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  1. 1. NEW APPROACH TO BACTERIAL DIAGNOSTICS: 2-METHYLBUTANAL AS A VOLATILE ORGANIC BIOMARKER FOR PROTEUS FOR DEVELOPING PROTEAL, A RAPID AND NON-INVASIVE DETECTION METHOD AND RATIONAL DESIGN OF ITS DIAGNOSTIC CULTURE MEDIUM A THESIS Submitted by AARTHI R in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY FACULTY OF TECHNOLOGY ANNA UNIVERSITY CHENNAI 600 025 JULY 2015
  2. 2. ii ANNA UNIVERSITY CHENNAI 600 025 CERTIFICATE The research work embodied in the present Thesis entitled “NEW APPROACH TO BACTERIAL DIAGNOSTICS: 2-METHYLBUTANAL AS A VOLATILE ORGANIC BIOMARKER FOR PROTEUS FOR DEVELOPING PROTEAL, A RAPID AND NON-INVASIVE DETECTION METHOD AND RATIONAL DESIGN OF ITS DIAGNOSTIC CULTURE MEDIUM” has been carried out in the Centre for Biotechnology, Anna University, Chennai - 600 025. The work reported herein is original and does not form part of any other thesis or dissertation on the basis of which a degree or award was conferred on an earlier occasion or to any other scholar. I understand the University’s policy on plagiarism and declare that the thesis and publications are my own work, except where specifically acknowledged and has not been copied from other sources or been previously submitted for award or assessment. AARTHI R Dr. K. SANKARAN RESEARCH SCHOLAR SUPERVISOR Professor Centre for Biotechnology Anna University Chennai – 600 025
  3. 3. iii ABSTRACT Control of infectious diseases through early identification of pathogens, or better still, surveillance to eradicate is becoming more and more meaningful with the emergence of Multi-drug-resistance (MDR) and spread of dangerous pathogenic forms from hospitals to communities. The most common and prevalent Urinary Tract Infections (UTI) are also one of the most neglected infectious diseases. The classical and current techniques for diagnosis are not effective for a variety of reasons including the nature of the diagnostic targets and methods. Hence, its treatment is quite challenging making it imperative to develop quick diagnosis and render antibiotic treatment effective. Taking one of the notorious nosocomial causative bacterium, Proteus, we have addressed the challenge making a paradigm shift in the approach of detecting the bacteria. In this regard, Volatile Organic Compounds (VOCs) which are secreted as defense against antagonists or as signalling molecules by the organisms under specific conditions through specific biochemical pathways were exploited. In the case of Proteus, 2-methylbutanal identified by GC-MS was found to be the characteristic volatile compound released in Luria Bertani (LB) broth. Using this compound we were able to develop a simple test in 96- well microplate format that can be directly applied to the 7 h culture of the bacterium to give a yes-or-no type of response for fluorimetric detection. The assay, named ProteAl, (Prote, “Proteus” & Al, “Aldehyde”) involves instant reaction of 5-dimethylaminonaphthalene-1-sulfonylhydrazine (DNSH) with
  4. 4. iv 2-methylbutanal under acidic condition to give green fluorescence (other organisms show orange fluorescence). This diagnostic assay has been tested using 39 standard and 56 known clinical strains representing frequently encountered uropathogens including {27 Proteus (both mirabilis and vulgaris), 27 E.coli, 8 Klebsiella, 10 Staphylococcus, 7 Pseudomonas}, 2 Enterobacter, 2 Citrobacter, 7 Salmonella, 4 Shigella and 200 environmental soil strains. The sensitivity and specificity of this high-throughput assay performed in 96-well format were 100% under laboratory conditions and therefore forms the basis for larger clinical validation. This cost-effective diagnostic tool will be useful in hospitals, peripheral clinics, epidemiological studies and environmental surveillance. Metabolic pathway and regulation studies (including qPCR) based on the limited reports available in a few other systems revealed the presence of functional pathway in Proteus and its regulation through Isoleucine (Ile) and Thiamine pyrophosphate (TPP). This led to the designing of LB-Ile medium with 15 mM isoleucine in LB to enhance the production of the biomarker 2.5 times more than normal. The growth in the rationally designed medium and ProteAl now would provide a convenient diagnostic tool for identifying this bacterium from clinical samples within 7 h. The expression of alpha-ketoacid decarboxylase (kdcA) of Proteus grown in LB-Ile medium revealed a seven-fold increase in expression compared to normal LB. This indicated to the operation of transcriptional control in Proteus and this is the first such report revealing the existence of isoleucine catabolism in Proteus (mirabilis and vulgaris).
  5. 5. v Though we have focused on Proteus associated with UTI, the method is genus specific and therefore can be used for other disease conditions. The development of such cost effective, non-invasive and non- destructive method has been shown to be readily amenable for simple imaging based instrumentation (like gel doc) for routine clinical use. In conclusion, we have taken a new approach towards next generation diagnostic method for infectious bacteria that can be readily adapted to instrumentation and automation.
