Pathogens and Disease ISSN 2049-632X
SHORT COMMUNICATION
In vivo imaging of bioluminescent Pseudomonas aeruginosa in an acute murine airway infection model €lbeling1, Jens Klockgether1, Lutz Wiehlmann1 & Burkhard Tu €mmler1,2 Antje Munder1, Florian Wo 1 Clinical Research Group, Hannover Medical School, Clinic for Pediatric Pneumology, Allergology and Neonatology, Hannover, Germany 2 Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research, Hannover, Germany
This short communication describes in vivo imaging of bioluminescent bacteria as a tool to monitor murine airway infection caused by Pseudomonas aeruginosa. Using a virulent strain of the bacterial pathogen the inoculation of a sufficiently high dose to make the infection visible is followed by rapid death. Therefore, the authors apply a strain with reduced virulence to monitor airway infection over time. This may provide a useful approach for other pathogens with the same potential problem.
Keywords bioluminescence; Pseudomonas aeruginosa; in vivo imaging; infection monitoring; transposon mutant; mouse model. Correspondence Antje Munder, Carl-Neuberg Strasse 1, Hannover D-30625, Germany. Tel.: +49 511/532-5889 fax: +49 511/532-6723 e-mail: [email protected] Received 6 March 2014; revised 29 April 2014; accepted 2 May 2014. Final version published online 30 June 2014.
Abstract Non-invasive bioluminescence imaging allows the analysis of infectious diseases in small animal models. In this study, an acute airway infection of C3H/HeN mice with luxCDABE transformed Pseudomonas aeruginosa TBCF10839 and an isogenic transposon mutant was followed by optical imaging in vivo. Using the disease-causing dose of 2.0 9 106 CFU of the cystic fibrosis airway isolate TBCF10839, subtle luminescence of the lungs was inconsistently visible for the first hour after infection. Conversely, using a 100-fold higher dose of the strongly virulence-attenuated transposon mutant, the robust signal of bioluminescent bacteria increased over 24 h. To monitor murine airway infections with P. aeruginosa in vivo by bioluminescence, one should select an attenuated mutant of a virulent strain or a wild type strain that naturally lacks virulence determinants and/ or that has acquired a low virulence persister phenotype by patho-adaptive mutations.
doi:10.1111/2049-632X.12184 Editor: Nicholas Carbonetti
Bioluminescence imaging allows the analysis of bacterial infectious diseases in animal models (Chang et al., 2011; Kong et al., 2011). During the last 20 years, non-invasive imaging studies have been performed with numerous micro-organisms including Pseudomonas aeruginosa (reviewed by Andreu et al., 2011). Here, we tested the suitability of the in vivo monitoring of acute lung infections with bioluminescent P. aeruginosa in a standardized murine airway infection model (Munder et al., 2011). The cystic fibrosis (CF) airway isolate TBCF10839 (Klockgether et al., 2013) whose virulence in murine airways had been estab€lbeling lished in previous studies (Munder et al., 2011; Wo et al., 2011), and its isogenic virulence-attenuated transposon mutant D8A6 that displays an inactivated ORF 5PG21 in genomic island PAGI-5 (Wiehlmann et al., 2007; Klockgether et al., 2013) were used in this study. Luminescent derivatives of the two strains were generated by integrating 74
a luxCDABE luciferase reporter gene operon into the bacterial chromosome. Therefore, a miniCTX-lac-lux plasmid was isolated from recombinant Escherichia coli Topo-one-shot CTX-lac-lux cells (DiGiandomenico et al., 2007) and integrated site-specifically to the attB-site of the P. aeruginosa chromosome via electroporation (Choi et al., 2006). The functional efficiency of the insertion was controlled by measuring luminescent signals with the IVIS system. To excise the miniCTX backbone, we performed a Flp-FRT recombination (Hoang et al., 1998; Hoang et al. 2000). The kinetics of the infection was followed by the sensitive IVISâ Imaging System 200 Series (Perkin Elmer, Waltham, MA; formerly manufactured by Xenogen, Alameda, CA). To monitor infections with P. aeruginosa in the mouse, the IVIS system has so far primarily applied to systemic (BitMansour et al., 2002), burn (Hamblin et al., 2003;
Pathogens and Disease (2014), 72, 74–77, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
