Three different P. aeruginosa isolates from tracheobronchial secretions of patients with sepsis due to VAP were used; a) the P. aeruginosa 6–11–19 isolate with minimal inhibitory concentration (MIC) of amikacin, ceftazidime, ciprofloxacin and meropenem >256, >512, >16 and 8 μg/ml respectively; b) the P. aeruginosa 19–2–45 isolate 2 with MIC of amikacin, ceftazidime, ciprofloxacin and meropenem >256, >512, >128 and >256 μg/ml respectively; and c) the P. aeruginosa 20–1–30 isolate with MIC of amikacin, ceftazidime, ciprofloxacin and meropenem >256, >512, >64 and >64 μg/ml respectively. Ιsolates were genetically distinct as defined by pulsed-field gel electrophoresis (PFGE) of their DNA to exclude similar isolates coming from horizontal spread (S3A Fig). Additionally, a K. pneumoniae 87Β producing carbapenemase KPC-2 isolate with MIC of amikacin, meropenem, aztreonam and colistin >512, >512, 128 and 32 μg/ml respectively. The MIC was measured with the microdilution method and the production of KPC-2 by polymerase chain reaction.

We used 406 male and female mice (7–8 weeks old). Mice were allowed to acclimate for seven days before beginning the experiments. They were housed in individually ventilated cages, up to 5 mice per cage on 12-h dark/light cycle and allowed free access to standard dry rodent diet and water, supplemented with 1g/l L- cysteine (AppliChem, Darmstadt, Germany) [11]. Analgesia was achieved with paracetamol suppositories, in order to avoid interactions with the immune system.

H2S synthesis is catalyzed by three different enzymatic systems, cystathionine γ- lyase (CSE), which is the key enzyme for H2S synthesis in the liver and kidney, cystathionine β- synthase (CBS), which is overexpressed in brain tissue and 3-mercaptopyruvate sulfurtransferase (3MST) which is ubiquitously expressed [12]. We investigated the role only of CSE and 3MST, due to the fact that homozygotic CBS deficiency is related to a neonatal mortality of over 90%. [13,14]. Cse -/- mice were created after breeding of Cse +/- mice (Friedrich Shiller University Hospital, Jena, Germany). The Cse+/- mice were generated as previously described [13]. In short chimeric male Cse +/- mice were produced after injection of Cth+/- stem cells into C57BL/6 blastocysts. These were then crossed with C57BL/6 female mice to obtain Cse +/- pups. Cse +/- pups were backcrossed multiple times to achieve high genetic homogenicity on C57BL/6 background. The Cse +/- males and females produced were finally bred to obtain wild- type (Cse +/+), Cse +/-, and Cse -/- littermates. 3Mst+/+ and 3Mst-/- mice were created, as described previously [15].

4 weeks after birth genomic DNA for genotyping mice was prepared from the tail tips by the rapid, hot sodium hydroxide and Tris (HotSHOT) [16]. A small tail punch (<2mm) was prepared using a sharp sterile surgical scissor and the tail biopsy was placed immediately into sterile polypropylene microfuge tube and stored at -80°C until DNA isolation and purification. The CSE null and wild-type alleles were detected by a three-primer PCR mild modified protocol [17] of tail-tip DNA: N1 (5′-TGC GAG GCC AGA GGC CAG TTG TGT AGC-3′), F1 (5′- TGT TCA TGG TAG GTT TGG CC-3′) and R1 (5′-TCA GAA CTC GCA GGG TAG AA -3′). These primers amplify a ~500 bp wild type band (from primers F1 and R1) and a ~450 bp targeted (null) band (from primers N1 and R1). All primers were purchased from IDT (IDT, San Jose, California, USA). The samples were spun down and 2 μl of the supernatant was used as a template in a 25-μl reaction using 12 μl BioMix (Bioline GmbH, Luckenwalde, Germany), 3.33 pmol/ml of each primer and 1.5–2.0 mM MgCl2. qRT-PCR was performed by the iQ5 cycler system (BioRad, Hercules, CA, USA). The thermal cycling conditions consisted of a 7 min initial denaturation cycle at 95°C; followed by 30 cycles of denaturation at 60°C for 75 s; annealing at 72°C for 2 min; extension at 95°C for 1 min. Finally, the reaction ended with 1 cycle at 60°C for 75 s and with a final extension at 72°C for 10 min. After this, samples were kept at 4°C. Reactions containing Molecular Grade Water (AppliChem, GmbH, Germany) were used as negative controls to evaluate for potential contamination. Specificity was confirmed by electrophoretic analysis of the reaction products by 2% w/v agarose electrophoresis gel in 0.5 × Tris–Borate EDTA (TBE) buffer (5V/cm) and 1 μg/mL ethidium bromide staining (AppliChem).