  6. 6. vi ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my guide Prof. K. Sankaran for providing me an excellent opportunity to work in this challenging field of research. I graciously thank him for all the stimulating scientific discussions and the constant encouragement to aim high scientific standards. I sincerely thank Prof. P. Gautham, Director, Centre for Biotechnology for his support during my Ph.D. I am also grateful to my doctoral committee members, Dr. Venkatesh Balasubramanian, IIT-Madras and Dr. M. Ramalingam (Retd.) Anna University, for their helpful suggestions. I profoundly thank Prof. G.M. Samuel Knight, Director CPDE for his support and encouragement. I am grateful to Mr. Suresh Lingham, M/s Trivitron Pvt Ltd. for clinical samples, Dr. Mathiyarasu and Sankararao, CECRI, Karaikudi, Dr. T. Sivakumar, Prof. B. Sivasankar Anna University, Prof. Mohanakrishnan, University of Madras, Dr. A. Alagumaruthanayagam and B. Palanisamy for analysis and analytical data. I would like to specifically thank my seniors, fellow colleagues and all scholars of CBT for their constant encouragement and support. I owe my sincere gratitude to all technical and non-technical staffs of CBT for their support. I thank UGC-BSR, CPEES and CSIR-SRF for their financial assistance during my research. Heartfelt thanks to my husband Mr. M. Thiruvengadam and my in-laws for their encouragement. Lastly, I must say that I would not be where I am without the unending support of my parents Late. Mr. S. Raju, Mrs. Mangai Raju and all others in my family. I am indebted to them. Their moral support all through these years of my research is the driving force behind this achievement. AARTHI R
  7. 7. vii TABLE OF CONTENTS CHAPTER NO. TITLE PAGE NO. ABSTRACT iii LIST OF TABLES xvi LIST OF FIGURES xviii LIST OF SYMBOLS AND ABBREVIATIONS xxvi 1 INTRODUCTION 1 1.1 INCREASING BURDEN AND THREAT OF INFECTIOUS DISEASES 1 1.1.1 Nosocomial Infections, Complicating Factor in the Control 8 1.1.2 Multi-drug-resistance is a Major Threat and Challenge 10 1.2 INADEQUACY OF CLASSICAL AND CURRENT DIAGNOSTIC METHODS AND LACK OF SCREENING AND SURVEILLANCE METHODS FOR PREVENTIVE HEALTHCARE 13 1.2.1 Limitations of Emerging Modern Methods 14 1.3 NEED FOR NEW APPROACHES TO DEVELOP NEXT GENERATION TOOL WITH MODERN KNOWLEDGE 17 1.3.1 Intra and Extracellular Targets for Non-invasive and Non-destructive Detection Methods 17 1.3.2 Volatile Organic Compounds (VOCs) as Extracellular Targets 19
  8. 8. viii CHAPTER NO. TITLE PAGE NO. 1.4 CURRENT METHODS FOR DETECTION OF VOLATILE ORGANIC COMPOUNDS (VOCs) 21 1.4.1 Colorimetric Sensor Array 21 1.4.2 Fluorescent Method for VOC Detection 22 1.4.3 Gas Chromatography and Mass Spectroscopy (GC-MS) 23 1.4.4 Biosensors 25 1.4.5 E-nose 26 1.5 REGULATION OF VOLATILE ORGANIC COMPOUND METABOLISM 27 1.6 RATIONAL DESIGN OF MEDIA FOR ENHANCED VOLATILE ORGANIC COMPOUND PRODUCTION 30 1.7 PROTEUS AS A MODEL STUDY ORGANISM 31 1.7.1 Proteus –General Introduction 32 1.7.2 Pathogenesis and Diseases Caused by Proteus 33 1.7.3 Proteus as a Nosocomial Organism 36 1.8 OVERVIEW OF THE THESIS 37 1.9 OBJECTIVES 39 2 MATERIALS AND METHODS 41 2. 1 MATERIALS USED IN THIS STUDY 41 2.1.1 Chemicals Used 41 2.1.2 Buffers used in this Study 44 2.1.3 Cheminformatic Analysis of Bacterial Volatile Organic Compound 45 2.1.4 Bacterial Strains used in the Study 45
  9. 9. ix CHAPTER NO. TITLE PAGE NO. 2.1.4.1 Standard strains 45 2.1.4.2 Clinical isolates 46 2.2 PREPARATION OF GROWTH MEDIUM AND TEST METHOD 48 2.2.1 Antibiogram Medium 48 2.2.2 Catalase Test 48 2.2.3 Cetrimide Agar Test 48 2.2.4 Eosin Methylene Blue Agar (EMB) Test 49 2.2.5 Luria Bertani Broth 49 2.2.6 Luria Bertani Agar 49 2.2.7 Methyl Red and Voges Proskauer (MR-VP) Test 49 2.2.8 Motility Test Agar 50 2.2.9 Nutrient Broth 50 2.2.10 Phenylalanine Deaminase Test 50 2.2.11 Salmonella Shigella Agar 51 2.2.12 Simmons’ Citrate Agar 51 2.2.13 Triple Sugar Iron Agar 51 2.2.14 Tryptone Soya Broth 51 2.2.15 Tryptone Broth 51 2.2.15.1 Indole test method 52 2.2.16 Urea Broth 52 2.3 GENOMIC DNA ISOLATION 52 2.3.1 Agarose Gel Electrophoresis 53 2.3.2 Polymerase Chain Reaction (PCR) 54 2.4 EXTRACTION OF VOLATILE ORGANIC COMPOUNDS (VOCS) FROM CULTURE 54
  10. 10. x CHAPTER NO. TITLE PAGE NO. 2.5 INSTRUMENTAL METHODS FOR VOC IDENTIFICATION 56 2.5.1 Gas Chromatographic (GC) Analysis 57 2.5.2 Gas Chromatography-Mass Spectroscopy (GC-MS) Analysis 57 2.5.2.1 GC 57 2.5.2.2 MS 57 2.5.3 Fourier Transform-Infrared (FT-IR) Analysis 58 2.5.4 Comparative Analysis of Pure Compound and the Characteristic VOC from Proteus using Gas Chromatography 58 2.6 DEVELOPMENT OF SURVEILLANCE METHOD FOR IDENTIFICATION OF CHARACTERISTIC VOC 58 2.6.1 Colorimetric Assay for Carbonyl Volatile Organic Compounds 59 2.6.2 Fluorescent Dye Reagent Specific for Carbonyl Compounds 59 2.7 STANDARDIZATION OF DNSH ASSAY FOR CARBONYL COMPOUNDS 60 2.8 FLUORESCENCE BASED DNSH ASSAY (PROTEAL) FOR DETECTION OF PROTEUS SPECIES 61 2.9 TESTING THE VOLATILITY OF 2-METHYLBUTANAL FROM CULTURE 62 2.10 LABORATORY VALIDATION OF THE PROTEAL ASSAY 62