A. Munder et al.
In vivo imaging of P. aeruginosa in mice
2002) with doses of 2.0 9 106 and 2.0 9 108 CFU of P. aeruginosa strains TBCF10839lux or D8A6lux. For survival experiments, mice were infected with 7.5 9 106 CFU, a dose which resulted in 50% lethality of the animals within the first 120 h of infection for strain TBCF10839. In contrast, the same number of D8A6 bacteria was not able to induce
Burkatovskaya et al., 2006; Barman et al., 2011) and biofilm (Kadurugamuwa et al., 2003), but less to airway infection models (DiGiandomenico et al., 2007; Riedel et al., 2007; Ramphal et al., 2008). For in vivo imaging, C3H/HeN mice were infected by view-controlled intratracheal instillation (i.t.) (Munder et al., (b)
Number of mice
10 8 6
LD 50
4 2 0
0
48
96
144
192 [h]
10
8 6 4 2 0 0
(d)
(e)
(g)
(h)
(i)
(j)
(k)
(c) Disease score [points]
Loss of body weight [g]
(a)
(l)
48
96
144
192 [h]
8 6 4 2 0
0
48
96
144
192 [h]
(f)
I
II
Color Bar Min = 23.82 Max = 116,9
Color Bar Min = 6900 Max = 20911
III
Color Bar Min = 85951 Max = 8.8e+06
(m)
Fig. 1 (a–c) Physiological parameters of C3H/HeN mice after i.t. infection with 7.5 9 106 CFU of Pseudomonas aeruginosa TBCF10839 (closed squares; n = 12) and the isogenic mutant D8A6 (open circles; n = 12). (a) Survival. The applied dose of 7.5 9 106 CFU was equivalent to a LD50 for strain TBCF10839, but did not cause any mortality in D8A6 infected mice. (b) Body weight. TB10839 infected mice displayed a dramatic loss of body weight under infection, D8A6 infected animals showed milder weight loss, started regaining of weight earlier (72 h p.i.) and were almost able to return to their initial weight within the observation period of 192 h. (c) Disease score. The body condition of mice was strongly impaired under TBCF10839 infection, in contrast, all D8A6 infected animals recovered within 48 h p.i. to normal body condition. (d) TBCF10839 shows a strong purulent inflammation in murine lungs with intra- and peribronchiolar infiltrates of leucocytes and perivascular oedema 48 h after i.t. infection with 7.5 9 106 CFU. (e) Only single leucocytes are seen 48 h after i.t. infection around the bronchi with the same dose of the isogenic mutant D8A6 (f) Vehicle control, 48 h post instillation of 30 lL PBS, original magnification 9 200, bar 50 lm, H&E. (g–m) In vivo monitoring of P. aeruginosa in acute murine airway infection with the IVISâ imaging system (binning 4, exposure time 2 m, f/stop 1, FOV 12,8 cm). Bacterial distribution was monitored 0 h (Fig. 1g, i and k), 6 h (Fig. 1h, j and l) and 24 h (Fig. 1m) after i.t. instillation with TBCF10839.lux and D8A6.lux in infected mice. Infection doses were 2.0 9 106 CFU (TBCF10839.lux: Fig. 1g/h; D8A6: Fig. 1i/j) and 2.0 9 108 CFU (for D8A6.lux exclusively; Fig. 1k–m). Animals were depiliated using hair removal cream, imaged ventrally and although under anaesthesia additionally fixed with adhesive tape to reduce movement artefacts during imaging. Luminescence is observed emanating from the nasopharynx, tracheas and lungs. The colour bars indicate the intensity of the luminescent signal, with red and blue serving as the high and low signals, respectively (Fig 1, bar I: g–k; bar II: l; bar III: m).
