Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosis
Abstract
:1. Introduction
2. Results
2.1. CRBSI Aetiological Agents
2.2. Antimicrobial Agent Resistance
2.3. Central Venous Catheter Colonization and CRBSI
2.4. Dual Biofilms of E. cloacae Complex (E. bugandensis) and C. parapsilosis
2.5. Host Factors and Biofilm
3. Discussion
4. Materials and Methods
4.1. Microorganism Isolation and Culture
4.2. Antimicrobial Susceptibility Tests
4.3. Biofilm Assay
4.4. Biofilms and Host Factors
4.5. Analysis of Biofilm Assembled In Vitro on Different Surfaces
- (i)
- Scanning electron microscopy (SEM). Samples were washed with PBS and fixed with 4% paraformaldehyde (Merck, Darmstadt, Germany) in PBS for 30 min at room temperature (RT) protected from light. The fixative was removed, and the samples were washed twice in PBS for 10 min and post-fixed with 1% osmium tetroxide (EMS, Hatfield, PA, USA) in the same buffer for 90 min on ice protected from the light. Then, the samples were washed twice for 10 min at RT with PBS and twice with deionized water. Dehydration was performed at RT using serial dilutions of ethanol as follows: once in 50%, 70%, 80% and 95% ethanol for 30 min and twice in 100% ethanol for 30 min each. Samples were then trimmed (CVC was cut longitudinally), allowed to dry at RT, mounted on top of double-sided carbon tape (CVC was mounted in such a way that both inner and outer surfaces were visible), coated with 20 nm thick gold-palladium film using a sputter coater QISOT ES (Quorum Technologies, Laughton, UK) and analyzed under a scanning electron microscope, JSM-7100F (JEOL, Tokyo, Japan) using secondary electron detector.
- (ii)
- Focused ion beam scanning electron microscopy (FIB-SEM) tomography. Samples were prepared as described previously for SEM until the second incubation with 100% ethanol. Then, Epoxy 812 resin (EMS) was added with the samples left uncapped overnight in a chemical chamber. The resin was then replaced by a new batch and further incubated for 3 h. Then, the sample and one drop of resin were compressed between 2 sheets of ACLAR film (EMS) allowing the sample to be encased in a thin resin layer. Samples were allowed to polymerize at 65 °C. FIB-SEM tomography was performed with an FEI HELIOS G4 instrument (Thermo Fisher Scientific, Waltham, MA, USA). Slicing was performed with a Ga+ ion beam accelerated at 30 kV and a current of 1 nA, while imaging was performed with an electron beam accelerated at 5 kV, a current of 1.6 nA and using the backscattered electron detector. A slice thickness of 30 nm was used. The acquisition operation was controlled through the Auto Slice & View 4.0 software package and reconstruction was carried out with Avizo Fire software package. Individual fungi and bacteria were manually labelled for intensity thresholding and 3D volume reconstruction.
- (iii)
- Fluorescence in situ hybridization (FISH)/laser scanning confocal microscopy. For biofilms assembled on glass coverslips, FISH was used to assess the distribution of fungi (C. parapsilosis-probe PF2) and bacteria (E. cloacae complex-probe EUB) using 5‘-labeled oligonucleotide probe fluorochromes FITC and Cy3, respectively (Biomers.net, Ulm, Germany). Non-sense probes labelled with the same fluorochromes were used as control. The probe sequences were previously described [56,57]. Samples were fixed as described for SEM, washed with PBS, permeabilized with 200 U/mL of lyticase (Sigma-Aldrich) followed by 10 mg/mL of lysozyme in hybridization buffer pH 8 (20 mM Tris-HCl pH8, 0.9 M sodium chloride, 20% formamide, 0.01% sodium dodecyl sulfate all from Sigma-Aldrich) for 15 min at 30 °C and incubated in a humidified chamber with 1 µM PF2 probe in the hybridization buffer for 3 h at 45 °C. After the first hybridization step, the unbound probe was washed off with a 45 °C pre-warmed washing buffer (20 mM Tris-HCl pH8, 0.212 M sodium chloride, 5 mM EDTA, 0.01% SDS) and water for 10 min each. Hybridization with 1 µM with EUB probe was performed for 2 h at 45 °C; the unbound probe was removed as previously. Coverslips were mounted by inversion on a drop of fluorescence-mounting medium previously placed on a glass slide. Samples were stored at 4 °C, protected from light, until visualization through a confocal microscope (Leica, SP2, Wetzlar, Germany) under an immersion objective of ×63 and an ocular objective of ×10.
4.6. Analysis of CVC Colonization
4.7. DNA Extraction and WGS
4.8. Genome Characterization
4.9. Isolation of RNA from C. parapsilosis
4.10. Evaluation of Gene Expression Related to FLU Resistance in C. parapsilosis
4.11. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
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Source | Central Venous Catheter | Hemoculture | ||
---|---|---|---|---|
Antibiotic | MIC (mg/L) | Phenotype 1 | MIC (mg/L) | Phenotype |
Ticarcillin | ≥128 | R | ≥128 | R |
Piperacillin/Tazobactam | ≥128 | R | ≥128 | R |
Ceftazidime | ≥64 | R | ≥64 | R |
Ceftolozane/Tazobactam | 2 | R | 1 | S |
Cefepime | 0.25 | S | ≤0.12 | S |
Aztreonam | 16 | R | 16 | R |
Imipenem | ≤0.25 | S | ≤0.25 | S |
Meropenem | ≤0.25 | S | ≤0.25 | S |
Amikacin | 2 | S | ≤1 | S |
Gentamicin | ≤1 | S | ≤1 | S |
Tobramycin | ≤1 | S | ≤1 | S |
Ciprofloxacin | ≤0.06 | S | ≤0.06 | S |
Levofloxacin | ≤0.12 | S | ≤0.12 | S |
Tigecyclin | ≤0.5 | S | ≤0.5 | S |
Trimethoprim/Sulfamethoxazole | ≤20 | S | ≤20 | S |
Colistin | >8 | R | >8 | R |
Source | Central Venous Catheter | Hemoculture | ||
---|---|---|---|---|
Antimicrobial Agents | MIC (mg/L) | Phenotype 1 | MIC (mg/L) | Phenotype |
Fluconazole | >256 | R | >256 | R |
Amphotericin B | 0.032 | S | 0.125 | S |
Anidulafungin | 0.50 | S | 0.50 | S |
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Štefánek, M.; Wenner, S.; Borges, V.; Pinto, M.; Gomes, J.P.; Rodrigues, J.; Faria, I.; Pessanha, M.A.; Martins, F.; Sabino, R.; et al. Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosis. Antibiotics 2022, 11, 1245. https://doi.org/10.3390/antibiotics11091245
Štefánek M, Wenner S, Borges V, Pinto M, Gomes JP, Rodrigues J, Faria I, Pessanha MA, Martins F, Sabino R, et al. Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosis. Antibiotics. 2022; 11(9):1245. https://doi.org/10.3390/antibiotics11091245
Chicago/Turabian StyleŠtefánek, Matúš, Sigurd Wenner, Vítor Borges, Miguel Pinto, João Paulo Gomes, João Rodrigues, Isabel Faria, Maria Ana Pessanha, Filomena Martins, Raquel Sabino, and et al. 2022. "Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosis" Antibiotics 11, no. 9: 1245. https://doi.org/10.3390/antibiotics11091245