns by gravity flow. Columns were washed with buffer QC. Subsequently, plasmid DNA was eluted with 3.5 ml buffer QF and precipitated by addition of 0.7 vol isopropanol. After centrifugation (13,000 rpm, 4 °C, 20 min), DNA pellets were washed with 70 % (v/v) ethanol, air-dried and resuspended in water.

Plasmid isolation using the QIAspin mini kit were performed in a similar way with some modifications: bacteria were harvested from 1-10 ml over night cultures, buffer N3

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