Pathogens and Disease (2014), 72, 74–77, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
75
In vivo imaging of P. aeruginosa in mice
any mortality (Fig. 1a). A similar picture emerged when looking on the loss of body weight, which was dramatically for TB10839 infected mice, whereas D8A6 infected animals showed milder weight loss and were able to compensate this within the observation period of 192 h (Fig. 1b). The multiparametric disease score of infected mice (Munder et al., 2005) displayed a strongly affected body condition in TB10839 infected mice, but D8A6 infected animals showed only slight impairment (Fig. 1c). Lung slices were examined pathohistologically for inflammatory changes. Whereas TBCF10839 had caused an acute catarrhalic-suppurating alveolar pneumonia in the murine lungs (Fig. 1d), only very slight signs of inflammation, such as a minor migration of PMNs and alveolar macrophages into the alveoli, could be observed in the lungs of D8A6 infected mice (Fig. 1e) compared with mock infected mice instilled with PBS (Fig. 1f). For in vivo monitoring with the IVIS system, imaging was started directly (0 h) after instillation of 2.0 9 106 CFU and 2.0 9 108 CFU of strains TBCF10839lux and D8A6lux and was repeated at 6, 12 and 24 h p.i. Prior to infection and to improve the luminescent signals, anaesthetized mice were depiliated ventrally and during imaging fixed with adhesive tape to minimize movement artefacts. Best results were achieved with a high resolution at binning 4 (that means four by four pixels had been grouped to form one larger pixel, thereby increasing the signal-to-noise ratio), a f/1 camera lens aperture and exposure times of 2–4 min. The Living Image Softwareâ 2.50 (Perkin Elmer, Waltham, MA) was utilized to acquire, store and analyse the images with the connected computer. Occasionally, luminescent signals were detected around the mouth and along the trachea of the mice at 0 h p.i. (Fig. 1i), indicating a contamination of the upper airways with bacteria during the instillation. These signals disappeared within the next few hours (Fig. 1h, j and l), but re-emerged as time passed, presumably caused by multiplying of the bacteria in the lower airways (Fig. 1m). In the lungs, luminescence either emerged immediately after instillation or at the next time point of 6 h (Fig. 1h and k–m). Using the disease-causing dose of 2.0 9 106 CFU of TBCF10839, subtle luminescence was observed over the left or right lung in several, but not all mice (Fig. 1h). The luminescent signal had disappeared by 24 h indicating that bacterial numbers had decreased below the levels of detection (data not shown). Conversely, using a 100-fold higher dose of the virulence-attenuated D8A6 mutant, the luminescent bacteria showed signals with the IVIS that increased over time during the observation period of 24 h (Fig. 1k). This study demonstrates that the monitoring of an acute airway infection of mice with bioluminescent P. aeruginosa is feasible. However, in case of virulent P. aeruginosa, the possible dose is too low to reliably follow the 5-day natural €lbeling course of the acute infection up to recovery (Wo et al., 2011) by in vivo imaging of the bacteria. A range of two orders of magnitude of the P. aeruginosa inoculum evokes the full spectrum of responses in the mouse from subtle signs of morbidity to full-blown lethality. According to our experience, the LD50 of most P. aeruginosa strains from 76
A. Munder et al.
environment or disease habitats will be in the range of 106–107 CFU. A dose of about 10% of the LD50 results in a solid clinical manifestation of infection without mortality €lbeling et al., 2011). These doses result in a satisfactory (Wo imaging of bioluminescent bacteria in explanted lungs ex vivo (Henken et al., 2010), but are too low for an imaging of the lower airways in vivo. As the bioluminescence signal increases with the number of organ-incorporated bacteria, P. aeruginosa strains should be selected that are attenuated in acute virulence but are still capable to persist in the murine lungs. Only then – as shown in this study for the D8A6 mutant – infection doses of more than 108 CFU become feasible. One may choose an attenuated mutant of a virulent strain – as shown in this study – or a wild type strain that naturally lacks virulence determinants such as the type III secretion system and/or that has acquired a low virulence persister phenotype by patho-adaptive mutations.