  11. 11. xi CHAPTER NO. TITLE PAGE NO. 2.11 SENSITIVITY AND SPECIFICITY CALCULATION 63 2.12 IDENTIFICATION OF THE METABOLIC PATHWAY USING BIOLOGICAL DATABASES 64 2.13 RATIONAL DESIGN OF GROWTH MEDIUM FOR ENHANCED 2-METHYLBUTANAL PRODUCTION 64 2.13.1 Study on the Effect of Branched Chain Amino Acids on 2-methylbutanal Production 65 2.13.2 Study on the Effect of TPP for 2-methylbutanal Production 65 2.14 REGULATION OF THE METABOLIC PATHWAY INVOLVED IN 2-METHYLBUTANAL PRODUCTION 66 2.14.1 Extraction of Total RNA from Proteus Culture 66 2.14.2 Conversion of RNA to cDNA 67 2.14.3 Quantification of Gene Expression using Real-time PCR (qPCR) 67 3 RESULTS 69 3.1 A NON-DESTRUCTIVE APPROACH FOR PATHOGEN DETECTION USING VOLATILE ORGANIC COMPOUNDS 69 3.1.1 VOC Biomarkers Found in Various Uropathogens 70
  12. 12. xii CHAPTER NO. TITLE PAGE NO. 3.1.2 Microbiological, Biochemical and Molecular Techniques Identifies the Uropathogens 77 3.2 SOLVENT EXTRACTION WAS THE SUITABLE METHOD FOR VOC EXTRACTION FROM CULTURE 79 3.3 GAS CHROMATOGRAM IDENTIFIED THE CHARACTERISTIC COMPOUNDS OF PROTEUS AND SALMONELLA CULTURE EXTRACT 80 3.3.1 Identification of 2-methylbutanal as Specific VOC for Proteus using GC-MS and FT-IR 82 3.3.2 Comparative Analysis of the Gas Chromatogram of 2-methylbutanal and DCM-extract of Proteus Confirmed 2-methylbutanal as the Characteristic VOC of Proteus 85 3.4 DETECTION OF VOLATILE CARBONYLS USING COLORIMETRIC AND FLUORIMETRIC REAGENTS 86 3.4.1 Colorimetric Reagent Detected Micromole Levels of VOCs 86 3.4.2 Standardization of the Fluorescent Reagent Showed Better Sensitivity 87 3.4.2.1 Identification of carbonyl compounds using fluorescent reagent 2,4-DNSH 88
  13. 13. xiii CHAPTER NO. TITLE PAGE NO. 3.4.2.2 Development of 96-well based fluorimetric assay for detection of carbonyl compounds using the optimized reagent 89 3.4.2.3 Fluorescence shift was observed between Proteus and non-Proteus organisms 90 3.4.2.4 ProteAl is found specific to Proteus among the commonly occurring Uropathogens 92 3.4.2.5 The amount of 2-methylbutanal from Proteus culture was quantified 93 3.4.2.6 The volatile component responsible for green fluorescence in ProteAl was confirmed to be 2-methylbutanal 95 3.4.2.7 The characteristic 2-methylbutanal was highly volatile 96 3.5 VALIDATION OF THE ASSAY USING VARIOUS CLINICAL UROPATHOGENS 97 3.6 RELEASE OF 2-METHYLBUTANAL BY PROTEUS THROUGH ISOLEUCINE METABOLIC PATHWAY 100 3.6.1 In Silico Analyses Revealed the Presence of the Enzymes of Isoleucine Catabolism in Proteus 101
  14. 14. xiv CHAPTER NO. TITLE PAGE NO. 3.6.2 Enhanced Fluorescence Due to Isoleucine Supplementation in the Growth Medium 104 3.6.3 Enhancement of 2-methubutanal Production using Thiamine Pyrophosphate Supplements 106 3.6.4 LB-Isoleucine (LB-Ile) Medium Enhanced 2-methylbutanal Production Compared to other Supplemented Medium 108 3.7 TOTAL RNA WAS EXTRACTED BY PHENOL- CHLOROFORM METHOD 109 3.7.1 Total RNA was Efficiently Reverse Transcribed to cDNA 110 3.7.2 Amplified Product Showed the Presence of α-ketoacid decarboxylase (kdcA) Gene Transcript 111 3.7.3 Gene Expression of Proteus Species in LB and LB Supplemented Growth Medium 113 3.7.3.1 Isoleucine (Ile) and Thiamine pyrophosphate (TPP) addition to LB medium alters the expression of α-ketoacid decarboxylase (kdcA) Gene in P. mirabilis 113 3.7.3.2 Isoleucine (Ile) and Thiamine pyrophosphate (TPP) addition to LB medium alters the expression of α-ketoacid decarboxylase (kdcA) Gene in P. vulgaris 115
  15. 15. xv CHAPTER NO. TITLE PAGE NO. 4 DISCUSSION 118 4.1 EXTRACELLULAR VOC HAS BEEN TARGETED FOR NON-DESTRUCTIVE DIAGNOSIS 119 4.1.1 Single Step Reaction to Provide a Sensitive Method 121 4.2 REGULATION OF THE METABOLIC PATHWAY IN PROTEUS 125 4.2.1 ProteAl is Useful in Identifying Multi-drug-resistance of Proteus 127 4.2.2 ProteAl is a Convenient Signal Generating Component of Simple and Affordable Imaging based Diagnostic and Surveillance Instrumentation 127 5 CONCLUSION 130 REFERENCES 133 LIST OF PUBLICATIONS 146
  16. 16. xvi LIST OF TABLES TABLE NO. TITLE PAGE NO. 1.1 Common infectious agents, symptoms and tests currently available for their detection 3 1.2 Advantages and disadvantages of molecular methods used for bacterial identification 16 1.3 Diseases and their odours 18 1.4 Advantages and disadvantages of some of the methods currently used for VOC analysis in clinical aspect 24 2.1 List of reagents, dyes and kits 41 2.2 List of buffers used and their composition 44 2.3 List of biochemical and microbiological tests to identify E. coli, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella and Staphylococcus 47 2.4 List of organisms and their 16S rRNA Primer sequence 53 2.5 List of environmental sample collection locations 63 2.6 Table for sensitivity and specificity calculation 63 2.7 List of genes and their primer sequences 68 3.1 Reported Volatile Organic Compounds released by various bacteria in different growth medium 71 3.2 Results of the tests performed for a few uropathogens 77 3.3 Comparative VOC profiles of Proteus with medium and negative control 81 3.4 Assay sensitivity for various carbonyl compounds 89 3.5 Validation of ProteAl using standard and clinical strains 98 3.6 Environmental sample details and the strains identified 99
