Axenic Long-Term Cultivation of Pneumocystis jirovecii
Abstract
:1. Introduction
2. Materials and Methods
2.1. Pneumocystis Culture Literature Review
2.2. Sample Collection, Transport, and Freezing Conditions
2.3. Pneumocystis Detection in BALF Samples Using Staining Methods
2.4. Pneumocystis Detection Using Electron Microscopy (EM)
2.5. Absolute Quantification of P. jirovecii by mtLSU rRNA Gene Real-Time qPCR
2.6. P. jirovecii Culture from Human BALF Samples
2.6.1. Preparation of P. jirovecii Inocula for All Culture Attempts
2.6.2. Initial P. jirovecii Culture with Isolates from a Single Patient in DMEM-C Medium
2.6.3. DMEM-C Medium Optimisation of Axenic Cultures with Mixed P. jirovecii Strains
2.6.4. Long-Term Axenic P. jirovecii Flask Cultures with DMEM-O2 Medium
2.6.5. DMEM-O2 Medium Optimization of Axenic Cultures with Mixed P. jirovecii Strains
2.6.6. Long-Term Axenic P. jirovecii Flask Culture with DMEM-O3 Medium and Cluster Size Measurement
2.7. Quantification of P. jirovecii Growth in Cultures
2.7.1. Microscopic Examination of P. jirovecii Clusters in Cultures
2.7.2. Absolute Quantification of P. jirovecii in Cultures Using mtLSU rRNA Gene Real-Time qPCR
3. Results
3.1. Pneumocystis Culture Literature Review
3.2. Initial P. jirovecii Culture with Isolates from a Single Patient in DMEM-C Medium
Year Pub-lished | Pneumocystis Strain and Host Species 1 | Feeder Cells | Medium | Supplements and Concentration | Culture Conditions | Duration | Start Inoculum and Pneumocystis Growth | Non-Successful Experiments /Cell Lines | References |
---|---|---|---|---|---|---|---|---|---|
(a) P. jirovecii culture attempts | |||||||||
1977 | P. carinii (human = P. jirovecii) | CEL | Medium 199 | FBS, human serum or no serum | 37 °C | 12 days with passages every 3rd day (Pc) or 2 passages every 7th day (P. jirovecii) | Inoculum: 9.1 × 104 cysts or unquantified number of trophic forms Growth: Murine Pc: 100-fold (2.7 × 105 to 2.3 × 107) P. jirovecii: 10-fold (5 × 103 to 7.6 × 104) | Less than 1 × 103 cysts/ 12.4 × 107 cells resulted in culture failure, No growth in any cell-free media | [18] |
1984 | P. carinii from human lung and BALF samples (=P. jirovecii) | A549 | DMEM | 10% FCS | 37 °C 25 cm2 and 75 cm2 flasks | 7 days, 14 days | Inoculum: 1 mL pellet in PBS from mined human lungs or BALF to 9 mL medium Growth: 8-fold increase of trophic forms after one week, 10-fold increase after 14 days | Growth only in one of ten human specimens (lung biopsy) | [13] |
1989 1990 | P. carinii of human origin (=P. jirovecii) | A549 Vero | RPMI 1640 DMEM | 0 or 10% FCS Penicillin 200 U/mL Streptomycin 200 µg/mL Miconazole 0.5 µg/mL L-glutamine 200 mM HEPES 25 mM | 37 °C 5 or 10% CO2 Some cultures: irradiation of A549 prior to infection 25 cm2 flasks chamber slides | 2–6 days | Inoculum: 0.1 to 0.5 mL BALF sediment to 5 mL medium Growth: optimal with A549 incubated at 37 °C and 10% FCS plus all additives; cyst density larger in irradiated A549 cultures | - | [17] |
1997 | P. carinii (human = P. jirovecii) | A549 | F12 | 10% FCS Penicillin 200 U/mL Streptomycin 200 mg/mL Miconazole 0.5 mg/mL | 37 °C | 4 days | Inoculum: from human lung, P. jirovecii number not mentioned Growth: only decline of P. jirovecii within 48–72 h, after 96 h no P. jirovecii detectable | No success | [14] |