Cse +/+ and Cse -/- mice were infected by intraperitoneally injecting 1x108 cfu/mouse of MDR P. aeruginosa isolate 6–11–19, concentration in which P. aeruginosa is characterized by a lethality of up to 60% in infection models in rodents [18]. Mice were then randomized via a typical randomization table in treatment groups receiving 200 μl subcutaneously of: a) 120 mg/kg of the H2S biosynthesis inhibitor aminooxyacetic acid AOAA (Sigma-Aldrich), which inhibits both CBS and CSE catalytic activity [19] diluted in 0.9% NaCl once per day for 4 days or 0.9% NaCl once daily for 4 days; b) 200 μmol/kg/mouse PAG (Sigma-Aldrich), which is a specific CSE inhibitor [20], diluted in 0,9% NaCl administrated intraperitoneally once per day for 4 days; c) 2 g/kg/mouse [21] sodium thiosulfate 10% (STS, Dr. Franz Köhler Chemie GmbH, Bensheim, Germany) diluted in WFI once per day for 4 days or WFI once per day for 4 days and d) 25mg/kg/mouse [22] GYY3147 (Sigma-Aldrich). Some experiments were repeated in CSE+/+ and CSE-/- mice after induction of neutropenia through i.p. injection of 150mg/kg and 100mg/kg of cyclophosphamide (Baxter, Chicago, Illinois, USA) 4 and 1 days before the MDR P. aeruginosa injection. Additional survival experiments were done using 1 x 108 cfu/mouse of the MDR P. aeruginosa isolates 19–2–45 and 20–1–30 and 1 x 108 cfu/mouse of the carbapenemase-producing Klebsiella pneumoniae isolate (Lee et al., 2015) as well as challenge with 10 or 30 mg/kg/mouse bacterial endotoxin (LPS) of E. coli O55:B5 (Sigma-Aldrich). Some experiments were repeated in 3Mst+/+ and 3Mst-/- mice. On each day of experimentation, we infected up to 3 mice from each group. Survival was recorded every 12 hours for 7 days. Analgesia was achieved by subcutaneous administration of meloxicam 5 mg/kg.

Cse +/+ and Cse /- mice were sacrificed at certain timepoints after infection with MDR P. aeruginosa isolate 6–11–19 by s.c. injection of 300 mg/kg ketamine, followed by cervical dislocation. Under sterile conditions a midline abdominal incision was performed, intestines were displaced to the left and the lower vena cava was punctured by a 20-gauge needle. 500 μl of whole blood was aspirated, collected into sterile and pyrogen- free tubes (Vacutainer, Cockeysville, MD, USA) and centrifuged. Serum was stored at -80°C. Then, segments of the liver and of the lower lobe of the right lung were excised and collected into sterile tubes with 1 ml NaCl 0.9%. The samples were weighted and homogenized. Additionally, segments of the spleen were stored in RPMI 1640 (Biochrom, Berlin, Germany) and of the lower lobe of the right lung in RNAlater (Qiagen, Hilden, Germany).