Acknowledgements The authors would like to thank Anna-Silke Limpert for the support in the construction of the D8A6 mutant and Joanna Goldberg for kindly providing us with the strain E. coli Topo-one-shot CTX-lac-lux and the plasmid pFLP2. This study was supported by grants from the Deutsche Forschungsgemeinschaft (IRTG 653; SFB 587, A9) and the German Center for Lung Research. References Andreu N, Zelmer A & Wiles S (2011) Noninvasive biophotonic imaging for studies of infectious disease. FEMS Microbiol Rev 35: 360–394. Barman TK, Rao M, Bhati A, Kishore K, Shukla G, Kumar M, Mathur T, Pandya M & Upadhyay DJ (2011) Non invasive real-time monitoring of bacterial infection & therapeutic effect of anti-microbials in five mouse models. Indian J Med Res 134: 688–695. BitMansour A, Burns SM, Traver D, Akashi K, Contag CH, Weissman IL & Brown JM (2002) Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation. Blood 100: 4660–4667. Burkatovskaya M, Tegos GP, Swietlik E, Demidova TN, P Castano A & Hamblin MR (2006) Use of chitosan bandage to prevent fatal infections developing from highly contaminated wounds in mice. Biomaterials 27: 4157–4164. Chang MH, Cirillo SL & Cirillo JD (2011) Using luciferase to image bacterial infections in mice. J Vis Exp 48: pii: 2547. Choi KH, Kumar A & Schweizer HP (2006) A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J Microbiol Methods 64: 391– 397. DiGiandomenico A, Rao J, Harcher K, Zaidi TS, Gardner J, Neely AN, Pier GB & Goldberg JB (2007) Intranasal immunization with heterologously expressed polysaccharide protects against multiple Pseudomonas aeruginosa infections. P Natl Acad Sci USA 104: 4624–4629. Hamblin MR, Zahra T, Contag CH, McManus AT & Hasan T (2003) Optical monitoring and treatment of potentially lethal wound infections in vivo. J Infect Dis 187: 1717–1725.
Pathogens and Disease (2014), 72, 74–77, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
A. Munder et al.
Henken S, Bohling J, Ogunniyi AD, Paton JC, Salisbury VC, Welte T & Maus UA (2010) Evaluation of biophotonic imaging to estimate bacterial burden in mice infected with highly virulent compared to less virulent Streptococcus pneumoniae serotypes. Antimicrob Agents Chemother 54: 3155–3160. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ & Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212: 77–86. Hoang TT, Kutchma AJ, Becher A & Schweizer HP (2000) Integration-proficient plasmids for Pseudomonas aeruginosa: site-specific integration and use for engineering of reporter and expression strains. Plasmid 1: 59–72. Kadurugamuwa JL, Sin L, Albert E, Yu J, Francis K, DeBoer M, Rubin M, Bellinger-Kawahara C, Parr Jr TR Jr & Contag PR (2003) Direct continuous method for monitoring biofilm infection in a mouse model. Infect Immun 71: 882–890. Klockgether J, Miethke N, Kubesch P et al. (2013) Intraclonal diversity of the Pseudomonas aeruginosa cystic fibrosis airway isolates TBCF10839 and TBCF121838: distinct signatures of transcriptome, proteome, metabolome, adherence and pathogenicity despite an almost identical genome sequence. Environ Microbiol 15: 191–210. Kong Y, Shi Y & Chang M (2011) Whole-body imaging of infection using bioluminescence. Curr Protoc Microbiol Chapter2: Unit 2C.4.
In vivo imaging of P. aeruginosa in mice
Munder A, Krusch S & Tschernig T (2002) Pulmonary microbial infection in mice: comparison of different application methods and correlation of bacterial numbers and histopathology. Exp Toxicol Pathol 54: 27–133. Munder A, Zelmer A & Schmiedl A (2005) Murine pulmonary infection with Listeria monocytogenes: differential susceptibility of BALB/c, C57BL/6 and DBA/2 mice. Microbes Infect 7: 600–611. €lbeling F, Kerber-Momot T, Wedekind D, Baumann U, Munder A, Wo €mmler B (2011) Acute intratracheal Pseudomonas Gulbins E & Tu aeruginosa infection in cystic fibrosis mice is age-independent. Respir Res 12: 148. Ramphal R, Balloy V, Jyot J, Verma A, Si-Tahar M & Chignard M (2008) Control of Pseudomonas aeruginosa in the lung requires the recognition of either lipopolysaccharide or flagellin. J Immunol 181: 586–592. Riedel CU, Casey PG, Mulcahy H, O’Gara F, Gahan CG & Hill C (2007) Construction of p16Slux, a novel vector for improved bioluminescent labeling of gram-negative bacteria. Appl Environ Microbiol 73: 7092–7095. Wiehlmann L, Munder A, Adamns T, Juhas M, Kolmar H, Salunkhe €mmler B (2007) Functional genomics of Pseudomonas P & Tu aeruginosa to identify habitat-specific determinants of pathogenicity. Int J Med Microbiol 297: 615–623. €lbeling F, Munder A, Kerber-Momot T, Neumann D, Hennig C, Wo €mmler B & Baumann U (2011) Lung function and Hansen G, Tu inflammation during murine Pseudomonas aeruginosa airway infection. Immunobiology 216: 901–908.