1999 | P. carinii (human = P. jirovecii) | Axenic | MEM with Earle’s salt | 20% horse serum 500 mg/mL S-adenosyl-L-methionine sulphate 80 mg/mL each of the following p-aminobenzoic acid, putrescine, ferric pyrophosphate, L-cysteine, L-glutamine, and N-acetyl-D-glucosamine 500 U/mL penicillin 500 µg/mL streptomycin | 31 °C pH 8.8–9 Transwell inserts | 50 days with 6 sub-cultures (weekly splitting) | Inoculum: 2 to 3 × 107 organisms/mL Pc doubling time 19–44 h Cultures with small inoculum (2 × 106) had faster growth 920-fold increase in 7 days (doubling time: 19 h) Growth of P. jirovecii in preliminary experiments! | No growth after dilutions to only 5–10 clusters/well | [30] |
2018 | P. jirovecii | CuFi-8 cells EpiAirway cells | Hams F-12 | No further supplements | 37 °C | 2–5 days | Inoculum: 10–150 µL P. jirovecii-positive BALF Growth: P. jirovecii copy number max. increase from 1 × 102 to 1 × 107 (measured by a mtLSU gene qPCR) | Cytopathic effect of P. jirovecii destroys CuFi-8 and EpiAirway cells quickly Commercially non-available CuFi-8 cells P. jirovecii culture was NOT reproducible by others | [15] reply: [16] reply: [19] |
2018 | P. jirovecii | CuFi-8 cells | Hams F-12 | No further supplements | 37 °C | min. 5 days | Inoculum: not mentioned Growth: no growth observed | P. jirovecii-positive BALF of 10 patients did not reveal any P. jirovecii growth (8 of 10 had no P. jirovecii copies after day 5 of culture), 2 of 10 rapid decline | [16] 2 reply: [19] |
(b) Important animal-derived Pneumocystis spp. culture attempts (shortened, see Supplementary Materials). | |||||||||
1977 | P. carinii (rat) | CEL | Medium 199 | FBS human serum or no serum | 37 °C | 12 days with 4 passages every 3rd day (Pc) or 2 passages every 7th day (P. jirovecii) | Inoculum: 9.1 × 104 cysts or unquantified number of trophozoites Growth: Murine Pc: 100-fold (2.7 × 105 to 2.3 × 107) Human P. jirovecii: 10-fold (5 × 103 to 7.6 × 104) | Less than 1 × 103 P. carinii cysts/12.4 × l07 cells resulted in culture failure, No growth in any cell-free media | [18] |
1990 | P. carinii (rat) | Axenic | DMEM and others 3 | DMEM medium: Penicillin-streptomycin Amphotericin B NPG medium (variable supplements and conditions): FCS: 10 or 20% 1% neopeptone 0.2% N-acetylglucosamine Cysteine, palmitate, oleate, linoleate, ergosterol or cholesterol 0.025 to 0.1 mg/L, Hemin 10.0 mg/mL | Solid agars: pH 4.0 and 7.0 NPG medium: pH 4.0 to 8.0 (optimal: pH 4.0) 4-25-31-35-37-41 °C (optimal: 35-37 °C) | 6–7 days | Inoculum: 2.5 × 106 to 1 × 108/mL, optimal inoculum: 2.5 × 106 to 1 × 107 nuclei/mL Optimal growth: 8 to 10-fold increase in NPG medium containing 1% neopeptone (wt/vol) and 0.2% (wt/vol) N-acetyl-D-glucosamine (GlcNAc) at a pH of 4.0 over 1 week Subculture: - without dilution of organisms: 2-fold increase after 7 days - with dilution to 1 × 107 organisms/mL: 7-fold increase after 7 days | No growth enhancement: - NPG medium with sugars other than GlcNAc, including glucose, another amino sugar, N-acetyl galactosamine, - NPG medium with fatty acids or sterols - Neopeptone alone - reduced oxygen tension | [38] |
1995 1999 | P. carinii (rat) | L2 Axenic | DMEM | 10% FCS antibiotics (penicillin/streptomycin) | 37 °C 5% CO2 | 4 days 7 days | Inoculum (4-day culture): ~2 × 106 (/mL?) Inoculum (7-day culture): 1 × 107/mL | Rat-derived Pc in vitro culture: no growth after day 4 in cultures with feeder cells, after day 2 in axenic cultures | [34,52] |