Survival experiments were repeated in Cse-/- and Cse +/+ mice after bone marrow transplantation [23]. Recipient Cse-/- and Cse +/+ mice were placed in an acrylic container (22 cm diameter, 12 cm depth) and irradiated with a single dose of 9.5 Gy at a dose rate pf 300cGy/min (Energy 6MV photons) in a VitalBeam irradiator (Varian, CA, USA). One day after the irradiation BMT was done using bone marrow from Cse+/+ mice. More precisely, mice were killed and both hind legs were removed under aseptic conditions. Bone marrow cells (BMCs) were collected by flushing the femurs with PBS using one 21G needle. Bone marrow was gently homogenized using a pipette through a 40μm cell strainer. BMCs were counted with a Neubauer plate and suspended in PBS to 1x 107 BMCs/ml. The irradiated recipient Cse-/- and Cse +/+ mice were then injected 1x 106 BMCs in 100 μl volume intravenously via a tail vein. Mice were allowed to recover for 1 month post-BMT. Reconstitution was confirmed by analysis of complete blood counts. Then, mice were infected by intraperitoneally injecting 1x108 cfu/mouse of MDR P. aeruginosa isolate 6–11–19. Survival was recorded for 7 days.

Complete blood count was performed with the ADVIA 2120i system (SIEMENS, Munich, Germany). Creatinine was measured via the Jaffe method, with lowest detection limit 0.03 mg/dl. Aspartate transaminase (AST) and alanine transaminase (ALT) were measured by the IFCC method with the ADVIA 1800 system (SIEMENS). Lowest detection limits were 0.8 U/l and 0.6 UI/l respectively.

Segments of mice spleens stored in RPMI 1640 were gently squeezed and passed through a sterile filter (250 mm, 12–13 cm, AlterChem Co, Athens, Greece) for the collection of splenocytes. After three serial washings, cells were counted on Neubauer plates with trypan blue for exclusion of dead cells. A total of 5 x 106 cells/ml were then incubated into sterile 24-well plates in RPMI-1640, supplemented with 2 mM glutamine, 10% fetal bovine serum, 100 U/ml of penicillin G and 0.1 mg/ml of streptomycin in the absence or presence of 10 ng/ml LPS of E. coli O55:B5 or 5 x 105 cfu/ml heat killed Candida αlbicans. After 24 hours or 5 days of incubation at 37°C in 5% CO2, the plates were centrifuged, and the supernatants were collected. Concentrations of TNFα was measured in duplicate in supernatants from stimulated splenocytes, in tissue supernatants and in serum via enzyme- linked immunosorbent assays (ThermoFisher Scientific, California, USA). The lower detection limit was 39 pg/ml. Additionally, IL-1α, IL-1β, IL-6, IL-10, IL-12, IL-27, IFNγ and IFNβ in serum were determined by the LEGENDplex mouse TH 1/2 cytokine panel (13-plex) (Biolegend, California, USA) according to the manufacturer’s instructions.

Tissue segments were homogenized with T-PER (ThermoFisher Scientific, Massachusetts, USA) and centrifuged at 10,000 rpm at 4°C. Then the homogenates were incubated in wells of a 96-well plate at 37°C with 4.2 mM tetramethylbenzidine (Serva, Heidelberg, Germany), 2.5 mM citrate, 5 mM NaH2PO4 and 1.18 mM H2O2 pH 5.0 at a final volume of 150 μl. After 5 minutes the reaction was terminated by adding 50 ml 0.18M H2SO4. Absorbance was read at 450 nm against blank wells. Results were adjusted for tissue sample protein content on Bradford assay (Sigma-Aldrich) and they were expressed as MPO units/mg protein/g.