Pathogens and Disease (2014), 72, 74–77, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
77
SHORT COMMUNICATION
In vivo imaging of bioluminescent Pseudomonas aeruginosa in an acute murine airway infection model €lbeling1, Jens Klockgether1, Lutz Wiehlmann1 & Burkhard Tu €mmler1,2 Antje Munder1, Florian Wo 1 Clinical Research Group, Hannover Medical School, Clinic for Pediatric Pneumology, Allergology and Neonatology, Hannover, Germany 2 Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research, Hannover, Germany
This short communication describes in vivo imaging of bioluminescent bacteria as a tool to monitor murine airway infection caused by Pseudomonas aeruginosa. Using a virulent strain of the bacterial pathogen the inoculation of a sufficiently high dose to make the infection visible is followed by rapid death. Therefore, the authors apply a strain with reduced virulence to monitor airway infection over time. This may provide a useful approach for other pathogens with the same potential problem.
Keywords bioluminescence; Pseudomonas aeruginosa; in vivo imaging; infection monitoring; transposon mutant; mouse model. Correspondence Antje Munder, Carl-Neuberg Strasse 1, Hannover D-30625, Germany. Tel.: +49 511/532-5889 fax: +49 511/532-6723 e-mail: [email protected] Received 6 March 2014; revised 29 April 2014; accepted 2 May 2014. Final version published online 30 June 2014.
Abstract Non-invasive bioluminescence imaging allows the analysis of infectious diseases in small animal models. In this study, an acute airway infection of C3H/HeN mice with luxCDABE transformed Pseudomonas aeruginosa TBCF10839 and an isogenic transposon mutant was followed by optical imaging in vivo. Using the disease-causing dose of 2.0 9 106 CFU of the cystic fibrosis airway isolate TBCF10839, subtle luminescence of the lungs was inconsistently visible for the first hour after infection. Conversely, using a 100-fold higher dose of the strongly virulence-attenuated transposon mutant, the robust signal of bioluminescent bacteria increased over 24 h. To monitor murine airway infections with P. aeruginosa in vivo by bioluminescence, one should select an attenuated mutant of a virulent strain or a wild type strain that naturally lacks virulence determinants and/ or that has acquired a low virulence persister phenotype by patho-adaptive mutations.
doi:10.1111/2049-632X.12184 Editor: Nicholas Carbonetti
Bioluminescence imaging allows the analysis of bacterial infectious diseases in animal models (Chang et al., 2011; Kong et al., 2011). During the last 20 years, non-invasive imaging studies have been performed with numerous micro-organisms including Pseudomonas aeruginosa (reviewed by Andreu et al., 2011). Here, we tested the suitability of the in vivo monitoring of acute lung infections with bioluminescent P. aeruginosa in a standardized murine airway infection model (Munder et al., 2011). The cystic fibrosis (CF) airway isolate TBCF10839 (Klockgether et al., 2013) whose virulence in murine airways had been estab€lbeling lished in previous studies (Munder et al., 2011; Wo et al., 2011), and its isogenic virulence-attenuated transposon mutant D8A6 that displays an inactivated ORF 5PG21 in genomic island PAGI-5 (Wiehlmann et al., 2007; Klockgether et al., 2013) were used in this study. Luminescent derivatives of the two strains were generated by integrating 74
a luxCDABE luciferase reporter gene operon into the bacterial chromosome. Therefore, a miniCTX-lac-lux plasmid was isolated from recombinant Escherichia coli Topo-one-shot CTX-lac-lux cells (DiGiandomenico et al., 2007) and integrated site-specifically to the attB-site of the P. aeruginosa chromosome via electroporation (Choi et al., 2006). The functional efficiency of the insertion was controlled by measuring luminescent signals with the IVIS system. To excise the miniCTX backbone, we performed a Flp-FRT recombination (Hoang et al., 1998; Hoang et al. 2000). The kinetics of the infection was followed by the sensitive IVISâ Imaging System 200 Series (Perkin Elmer, Waltham, MA; formerly manufactured by Xenogen, Alameda, CA). To monitor infections with P. aeruginosa in the mouse, the IVIS system has so far primarily applied to systemic (BitMansour et al., 2002), burn (Hamblin et al., 2003;
Pathogens and Disease (2014), 72, 74–77, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