1999 | P. carinii (rat) | Axenic | MEM with Earle’s salt | 20% horse serum 500 mg/mL S-adenosyl- L-methionine sulphate 80 mg/mL each of the following p-aminobenzoic acid, putrescine, ferric pyrophosphate, L-cysteine, L-glutamine, and N-acetyl-D-glucosamine, 500 U/mL penicillin, 500 µg/mL streptomycin | 31 °C pH 8.8–9 Transwell inserts | 50 days with 6 sub-cultures (weekly splitting) | Inoculum: 2 to 30 × 106 organisms/mL Pc doubling time 19–44 h Cultures with small inoculum (2 × 106) had faster growth 920-fold increase in 7 days with a doubling time of 19 h | no growth was observed after dilutions to only 5–10 clusters/well | [30] |
2000 | P. carinii (rat) | Axenic | MEM with Earle’s salts | 20% horse serum Putrescine 80 µg/mL Ferric pyrophosphate 80 µg/mL L-cysteine 80 µg/mL Glutamine 80 µg/mL s-adenosyl-L-methionine (SAM) 500 µM | 31 °C Transwell, collagen-coated | 21 (27?) days | Inoculum: 3 × 106 cells/mL Growth: 24 × 107 nuclei per Giemsa-stained slide (culture volume/slide not mentioned) on day 21 of culture | Decline of Pc in medium without SAM after 15 days | [40] |
2001 | P. carinii (rat) | Axenic? No cells Mentioned | RPMI-1640 | 20% FCS 1× MEM vitamins 1× NEAA, L-glutamine (conc. not mentioned) 100 IU penicillin 100 µg/mL streptomycin | Temp. + CO2/O2 not mentioned Every 24 h agitation of samples, 50% medium exchange | 7 (9?) days | Inoculum: 1 × 108 to 2 × 108 nuclei/mL Growth: Slightly increase in media without pentamidine until day 2 (9.36 × 107 to 9.93 × 107 nuclei) | Decrease of Pc nuclei in media with and without pentamidine over the whole culture period of 7 days | [53] |
1978 | P. carinii (rat) | VERO | MEM Medium 199 | 2% FBS | 37 °C | 7 days Passage every 24 h | Inoculum: 1.3 × 105 to 8.5 × 105 cysts/culture of 1–2 mL Growth: Max. 11-fold increase in cysts 72h post inoculation | Little growth (3-fold increase) in owl monkey kidney, baby hamster kidney, and AV-3 cell cultures, and no growth in WI-38 cells and secondary chicken fibroblast cultures Maximum 3–4 passages, then decline | [54] |
1979 | P. carinii (rat) | WI-38 MRC-5 | Eagle medium | 2–10% FCS 50 µg/mL streptomycin 100 U/mL penicillin G potassium 10 U/mL nystatin | 35 °C No CO2 | 10 days Subcultures from 4–5 days old cultures | Inoculum: 1 mL supernatant derived from 1 cm3 ground rat lung; number of Pneumocystis in inoculum not measured Growth: 117.9 × 106 | High variances between growth of rat isolates even if the rat lung used for culture had high organism loads | [55] |
1985 | P. carinii (rat) | A549 WI-38 | A549: DMEM WI-38: HMEM | 25 mM HEPES 20% HyClone FBS/serum (rat, chicken, swine, human, horse) from several companies 1000 U penicillin 1000 µg streptomycin 0.5 µg amphotericin B NCTC vitamin mixture 107 formula no. 78-0776 or no vitamins | Room temp: 30-35-37-41 °C (optimal: 30-37 °C for Pc on A549 cells); Stationary or rocking 6 rpm; With/without 5%CO2 | 20 days | Inoculum: Optimal: 1 × 106 organisms/mL Maximal: 1 × 108 organisms/mL Minimum: 1 × 105 organisms/mL Growth: in both cell lines Subculture/passaging: 3 successful passages 7 days after start with 12-fold organism increase, or 14 days after start resulting in a steady-state or 21 days after start with 4-fold organism increase | Inhibitory effect (in both cell lines): - temperature 41 °C - 0.05% saponin - rocking/moving the cultures Source of serum (rat, swine, human) or temperature led to a decrease in growth in VA13 cells | [29] |