  17. 17. xvii TABLE NO. TITLE PAGE NO. 3.7 Multiple sequence alignment of aminotransferase in Lactococcus lactis and Proteus mirabilis sequence 102 3.8 Multiple sequence alignment of alpha-ketoacid decarboxylase in Lactococcus lactis and Proteus mirabilis sequence 103 3.9 Concentration of isoleucine and the fluorescence response of ProteAl 104 3.10 Concentration of Thiamine pyrophosphate and the fluorescence response of ProteAl 107 3.11 The fluorescence value of different supplemented growth medium obtained in three trials 109 3.12 Calculation of fold difference in P. mirabilis using 2-ΔΔCT method 114 3.13 Calculation of fold difference in P. vulgaris using 2-ΔΔCT method 115
  18. 18. xviii LIST OF FIGURES FIGURE NO. TITLE PAGE NO. 1.1 The percentage of death in developing countries caused by communicable and non-communicable diseases are represented in the pie chart. Communicable diseases account to 31% of deaths worldwide 2 1.2 The global market for treatment of infectious diseases shows an increase in economic burden due to viral and bacterial infections from 2008 to 2014 7 1.3 Different sources that cause hospital acquired infections 10 1.4 Colorimetric sensor array using metalloporphyrins, metal nanoparticles and acid-base indicators showing different coloured spots when reacted with VOC 22 1.5 Representative VOC metabolic pathway involving amino acids 29 1.6 A schematic diagram showing proteins produced by P. mirabilis that are known or hypothesized to be virulence factors important in urinary tract infections 34 1.7 A schematic diagram of the urinary tract showing urethra, bladder, ureters & kidneys and the indicating (red spots) are the diseases that are associated with Proteus. The virulence factors listed under each infection contribute to their pathogenicity 35 2.1 Charcoal adsorbant contained in a tissue paper bag was kept hanging above the culture or pure compound containing medium to facilitate adsorption for further analysis 55
  19. 19. xix FIGURE NO. TITLE PAGE NO. 2.2 Silica discs were used as VOC adsorbant as shown in pictures a-c. The adsorbed VOC were eluted using suitable solvent from the silica disc a) Silica disc cut to the size of inner dimension of the Vial cap b) Silica disc placed inside of the vial cap c) Silica disc covering the mouth of the conical flask 55 2.3 Simple VOC extraction setup using a syringe, needle and a capillary tube as shown in pictures a-c. The solvent phase which collects the VOC contained in the syringe and vial were analysed using GC-MS a) shows the VOC collection using a syringe from 1.5ml vial b) shows the VOC collection with the syringe set-up from 15ml centrifuge tube c) shows the VOC collection using a capillary tube 56 3.1 The gas chromatogram of Dichloromethane extracts of LB (media control), Proteus (positive sample) and Salmonella (negative control) cultures. The unique peak for Proteus culture at 8.227 min is denoted by an arrow 82 3.2 GC analysis of DCM extract from Proteus culture and the mass spectrum of the sample at retention time 1.78 min (a) shows the gas chromatograms of volatile organic compounds in the DCM extracts of Proteus. The characteristic peak at 1.78 min in Proteus was further analyzed for identification of mass (b) is the mass spectrum of the unique compound for Proteus at Rt. 1.78 min in GC. The fragment peak at 57 m/z is the base peak showing 100% abundance and corresponding to 2-methylbutanal. No other carbonyl compound was detected from the other peaks 83
  20. 20. xx FIGURE NO. TITLE PAGE NO. 3.3 FT-IR spectra of P. mirabilis and P. vulgaris solvent extract in comparison with 2-methylbutanal and medium blank. The Proteus samples showed the presence of carbonyl group along with the =C-H stretch corresponding to an aldehyde which is similar to the standard 2-methylbutanal. Together, the analysis was suggestive of the presence of 2-methylbutanal as the volatile organic compound in low abundance in the cultures of Proteus grown in LB 85 3.4 Comparative chromatogram of the culture extract of Proteus and standard 2-methylbutanal. The gas chromatographic peak at 2.3 min from Proteus culture extract matched with the peak for 2-methylbutanal 86 3.5 Spot detection of 2-methylbutanal vapours with 2,4 DNPH produced a bright yellow coloured product while with alcohol and blank no bright yellow coloured product was formed. Standard 2-methylbutanal ranging from 20-50 µmoles were spotted using 2,4 DNPH 87 3.6 Comparative fluorescence response of DNSH reacting with carbonyl compounds (positive) and non-carbonyl compounds (negatives) or DNSH reacting under acidic condition. The signal-to-noise ratio was high when DNSH reacts under acidic conditions. This formed the basis of the DNSH reagent preparation 88 3.7 The picture shows the fluorescence obtained from the reaction of DNSH with pure compounds. The DNSH reagent reacted with the carbonyl compounds to form respectively hydrazones showing green fluorescence while blank and acids form no product retaining the reagent’s orange fluorescence 89
