Renaissance of the Goldstandard

Transcription

1 Renaissance of the Goldstandard (?) Fluorogenic Enzyme Substrates in the Detection and Identification of Bacteria Linda Varadi CSIRO Manufacturing Contact Prof Paul Groundwater Faculty of Pharmacy The University of Sydney Methods for the detection and identification of pathogenic bacteria: past, present, and future Chem. Soc. Rev., 2017, 46, 4811.

2 The World is facing an Antibiotic Apocalypse Prof Sally Davies Deaths per annum attributable to AMR J O Neill et al. Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. The Review on Antimicrobial Resistance Predicted loss in global output if unchanged: $

3 Does Surveillance Work? Study: MRSA clinical disease rates in 3 consecutive periods in hospitals of >40,000 admissions p.a. (US) PCR-based nasal screening for MRSA topical decolonisation Contact Isolation of MRSA + patients Prevalence Density of MRSA cases / 10,000 patient days No Surveillance 8.9 MRSA Surveillance on ICU admissions 7.4 Robicsek et al. Ann Intern Med 2008, 148, 409. MRSE Surveillance on ALL admissions

4 Does Surveillance Work? YES Study: MRSA clinical disease rates in 3 consecutive periods in hospitals of >40,000 admissions p.a. (US) PCR-based nasal screening for MRSA topical decolonisation Contact Isolation of MRSA + patients Prevalence Density Additional of MRSA cases benefits: / 10,000 patient days No Surveillance 1319 less transmissions of MRSA reduction in disease occurring up to 30 days after discharge 8.9 MRSA Surveillance on ICU admissions 7.4 Robicsek et al. Ann Intern Med 2008, 148, 409. MRSE Surveillance on ALL admissions

5 Methods for the detection and identification of pathogenic bacteria culture media : - fluorogenic enzyme substrates for detection - suicide substrates for L-alanine racemase for selective growth inhibition / enrichment

6 Detection of bacteria using fluorogenic enzyme substrates Culture media based application on clinical isolates Fl. Substrate Non-fluorescent OR significantly shorter λ em relative to the fluorophore to be released Soluble in the detection media Ready uptake by bacteria Only hydrolysed by bacteria of interest Structural stability under storage and incubation conditions Ideal properties Released fluorophore Strong fluorescence OR significant red shift of λ em relative substrate (80-100nm) Large Stokes-shift (>80 nm) for minimal background Exists in fluorescent form at physiological ph Retained by the bacteria (solid media) for localisation Lasting photochemical properties

7 Examples of target enzymes in the detection of bacteria Esterases / Phosphatases / Glycosidases / β-lactamases AMR screening Greater rate of fluorescence at physiological ph Aminopeptidases Identification Nitroreductases General bacterial presence Specificity

8 Amino-fluorophores as aminopeptidase substrates Aminopeptidases Identification Specificity Proof-of-principle: Specific target enzyme: Pseudomonas aeruginosa β-alanyl aminopeptidase

9 Aminonaphthyridines β-alanine aminopeptidase (BAP) substrates for the detection of BAP producer Pseudomonas aeruginosa in clinical isolates + P. aeruginosa NCTC E. coli NCTC Selectively hydrolysed by BAP producers - Emission overlap with natural cell fluorescence Varadi et al. Org Biom Chem 2012, 10, 2578.

10 Aminonaphthyridines Substitution on fluorophores defines signal release >5 B. cepacia LMG 1222 P. rettgeri NCTC 7475 M. morganii Emission intensity increases after hydrolysis Wavelength unchanged Varadi et al. Org Biom Chem 2012, 10, Intensity unchanged before and after hydrolysis Wavelength shifts Pre-hydrolysis: 389 nm Post-hydrolysis: 428 nm Stokes shift: 39 nm Growth inhibition of polymixin susceptible cells lines longer lipophilic chains cell wall disruption

11 Aminonaphthyridines Substitution on fluorophores defines signal release >5 B. cepacia LMG 1222 Amino fluorophores, although selectively hydrolysed, they exist in a mixture of FLUORESCENT AND NON-FLUORESCENT FORMS under physiological ph. P. rettgeri NCTC 7475 Thus, DELAYED DETECTION of the enzyme activity will occur due M. morganii to greater amount of enzymatic hydrolysis required to reach detectable signal release. Emission intensity increases after hydrolysis Wavelength unchanged Varadi et al. Org Biom Chem 2012, 10, Intensity unchanged before and after hydrolysis Wavelength shifts Pre-hydrolysis: 389 nm Post-hydrolysis: 428 nm Stokes shift: 39 nm Growth inhibition of polymixin susceptible cells lines longer lipophilic chains cell wall disruption

12 How to combine specificity of aminopeptidases with improved signal release of phenolic fluorophores? Aminopeptidases Identification Greater rate of fluorescence at physiological ph Specificity

13 Phenolic fluorophores as aminopeptidase substrates Improving detection time via increased signal intensity Aminopeptidase requires: - N-terminal β-alanine - Amide bond on C-terminal to fluorophore Detection requires: - Robust fluorescence on physiological ph after bond cleavage between β- alanine and fluorophore

14 Relative Fluorescence Units 7-aminocoumarin vs. 7-hydroxycoumarin Sensitive and selective detection of β-alanine aminopeptidase activity in both solid and liquid culture media Currently-in-use control <7 hours Pseudomonas aeruginosa β-alanyl aminopeptidase producers Lack of β-alanyl aminopeptidase Number of cycles (1 cycle = 15 minutes) Varadi et al. RSC Advances 2016, 6,

15 Shifting gears Longer wavelength fluorophores Aminonaphthalenesulfonamides aimed for the detection of β-alanine aminopeptidase activity In Organic Solvent - THF F lu o re s c e n c e In te n s ity (a.u.) W a v e le n g th (n m ) λex (nm) λem (nm) Stokes shift Substrate Fluorophore In Aqueous Growth Media Luo et al. Dyes and Pigments 2016, 125, 15.