1985 | P. carinii (rat) | WI-38 MRC-5 | Eagle medium | 2–10% FCS 50 µg/mL streptomycin 100 U/mL penicillin G potassium 10 U/mL nystatin | 5% O2 5–10% CO2 35 °C | 10 days | Inoculum: 1 mL supernatant derived from 1 cm3 ground rat lung; number of Pneumocystis inoculum not measured Growth: peak at day 6 max organism number (peak day) 1.8 to 17.5 × 106 in P. carinii on WI-38 cells and 0.7 to 16.1 × 106 in P. carinii on MRC-5 cells more constant growth on WI-38 cells | 2 to 3 passages/harvests possible Growth decreased in subcultures | [56] |
1993 | P. carinii (rat) | HEL (A549 and L-132 gave no good results) | Eagle MEM (DMEM, RPMI-1640, medium 199 gave no good results) | 10% FCS Antibiotics (penicillin, streptomycin, amphotericin B, no concentration mentioned) Medium exchange every 3rd day Passaging every 3–5 days | 5% CO2 37 °C | 42 days | Inoculum: 1 to 3 × 107 P. carinii/mL in 24-well tissue culture plates Growth: peak at day 6 to 9 or 10, but after that decline | Cell-bound and supernatant P. carinii were counted separately (30% were adherent to cells) Infection of rats after 41 days of in vitro culture was successful | [39] |
2009 2011 | P. carinii (rat) | axenic | RPMI-1640 | 20% calf serum penicillin (200 U/mL) streptomycin (200 µg/mL) amphotericin B (0.5 µg/mL) vancomycin (5 µg/mL) S-adenosyl-L-methionine farnesol vitamins and amino acids as described before PET track-etched membrane cell culture inserts Transwell inserts Millicell-CM hydrophilic PTFE membranes Millicell-HA insert | 36 °C 5% CO2 | 7–14 (21) days | Inoculum: 1 × 106 to 1 × 108 Growth: Less extensive biofilm formation than P. murina from mice (thickness of <15 µm) | S-adenosyl-L-methionine led to a dramatic decrease of viability within 24 h in P. carinii and P. murina Farnesol led to decreased viability in P. carinii | [31,43] |
2006 | P. carinii (rat) | axenic | RPMI-1640 | 20% calf serum 1× MEM vitamins non-essential amino acids (conc. not mentioned), L-glutamine (conc. not mentioned), 100 IU penicillin, and 100 mg/mL streptomycin Start inoculum: 5 × 107 organisms/mL | Standard: 21% O2 and 5% CO2 microaerophilic:10–15% O2, 7–15% CO2 anaerobic: <1% O2, 10% CO2 | 7 days | Inoculum: 5 × 107 P. carinii/mL Growth: ATP measurement only: 15,000 RLU under microaerophilic conditions vs 3000 RLU under standard conditions after 7 days | Anaerobic conditions: decline after the first day | [57] |
2013 | P. carinii (rat) | axenic | DMEM | 10% FBS 100 U/mL penicillin 100 μg/mL streptomycin | 37 °C 5% mio | 2–4 days | Inoculum: 0.75 × 104 P. carinii/mL or 2.5 × 105 P. carinii/mL Growth: from 6 × 105 to 10 × 105 organisms within 40 h, then decline | Decline of Pneumocystis life stages after 2 days in all cultures | [42] |
Culture Conditions: | |
37 °C 5% CO2 1 mL/well in 6-well plates Duration: 14 days Medium exchange and sampling every day | |
Medium Ingredients: | |
DMEM low glucose | |
Sodium bicarbonate | 3.7 g/L |
Amphotericin B | 5 µg/mL |
Penicillin G | 200 U/mL |
Streptomycin | 200 µg/mL |
FCS | 10% |
S-adenosyl-L methionine | 500 µg/mL |
Para-aminobenzoic acid | 80 µg/mL |
L-glutamine | 80 µg/mL |
3.3. DMEM-C Medium Optimization of Axenic Cultures with Mixed P. jirovecii Strains
3.3.1. The Addition of Mono- and Disaccharides to the DMEM-C Medium Increased the Growth of P. jirovecii under Cell-Free Conditions
3.3.2. Addition of Micronutrients to the Culture Medium Promoted the Growth of P. jirovecii