  21. 21. xxi FIGURE NO. TITLE PAGE NO. 3.8 Differentiation of carbonyl (green fluorescence) and non-carbonyl compounds (orange fluorescence). Carbonyl Compounds used: Hexanal, Nonanal, 2-methylbutanal, Benzaldehyde, Decanal, 2-nonanone, 2-tridecanone, 2-heptanone, 2-undecanone, 2-pentanone, Acetophenone, Non-carbonyl compounds- alcohols: Propanol, Ethanol, Methanol, Butanol and acids: Propionic acid, Phosphoric acid and Butyric acid all added in duplicates 90 3.9 Determination of Ex. /Em. λmax for pure compounds and bacterial cultures. The emission spectra on the left (excitation 336 nm) (a) are of pure carbonyl (hexanal and 2-heptanone), acid (propionic acid) and alcohol (butanol) compounds after reaction with DNSH under the assay conditions. The emission spectra on the right (b) are of the cultures of Proteus, UPEC and Salmonella after reaction with DNSH under the assay conditions 91 3.10 Performance of DNSH reagent on a set of standard strains distinguishing Proteus (A2 to A11& B2 to B11) with green fluorescence from the LB medium blank (A1&B1) and negatives UPEC (A12&B12, D1 to D3 & E1 to E3), Klebsiella (D4, E4, D5 & E5), E. coli (D6 to D9 & E6 to E9) and Salmonella (D10 to D12 & E10 to E12) showing orange fluorescence 92 3.11 Proteus cultures grown in LB medium showed higher fluorescence response compared to the blank and other common growth media NB, AB, and TSB 93
  22. 22. xxii FIGURE NO. TITLE PAGE NO. 3.12 The fluorescence response of Proteus and other organisms after ProteAl. Proteus species showed maximum fluorescence compared to the medium blank and other bacteria, which have comparable response levels 94 3.13 The set of data in this composite figure compares the properties of pure 2-methylbutanal with those of DCM-extract from the Proteus culture (a) shows the fluorescence emission spectra of DNSH reacted with 2-methylbutanl matched with that of the spectrum obtained from the reaction of DNSH with the culture (b) is the standard graph for 2-methylbutanal using ProteAl assay showing sensitivity up to 1 nmol and good linearity up to 20 nmol (c) shows the graph of the fluorescence response for bacterial cultures using ProteAl performed every hour up to 24 h 95 3.14 2-metyhylbutanal is seen as a secretary VOC product as only the culture supernatant but not the cells of Proteus yielded green fluorescence (wells 7&8) after ProteAl 96 3.15 Volatility of 2-methylbutanal released by Proteus in comparison with pure compound. (a) shows that the fluorescence intensity of DNSH-derivatized carbonyl compound(s) in the Proteus cultures kept at room temperature (27 ºC), fridge (4 ºC) and on ice (0 ºC) reduces drastically as a function of temperature as well as duration of storage indicating volatile nature. (b) shows the fluorescence intensity of standard 2-methylbutanal experimented similar to Proteus culture at different temperatures 96
  23. 23. xxiii FIGURE NO. TITLE PAGE NO. 3.16 Validation of ProteAl using 39 standard strains and 56 clinical isolates as given in table 3.5. Out of the 95 strains screened, 27 strains gave positive results indicated by bright green fluorescence. Others including uropathogenic strains showed the background orange fluorescence 97 3.17 Validation of environmental strains. Wells G 4, 5 and H 4, 5 are duplicates of standard positive control, P. mirabilis and P. vulgaris respectively. Only Proteus strains were identified by the green fluorescence while the others gave orange fluorescence 100 3.18 The putative isoleucine catabolic pathway involved in the production of 2-methylbutanal in Proteus. The metabolic pathway uses the enzymes aminotransferase and α-ketoacid decarboxylase for conversion of acid to an aldehyde 101 3.19 Fluorescence response for only Proteus increased after addition of isoleucine in the LB medium while the negatives and blank did not show any distinct effect. The profile shows that the addition of isoleucine beyond 15mM (peak concentration) actually led to the reduction in the enzyme activity 105 3.20 The bar-diagram indicates specific increase in fluorescence of Proteus to ProteAl in LB -Ile medium compared to LB or its supplementation with related branched chain amino acids. It evidently shows that only isoleucine enhances 2-methylbutanal production 106
  24. 24. xxiv FIGURE NO. TITLE PAGE NO. 3.21 Fluorescence increased as a function of Thiamine pyrophosphate supplementation in the LB medium for Proteus. The peak indicates the concentration (2 mM) of TPP for maximal production of 2-methylbutanal. Beyond 2 mM of TPP there is a drastic reduction in 2-methylbutanal production 107 3.22 The picture shows the yield of 2-methylbutanal under growth in LB, LB-Ile, LB-TPP, LB-Ile-TPP. While LB-Ile showed the maximum 2-methylbutanal production in all the three trials 108 3.23 Ethidium bromide stained 1.5 % agarose gel shows the total RNA extracted from Proteus. Lane 1 contains a 1Kb DNA ladder. Lanes 2-4 and 5-7 contains RNA of Proteus mirabilis and Proteus vulgaris respectively 109 3.24 cDNA was synthesized from the total RNA of P. mirabilis and P. vulgaris grown in LB or LB supplemented with Ile or TPP. The cDNA preparations, which appear as smears in agarose gel electrophoresis, was used as template for qPCR amplification 110 3.25 The PCR amplified product shows distinct bands corresponding to the size of alpha-ketoacid decarboxylase gene transcript at approximately 225 bp in P. mirabilis (Fig. (a) lane 1 and Fig. (b) lanes 2&3) and P. vulgaris (Fig. (a) lane 2 and Fig. (b) lanes 4&5) 111 3.26 Sequencing results of alpha-ketoacid decarboxylase gene transcript. The red coloured basepairs denotes the sequence of kdcA gene transcript after sequencing in P. mirabilis and P. vulgaris 112