RNA was isolated from mice lung segments stored in RNAlater (Qiagen, Hilden, Germany), following the RNeasy mini kit protocol (QIAGEN) according to the manufacturer’s recommendations. RNA concentration was measured spectrophotometrically using the absorbance ratio of 260/280 nm. Gel electrophoresis was used to check the purity and integrity of the total RNA. Additionally, the fragmented bacterial RNA was analyzed using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Waldbronn, Germany) following the manufacturer’s protocol. 1 μg isolated RNA from P. aeruginosa was used as template, with the first strand iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) for the reverse transcription for cDNA synthesis; conditions were 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C. Quantitative real-time PCR (qRT-PCR) analysis of the expression of quorum sensing (QS) genes rhll, rhlR, lasl, lasR, pqsA and pqsR was carried out using the P. aeruginosa specific primers described [24] (Table C in S1 Text). All primers were purchased from IDT (IDT, San Jose, California, U.S.A). qRT- PCR was performed with the iQ5 cycler system (BioRad, Hercules, CA, USA) using 2 μl cDNA, 10 μl iTaq Universal SYBR Green Supermix, (BioRad), 6μl Molecular Grade Water (AppliChem) and 0.1 pmol/ml sense and antisense primers to a final volume of 20-μl on duplicate samples in 96-well plate. The thermal cycler profile involved a preliminary denaturation at 95°C for 1 min; followed by 40 cycles of denaturation at 95°C for 30 s; annealing at 52°C for 30 s; and extension for at 72°C 1 min.; followed by cooling at 4°C. Amplification was followed by a melting curve from 55°C to 95°C increasing of 0.5 each time for verification of the amplified product. The threshold was adjusted according to the amplification curves of all evaluated genes. No reverse transcriptase controls were prepared from total RNA and no template controls were prepared with Molecular Grade Water (AppliChem) in place of total RNA to indicate potential genomic DNA contamination in isolated bacterial RNA and contamination of reagents, respectively. Specificity was confirmed by electrophoretic analysis in 3% w/v agarose electrophoresis gel (5V/cm) and ethidium bromide staining (AppliChem). Analysis of relative gene expression was achieved according to the ΔΔ CT method [25]. The number of transcripts of QS genes in the lung was normalized to the respective number of bacteria in the lung at the time of sacrifice [26].

Additionally, each MDR P. aeruginosa isolate was incubated at a starting inoculum of 1 x 107 cfu/ml in tubes with Mueller-Hinton broth with and without 1mM of the H2S donor GYY4137 (Sigma-Aldrich) and with and without 1mM of the AOAA (Sigma-Aldrich). After 2 and 6 hours of incubation RNA was isolated from 1ml of each dilution, using the same methodology as described above. RNA was then used for measurement of expression of quorum sensing genes. An aliquot of 0.1ml of each dilution was diluted 1:10 into Mueller-Hinton broth (Becton Dickinson) six consecutive times; 0.1 ml of each dilution was plated onto MacConkey agar (Becton Dickinson). After incubation for 24 hours at 37°C, the number of viable colonies was counted. The results were expressed as log10 of colony forming units per ml (cfu/ml). The number of transcripts of QS genes was normalized to the respective bacterial load. The experiment was performed in duplicate.

Isolated spleen cells were incubated for 15 min in the dark with the monoclonal antibodies anti-CD45 PC5 (ThermoFisher Scientific), anti-CD11b PE (phycoerythrin, emission 575nm, ThermoFisher Scientific), anti-CD11c FITC (fluorescein isothiocyanate, emission 525 nm, ThermoFisher Scientific). Cells were analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co, Miami, Florida). Results were expressed as percentages.

A log-phase culture (1 × 105 cfu/ml) of MDR P. aeruginosa was opsonized with 5% mouse serum (Sigma-Aldrich) at room temperature for 15 min. 5 x 106 leukocytes/ ml were isolated from mice spleens as described above, and were incubated at 37°C in 5% CO2 for 30 min into sterile 24-well plates in RPMI 1640 supplemented with 10% FBS with and without the H2S donor 1mM GYY3147 (Sigma-Aldrich) [27]. Then, 1 × 105 cfu/ml bacteria were added in each well and the plates were incubated at 37°C. ii) in vivo. One aliquot of 0.1 ml of the tissue homogenates was diluted 1:10 into Mueller-Hinton broth six consecutive times; 0.1 ml of each dilution was plated onto MacConkey agar (Becton Dickinson). After incubation for 24 hours at 37°C, the number of viable colonies was counted. The results were expressed as log10 of colony forming units per gram tissue (cfu/g).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.