A. Munder et al.
In vivo imaging of P. aeruginosa in mice
2002) with doses of 2.0 9 106 and 2.0 9 108 CFU of P. aeruginosa strains TBCF10839lux or D8A6lux. For survival experiments, mice were infected with 7.5 9 106 CFU, a dose which resulted in 50% lethality of the animals within the first 120 h of infection for strain TBCF10839. In contrast, the same number of D8A6 bacteria was not able to induce
Burkatovskaya et al., 2006; Barman et al., 2011) and biofilm (Kadurugamuwa et al., 2003), but less to airway infection models (DiGiandomenico et al., 2007; Riedel et al., 2007; Ramphal et al., 2008). For in vivo imaging, C3H/HeN mice were infected by view-controlled intratracheal instillation (i.t.) (Munder et al., (b)
Number of mice
10 8 6
LD 50
4 2 0
0
48
96
144
192 [h]
10
8 6 4 2 0 0
(d)
(e)
(g)
(h)
(i)
(j)
(k)
(c) Disease score [points]
Loss of body weight [g]
(a)
(l)
48
96
144
192 [h]
8 6 4 2 0
0
48
96
144
192 [h]
(f)
I
II
Color Bar Min = 23.82 Max = 116,9
Color Bar Min = 6900 Max = 20911
III
Color Bar Min = 85951 Max = 8.8e+06
(m)
Fig. 1 (a–c) Physiological parameters of C3H/HeN mice after i.t. infection with 7.5 9 106 CFU of Pseudomonas aeruginosa TBCF10839 (closed squares; n = 12) and the isogenic mutant D8A6 (open circles; n = 12). (a) Survival. The applied dose of 7.5 9 106 CFU was equivalent to a LD50 for strain TBCF10839, but did not cause any mortality in D8A6 infected mice. (b) Body weight. TB10839 infected mice displayed a dramatic loss of body weight under infection, D8A6 infected animals showed milder weight loss, started regaining of weight earlier (72 h p.i.) and were almost able to return to their initial weight within the observation period of 192 h. (c) Disease score. The body condition of mice was strongly impaired under TBCF10839 infection, in contrast, all D8A6 infected animals recovered within 48 h p.i. to normal body condition. (d) TBCF10839 shows a strong purulent inflammation in murine lungs with intra- and peribronchiolar infiltrates of leucocytes and perivascular oedema 48 h after i.t. infection with 7.5 9 106 CFU. (e) Only single leucocytes are seen 48 h after i.t. infection around the bronchi with the same dose of the isogenic mutant D8A6 (f) Vehicle control, 48 h post instillation of 30 lL PBS, original magnification 9 200, bar 50 lm, H&E. (g–m) In vivo monitoring of P. aeruginosa in acute murine airway infection with the IVISâ imaging system (binning 4, exposure time 2 m, f/stop 1, FOV 12,8 cm). Bacterial distribution was monitored 0 h (Fig. 1g, i and k), 6 h (Fig. 1h, j and l) and 24 h (Fig. 1m) after i.t. instillation with TBCF10839.lux and D8A6.lux in infected mice. Infection doses were 2.0 9 106 CFU (TBCF10839.lux: Fig. 1g/h; D8A6: Fig. 1i/j) and 2.0 9 108 CFU (for D8A6.lux exclusively; Fig. 1k–m). Animals were depiliated using hair removal cream, imaged ventrally and although under anaesthesia additionally fixed with adhesive tape to reduce movement artefacts during imaging. Luminescence is observed emanating from the nasopharynx, tracheas and lungs. The colour bars indicate the intensity of the luminescent signal, with red and blue serving as the high and low signals, respectively (Fig 1, bar I: g–k; bar II: l; bar III: m).