  25. 25. xxv FIGURE NO. TITLE PAGE NO. 3.27 The fold difference in PCR template from Proteus cells growing in LB, LB-Ile and LB-Ile-TPP was calculated using the 2-ΔΔCT method. The expression of α-ketoacid decarboxylase of P. mirabilis grown in LB-Ile was found to be maximum compared to LB and LB-Ile-TPP medium corroborating with enzymatic activity data 114 3.28 The expression of α-ketoacid decarboxylase of P. vulgaris grown in LB-Ile was found to be maximum compared to LB and LB-Ile-TPP medium 116 3.29 Concept diagram showing positive feedback regulation of kdcA gene through isoleucine 117 4.1 Schematic Overview of the thesis 129
  26. 26. xxvi LIST OF SYMBOLS AND ABBREVIATIONS Symbols α - Alpha cm - Centimeter o C - Degree Celsius eV - Electron Volt g - Gram h - Hour λmax - Lambda max L - Litre m/z - Mass-to-charge ratio m - Meter µg - Microgram µl - Microlitre µm - Micrometer µM - Micromolar µmol - Micromole mg - Milligram ml - Milliliter mm - millimeter mM - Millimolar min - Minute M - Molar ng - Nanogram nm - Nanometer nM - Nanomolar
  27. 27. xxvii nmol - Nanomole N - Normality % - Percentage pmole - Picomole sec - Seconds U - Unit Abbreviations DNSH - 1-Dimethylaminonaphthalene- 5-sulfonylhydrazide MDNPH - 1-methyl-1-(2,4-dinitrophenyl)hydrazine TCPH - 2,4,6-trichlorophenylhydrazine DNPH - 2,4-dinitrophenylhydrazine DAIH - 2-diphenylacetyl-1,3-indandione-1-hydrazone pNPH - 4-nitrophenylhydrazine AIDS - Acquired Immuno Deficiency Syndrome ALT - Alanine transaminase kdcA - Alpha-keto decarboxylase ABD - Aminosulfonylgroup Ap–Sm–Su–Tc–Tp - Ampicillin - streptomycin – sulfamethoxazoletetracycline- trimethoprim AB - Antibiogram medium Ab - Antibody BVOCs - Bacterial Volatile Organic Compounds Bp - Base pair BLAST - Basic Local Alignment Search Tool BCATs - Branched chain aminotransferases BAW - Bulk Acoustic Wave
  28. 28. xxviii CDC - Centre of Disease Control CAGR - Compounded annual growth rate CP - Conductive Polymer composite chemiresistors dNTP - Deoxy Nucleotide Triphosphate DNA - Deoxy Ribonucleic Acid DCM - Dichloromethane DEPC - Diethyl pyrocarbonate DBD - Dimethylaminosulfonyl group DHE - Dynamic headspace extraction EI - Electron ionization EHEC - Enterohemorrhagic Escherichia coli ETEC - Enterotoxigenic Escherichia coli EIA - Enzyme immunoassay ELISA - Enzyme linked immune sorbent assay EMB - Eosin methylene blue E. coli - Escherchia coli EDTA - Ethylene Diamine Tetra Acetic acid, di sodium salt Ex/Em - Excitation and emission wavelengths ESBL - Extended-spectrum betalactamase FID - Flame ionization detection FT-IR - Fourier Transform-Infrared GC-MS - Gas Chromatography and Mass Spectroscopy GASFET - Gas sensitive field effect transistor sensors HIV - Human Immunodeficiency Virus IgM - Immunoglobulin M IMViC - Indole, methyl red, Voges-Proskauer and citrate ICUs - Intensive care units
  29. 29. xxix ICH &HC Institute of Child Health and Hospital for Children ICP - Intrinsically conductive polymer chemiresistors IMS - Ion mobility spectrometry Ile - Isoleucine kb - Kilobase KPa - Kilopascal KEGG - Kyoto Encyclopedia of Genes and Genomes Leu - Leucine LED - Light emitting diode LB - Luria Bertani MOSFET - Metal oxide semiconductor field effect transistors MOS - Metal oxide semiconductors MDR - Multi-drug-resistance NCBI - National Center for Biotechnology Information NBD - Nitrobenzooxadiazole NMR - Nuclear Magnetic Resonance NASBA - Nucleic Acid Sequence Based Amplification NB - Nutrient broth ORF - Open Reading Frame OD - Optical Density PPM - Parts per million PFPH - Pentafluorophenylhydrazine PBS - Phosphate Buffer Saline PID - Photoionization detection PCR - Polymerase Chain Reaction
  30. 30. xxx DPO - Polymer-Deposited Optical sensors PTR-MS - Proton-transfer-reaction mass spectrometry qPCR - Quantitative PCR QCM - Quartz crystal microbalance RFU - Relative Fluorescent Unit Rt - Retention time RT-PCR - Reverse Transcriptase Polymerase Chain Reaction RNaseA - RibonucleaseA RNA - Ribonucleic acid rpm - Rotations per minute SS agar - Salmonella-Shigella agar SEB - Self-encoded bead SDS - Sodium dodecyl sulfate SHE - Static Headspace Extraction SAW - Surface Acoustic Wave TPP - Thiamine pyrophosphate TSM - Thickness-shear mode TSI - Triple sugar iron test TBE - Tris Borate EDTA Tris - Tris-[Tris-(hydroxy methyl) amino methane] TSB - Tryptone Soya broth UTI - Urinary Tract Infections UPEC - Uropathogenic Escherichia coli Val - Valine VNC - Viable-but-nonculturable VOCs - Volatile Organic Compounds WBCs - White blood cells WHO - World Health Organization