16 Shifting gears Longer wavelength fluorophores Naphthalimides fluorescence Indication of GENERAL BACTERIAL PRESENCE due to localised No Substrate λ em λ ex Stokes shift 443 nm 510 nm 67 nm Luo et al. Dyes and Pigments 2016, 125, 15. Spot Organism Spot Organism 1 Escherichia coli 11 Steptococcus pyogenes 2 Klebsiella pneumoniae 12 MRSA 3 Providencia rettgeri 13 Staphylococcus aureus 4 Enterobacter cloacae 14 Staphylococcus epidermidis 5 Serratia marcescens 15 Listeria monocytogenes 6 Salmonella typhimurium 16 Enterococcus faecium 7 Pseudomonas aeruginosa 17 Enterococcus faecalis 8 Yersinia enterocolitica 18 Bacillus subtilis 9 Burkholderia cepacia 19 Candida albicans 10 Acinetobacter baumannii 20 Candida glabrata With Substrate

17 Effect of substituents on growth Longer, more lypophilic chains result in broader inhibitory profile CONTROL 1 E. coli 11 S. pyogenes 2 K. pneumoniae 12 S. aureus (MRSA) 3 P. rettgeri 13 S. aureus 4 E. cloacae 14 S. epidermidis 5 S. marcescens 15 L. monocytogenes 6 S. typhimurium 16 E. faecium 7 P. aeruginosa 17 E. faecalis 8 Y. enterocolitica 18 B. subtilis 9 B. cepacia 19 C. albicans 10 A. baumannii 20 C. glabrata

18 Fuorescence Intensity (a.u) Shifting gears Longer wavelength fluorophores Gram-negative organisms Styryl-coumarin derivative detecting P. AERUGINOSA Untouched fluorescence via in non-bap quenching producers by hydrolysis Substrate hydrolysed by BAP-producer(s) λ em λ ex Stokes shift 482 nm 522 nm 40 nm E. E. coli S. enteritidis S. P. aeruginosa P. BAP producer SUBSTRATE BEFORE HYDROLYSIS Gram-positive organisms Growth inhibitory effect of substrate on Gram-positive organisms Unpublished results FLUOROPHORE AFTER HYDROLYSIS Wavelength (nm) S. aureus E. faecalis

19 Shifting gears Longer wavelength fluorophores Styryl-coumarin derivative for the detection of GRAM-NEGATIVE BACTERIA Control Visible Light 365 nm Gram-negative: Localised green fluorescence a b c d Unpublished results 1 E. coli 11 S. pyogenes 2 K. pneumoniae 12 S. aureus (MRSA) 3 P. rettgeri 13 S. aureus 4 E. cloacae 14 S. epidermidis 5 S. marcescens 15 L. monocytogenes 6 S. typhimurium 16 E. faecium 7 P. aeruginosa 17 E. faecalis 8 Y. enterocolitica 18 B. subtilis 9 B. cepacia 19 C. albicans 10 A. baumannii 20 C. glabrata Gram-positive and yeasts: Growth inhibition

20 Shifting gears Longer wavelength fluorophores 3-Hydroxyflavone derivative with blue to yellow emission change upon hydrolysis by BAP PRODUCERS and GROWTH INHIBITION of some cell lines CONTROL VISIBLE LIGHT 365 NM BAP producers: yellow Non-BAP: blue Unpublished results Organism Organism 1 E. coli 11 S. pyogenes 2 K. pneumoniae 12 S. aureus (MRSA) 3 P. rettgeri 13 S. aureus 4 E. cloacae 14 S. epidermidis 5 S. marcescens 15 L. monocytogenes 6 S. typhimurium 16 E. faecium 7 P. aeruginosa 17 E. faecalis 8 Y. enterocolitica 18 B. subtilis 9 B. cepacia 19 C. albicans 10 A. baumannii 20 C. glabrata Inhibition of some MDR cell lines

21 Hidden gems Testing synthetic intermediates Styryl-phthalate displays blue fluorescence in MRSA and inhibits growth of MSSA, S. EPIDERMIS, E. FAECALIS Unpublished results Organism Organism 1 E. coli 11 S. pyogenes 2 K. pneumoniae 12 S. aureus (MRSA) 3 P. rettgeri 13 S. aureus 4 E. cloacae 14 S. epidermidis 5 S. marcescens 15 L. monocytogenes 6 S. typhimurium 16 E. faecium 7 P. aeruginosa 17 E. faecalis 8 Y. enterocolitica 18 B. subtilis 9 B. cepacia 19 C. albicans 10 A. baumannii 20 C. glabrata Inhibition of some MDR cell lines but MRSA Potential enrichment agent

22 Summary Customisable fluorescent enzyme substrates FLUOROPHORE + LINKER + TARGETING MOLECULE ALTERED TO NEEDS Use of phenolic fluorophores as aminopeptidase substrates for enhanced fluorescence resulting in reduced detection time Longer, lypophilic substrates also act as selective growth inhibitors Unexpected activity profiles inspire new directions

23 Acknowledgements Prof Paul Groundwater Prof Dai Hibbs Dr Ramesh Mamidi Dr Jia Lin Luo Mr Terry Jin Mr Jin Tan Mr Miaoyi Wang Prof John D. Perry Dr Sylvain Orenga Prof Roz Anderson Dr Andy Hall Dr Mark Gray