Pathogens and Disease (2014), 72, 74–77, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved
75
In vivo imaging of P. aeruginosa in mice
any mortality (Fig. 1a). A similar picture emerged when looking on the loss of body weight, which was dramatically for TB10839 infected mice, whereas D8A6 infected animals showed milder weight loss and were able to compensate this within the observation period of 192 h (Fig. 1b). The multiparametric disease score of infected mice (Munder et al., 2005) displayed a strongly affected body condition in TB10839 infected mice, but D8A6 infected animals showed only slight impairment (Fig. 1c). Lung slices were examined pathohistologically for inflammatory changes. Whereas TBCF10839 had caused an acute catarrhalic-suppurating alveolar pneumonia in the murine lungs (Fig. 1d), only very slight signs of inflammation, such as a minor migration of PMNs and alveolar macrophages into the alveoli, could be observed in the lungs of D8A6 infected mice (Fig. 1e) compared with mock infected mice instilled with PBS (Fig. 1f). For in vivo monitoring with the IVIS system, imaging was started directly (0 h) after instillation of 2.0 9 106 CFU and 2.0 9 108 CFU of strains TBCF10839lux and D8A6lux and was repeated at 6, 12 and 24 h p.i. Prior to infection and to improve the luminescent signals, anaesthetized mice were depiliated ventrally and during imaging fixed with adhesive tape to minimize movement artefacts. Best results were achieved with a high resolution at binning 4 (that means four by four pixels had been grouped to form one larger pixel, thereby increasing the signal-to-noise ratio), a f/1 camera lens aperture and exposure times of 2–4 min. The Living Image Softwareâ 2.50 (Perkin Elmer, Waltham, MA) was utilized to acquire, store and analyse the images with the connected computer. Occasionally, luminescent signals were detected around the mouth and along the trachea of the mice at 0 h p.i. (Fig. 1i), indicating a contamination of the upper airways with bacteria during the instillation. These signals disappeared within the next few hours (Fig. 1h, j and l), but re-emerged as time passed, presumably caused by multiplying of the bacteria in the lower airways (Fig. 1m). In the lungs, luminescence either emerged immediately after instillation or at the next time point of 6 h (Fig. 1h and k–m). Using the disease-causing dose of 2.0 9 106 CFU of TBCF10839, subtle luminescence was observed over the left or right lung in several, but not all mice (Fig. 1h). The luminescent signal had disappeared by 24 h indicating that bacterial numbers had decreased below the levels of detection (data not shown). Conversely, using a 100-fold higher dose of the virulence-attenuated D8A6 mutant, the luminescent bacteria showed signals with the IVIS that increased over time during the observation period of 24 h (Fig. 1k). This study demonstrates that the monitoring of an acute airway infection of mice with bioluminescent P. aeruginosa is feasible. However, in case of virulent P. aeruginosa, the possible dose is too low to reliably follow the 5-day natural €lbeling course of the acute infection up to recovery (Wo et al., 2011) by in vivo imaging of the bacteria. A range of two orders of magnitude of the P. aeruginosa inoculum evokes the full spectrum of responses in the mouse from subtle signs of morbidity to full-blown lethality. According to our experience, the LD50 of most P. aeruginosa strains from 76
A. Munder et al.
environment or disease habitats will be in the range of 106–107 CFU. A dose of about 10% of the LD50 results in a solid clinical manifestation of infection without mortality €lbeling et al., 2011). These doses result in a satisfactory (Wo imaging of bioluminescent bacteria in explanted lungs ex vivo (Henken et al., 2010), but are too low for an imaging of the lower airways in vivo. As the bioluminescence signal increases with the number of organ-incorporated bacteria, P. aeruginosa strains should be selected that are attenuated in acute virulence but are still capable to persist in the murine lungs. Only then – as shown in this study for the D8A6 mutant – infection doses of more than 108 CFU become feasible. One may choose an attenuated mutant of a virulent strain – as shown in this study – or a wild type strain that naturally lacks virulence determinants such as the type III secretion system and/or that has acquired a low virulence persister phenotype by patho-adaptive mutations.
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