  31. 31. 1 CHAPTER 1 INTRODUCTION 1.1 INCREASING BURDEN AND THREAT OF INFECTIOUS DISEASES Technical advancements not with-standing, infectious diseases spread by microorganisms including bacteria, fungi, viruses or parasites directly or indirectly result in epidemics and pandemics. Zoonotic diseases are stoically persistent due to animal-human cohabitation and emergence of virulent variants. Non-communicable diseases, malnourishment, therapeutic interventions like chemotherapy compromise immunity and make us prone to opportunistic microbial infections. Several such factors, both due to our dominance on earth and purely man-made factors, keep us constantly on our toes to combat infectious diseases and compel us to look for new approaches against evolving threats. There is a constant battle between technical advancement including the understanding of pathogenesis at molecular level and the capability of microbial pathogens in overcoming host defense, colonize and spread. Despite the remarkable advances in research and treatments during the 20th century, infectious diseases remain among the leading causes of death worldwide (WHO report 2012) for three main reasons: (a) emerging of new infectious diseases; (b) re-emerging of the old infectious diseases; and (c) Persistence of the intractable infectious diseases (Obi et al 2010). Influenza, HIV/AIDS, cholera, tuberculosis, diphtheria, malaria etc have exploded globally and re-emerging diseases such as plague, yellow fever, dengue are on the surge (Lashley 2003). The WHO reported in
  32. 32. 2 2010 that 31% of deaths in developing countries are caused by communicable disease, while the remaining deaths are caused by other non-communicable diseases as shown in Figure 1.1. Figure 1.1 The percentage of death in developing countries caused by communicable and non-communicable diseases are represented in the pie chart. Communicable diseases account to 31% of deaths worldwide. (Reproduced from (https://mikesnexus.files.wordpress.com/2015/02/causeofdea thdevelopingcountries.jpg?w=676) Past three decades of intense research in the molecular pathogenesis, especially using modern genetics and molecular biology, have unraveled stepwise progression involving entry and adherence of pathogens to specific host cells, colonization in tissues, and the damage, which is then diagnosed as the disease. Pathogens enter the host through the orifices in our body such as eyes, mouth, genital openings or wounds that breaches the skin barrier. Though some pathogens grow at the entry site, many pathogens travel to their specific host cells and colonize, either after intracellular or extracellular invasion. Pathogens apart from growing in the host, cause severe tissue damage and diseases through the release of destructive enzymes or
  33. 33. 3 toxins. Despite such detailed understanding at the molecular level, our inability to combat these diseases effectively is still a challenge, as the application of emerging technologies is outsmarted by the evolution and emergence of new infectious agents to changes in the human demographics, behavior, land use and changes in the transmission dynamics. Table 1.1 provides the currently prevalent infectious agent, signs and symptoms and diagnosis available for their detection. Table 1.1 Common infectious agents, symptoms and tests currently available for their detection Causative agents by type Signs and symptoms Laboratory testing Viral Hepatitis A Diarrhea, dark urine, jaundice and flu-like symptoms i.e. fever, headache, nausea and abdominal pain. Increase in ALT, bilirubin. Positive IgM and antihepatitis A antibodies. Noroviruses Nausea, vomiting, abdominal cramping, diarrhea, fever and myalgia. Routine RT-PCR. Clinical diagnosis. Stool is negative for WBCs. Rotavirus Vomiting, watery diarrhea, low- grade fever. Temporary lactose intolerance may occur. Infants and children, elderly and immunocompromised are especially vulnerable. Identification of virus in stool via immunoassay. Other viral agents (astroviruses, adenoviruses, parvoviruses) Nausea, vomiting, diarrhea, malaise, abdominal pain, headache and fever. Identification of the virus in early acute stool samples. Serology. Commercial ELISA kits are now available for adenoviruses and astroviruses
  34. 34. 4 Table 1.1 (Continued) Causative agents by type Signs and symptoms Laboratory testing Bacteria Bacillus anthracis Nausea, vomiting, malaise, bloody diarrhea, acute abdominal pain. Blood test. Bacillus cereus Sudden onset of severe nausea and vomiting. Diarrhea may be present. Normally a clinical diagnosis. Clinical laboratories do not routinely identify this organism. If indicated, send stool and food specimens to reference laboratory for culture and toxin identification. Campylobacter jejuni Diarrhea, cramps, fever, and vomiting; diarrhea may be bloody. Routine stool culture; Campylobacter requires special media and incubation at 42°C to grow Enterohemorrhagic E. coli (EHEC) including E. coli O157:H7 and other Shiga toxin- producing E. coli (STEC) Severe diarrhea that is often bloody, abdominal pain and vomiting. Usually, little or no fever is present. More common in children Stool culture; E. coli O157:H7 requires special media to grow. If E. coli O157:H7 is suspected, specific testing must be requested. Shiga toxin testing may be done using commercial kits; positive isolates should be forwarded to public health laboratories for confirmation and serotyping. Enterotoxigenic E. coli (ETEC) Watery diarrhea, abdominal cramps, some vomiting. Stool culture. ETEC requires special laboratory techniques for identification. If suspected, must request specific testing.