3.3.3. Supplementing the DMEM-O1 Culture Medium with Different Amino Acids and Varying Concentrations of FCS Showed Differential Effects on the Growth of P. jirovecii
3.4. Long-Term Axenic P. jirovecii Flask Cultures with DMEM-O2 Medium
3.5. Long-Term Axenic P. jirovecii Flask Culture with DMEM-O3 Medium and Cluster Size Measurement
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Conditions and Additives | Conditions Tested | P. jirovecii Growth in Axenic Culture |
---|---|---|
pH | 7.0 | − |
7.5 | + | |
8.0 | +++ | |
8.5 | + | |
9.0 | − | |
Temperature | 31 °C | − |
35 °C | (+) | |
37 °C | +++ | |
Coated plates | Gelatine | − |
Poly-L-lysin | − | |
Transwell plates | − | |
Medium exchange | every 2 days | +++ |
every 6 days | + | |
None | − |
Culture Conditions: | |
37 °C 5% CO2 1 mL/well in 6-well plates Duration: 14 days Medium exchange and sampling every 2nd day | |
Medium Ingredients: | |
DMEM low glucose | |
Sodium bicarbonate | 3.7 g/L |
Amphotericin B | 5 µg/mL |
Penicillin G | 200 U/mL |
Streptomycin | 200 µg/mL |
FCS | 20% |
S-adenosyl-L methionine | 500 µg/mL |
Glutamax | 20 mL/L |
Glucose | 3.4 mg/mL |
Ferric pyrophosphate | 80 µg/mL |
Putrescine | 8 µg/mL |
2-mercaptoethanol | 100 µg/mL |
HEPES | 12.6 µg/mL |
Culture Conditions: | |
37 °C 5% CO2 1 mL/well in 6-well plates 10 mL in 25 cm2 flasks Duration: up to 75 days Medium fed-batch addition and sampling every 6 days | |
Medium Ingredients: | |
DMEM low glucose | |
Sodium bicarbonate | 3.7 g/L |
Amphotericin B | 5 µg/mL |
Penicillin G | 200 U/mL |
Streptomycin | 200 µg/mL |
FCS | 30% |
Glutamax | 20 mL/L |
Glucose | 3.4 mg/mL |
Galactose | 0.9 mg/mL |
Maltose | 10.8 mg/mL |
Sucrose | 1.3 mg/mL |
Ferric pyrophosphate | 160 µg/mL |
HEPES | 12.6 µg/mL |
MEM essential amino acids solution (50×) | 2% |
MEM non-essential amino acids solution (100×) | 2% |
Culture Conditions: | |
37 °C 5% CO2 1 mL/well in 6-well plates 10 mL in 25 cm2 flasks Duration: up to 75 days Medium fed-batch addition and sampling every 6 days | |
Medium Ingredients: | |
DMEM low glucose | |
Sodium bicarbonate | 3.7 g/L |
Amphotericin B | 5 µg/mL |
Penicillin G | 200 U/mL |
Streptomycin | 200 µg/mL |
FCS | 30% |
Glutamax | 20 mL/L |
Glucose | 3.4 mg/mL |
Galactose | 0.9 mg/mL |
Maltose | 10.8 mg/mL |
Sucrose | 1.3 mg/mL |
Ferric pyrophosphate | 160 µg/mL |
HEPES | 12.6 µg/mL |
MEM essential amino acids solution (50×) | 2% |
MEM non-essential amino acids solution (100×) | 2% |
L-alanine | 15 µg/mL |
L-cystine | 15 µg/mL |
Copper(II) sulphate pentahydrate | 100 µg/mL |
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Riebold, D.; Mahnkopf, M.; Wicht, K.; Zubiria-Barrera, C.; Heise, J.; Frank, M.; Misch, D.; Bauer, T.; Stocker, H.; Slevogt, H. Axenic Long-Term Cultivation of Pneumocystis jirovecii. J. Fungi 2023, 9, 903. https://0-doi-org.brum.beds.ac.uk/10.3390/jof9090903
Riebold D, Mahnkopf M, Wicht K, Zubiria-Barrera C, Heise J, Frank M, Misch D, Bauer T, Stocker H, Slevogt H. Axenic Long-Term Cultivation of Pneumocystis jirovecii. Journal of Fungi. 2023; 9(9):903. https://0-doi-org.brum.beds.ac.uk/10.3390/jof9090903
Chicago/Turabian StyleRiebold, Diana, Marie Mahnkopf, Kristina Wicht, Cristina Zubiria-Barrera, Jan Heise, Marcus Frank, Daniel Misch, Torsten Bauer, Hartmut Stocker, and Hortense Slevogt. 2023. "Axenic Long-Term Cultivation of Pneumocystis jirovecii" Journal of Fungi 9, no. 9: 903. https://0-doi-org.brum.beds.ac.uk/10.3390/jof9090903