  35. 35. 5 Table 1.1 (Continued) Causative agents by type Signs and symptoms Laboratory testing Bacteria Listeria monocytogenes Fever, muscle aches, and nausea or diarrhea. Pregnant women may have mild flu-like illness, and infection can lead to premature delivery or stillbirth. Elderly or immunocompromised patients may have bacteremia or meningitis. Blood or cerebrospinal fluid cultures. Asymptomatic fecal carriage occurs; therefore, stool culture usually not helpful. Antibody to listerolysin O may be helpful to identify outbreak retrospectively Salmonella spp Diarrhea, fever, abdominal cramps, vomiting. S. typhi and S. Paratyphi produce typhoid with insidious onset characterized by fever, headache, constipation, malaise, chills, and myalgia; diarrhea is uncommon, and vomiting is not usually severe. Routine stool cultures Shigella spp. Abdominal cramps, fever, and diarrhea. Stools may contain blood and mucus. Routine stool cultures. Staphylococcus aureus Sudden onset of severe nausea and vomiting. Abdominal cramps. Diarrhea and fever may be present. Normally a clinical diagnosis. Stool, vomitus, and food can be tested for toxin and cultured if indicated. Vibrio cholera Profuse watery diarrhea and vomiting, which can lead to severe dehydration and death within hours. Stool culture; Vibrio cholerae requires special media to grow. If V. cholerae is suspected, must request specific testing.
  36. 36. 6 Table 1.1 (Continued) Causative agents by type Signs and symptoms Laboratory testing Parasites Cryptosporidium Diarrhea (usually watery), stomach cramps, upset stomach, slight fever. Request specific examination of the stool for Cryptosporidium. May need to examine water or food. Cyclospora cayetanensis Diarrhea (usually watery), loss of appetite, substantial loss of weight, stomach cramps, nausea, vomiting, fatigue. Request specific examination of the stool for Cyclospora. May need to examine water or food. Entamoeba histolytica Diarrhea (often bloody), frequent bowel movements, lower abdominal pain. Examination of stool for cysts and parasites—may need at least 3 samples. Serology for long-term infections. Trichinella spiralis Acute: nausea, diarrhea, vomiting, fatigue, fever, abdominal discomfort followed by muscle soreness, weakness, and occasional cardiac and neurologic complications Positive serology or demonstration of larvae via muscle biopsy. Increase in eosinophils. (Adapted from http://www.fda.gov/Food/FoodborneIllnessContaminants/ FoodborneIllnessesNeedToKnow/default.htm) The huge expenditure involved in the treatment of infectious diseases proves to be a drain on global economic resources. Figure 1.2 shows the expenditure on infectious diseases in 2008, valued to be $90.4 billion and this is expected to increase at a compounded annual growth rate (CAGR) of 8.8% and reach $138 billion in 2014. Out of the total expenditure, 53% is spent on antibiotic treatment for bacterial and fungal diseases. As bulk of it is for bacterial diseases, mainly due to a limited number of bacteria like
  37. 37. 7 Mycobacterium tuberculosis, Salmonella typhi, Shigella spp, E. coli, Streptococcus, Pseudomonas, Proteus, Klebsiella and Camphylobacter our interest is in bringing down the bacterial diseases treatment cost which increased from $40 billion in 2009 to $50 billion in 2014. Viral disease treatments see the fastest CAGR of 12.1%, increasing from nearly $45 billion in 2009 to $79 billion in 2014, but a significant portion of this expenditure is for treating the secondary bacterial infections (Infectious Disease Treatments report 2010). Figure 1.2 The global market for treatment of infectious diseases shows an increase in economic burden due to viral and bacterial infections from 2008 to 2014 (Adapted from Infectious Disease Treatments: Global Markets BCC research market forecasting 2010) Approximately 26% of annual deaths worldwide are caused by emerging infectious diseases. The people in developing countries particularly infants and children face a heavier burden of mortality and morbidity associated with infectious diseases (diarrhoeal diseases and malaria alone is estimated to cause about three million deaths each year) (Fauci 2001, Taylor et al 2001). Developing countries like India suffer excessively from the triple burden of infectious diseases: emergence of new pathogens, communicable diseases and non-communicable diseases that are linked with lifestyle and infrastructural changes (Quigley 2006).
  38. 38. 8 Nearly half of India’s disease burden is due to communicable diseases mainly because of improper sanitation, contaminated food, lack of basic health services and inadequate personal hygiene (Ministry of Health, Government of India 2005). Other demographical, environmental, and socio- economic factors also put India at risk of severe epidemics of new infections. An important take-home message for developing countries like India is to work on prevention and control of bacterial infectious diseases than spending huge amounts of money on treatment. As can be seen, the common denominator in our inability to combat these diseases is lack of field-deployable simple, inexpensive and high- throughput methodologies that have to be addressed in future developments. 1.1.1 Nosocomial Infections, Complicating Factor in the Control Despite a widespread awareness in both public health and hospital care, nosocomial infections continue to develop. Factors like increased medical procedures, decreased immunity among patients and invasive techniques create potential routes of infection, transmission of drug-resistant bacteria and ineffective control practices promote infection among hospital populations (Meenakshi 2012). Sources of hospital acquired infections are listed in Figure 1.3. A survey on the prevalence of nosocomial infections were conducted by World Health Organisation (WHO) in 55 hospitals in 14 countries representing 4 WHO Regions (Europe, Eastern Mediterranean, South-East Asia and Western Pacific). It reported an average of 8.7% of hospital patients with nosocomial infections. An estimation showed that over 1.4 million people suffer from hospital acquired complications worldwide (Tikhomirov 1987, Ginawi et al 2014).
  39. 39. 9 The highest frequencies of nosocomial infections were reported from hospitals in the East Mediterranean (11.8%) and South-East Asia Regions (10.0%), with a prevalence of 7.7% and 9.0% respectively in the European and Western Pacific (Mayon et al 1988). The urinary tract infections (UTI), infections of surgical wounds and lower respiratory tract infections are the most frequent nosocomial infections. The WHO and other studies have also reported that the highest prevalence of nosocomial infections occurs in Intensive care units (ICUs) and in orthopaedic and acute surgical wards. Infection rates are higher among patients undergoing chemotherapy and increased susceptibility due to old age (Ginawi et al 2014). Hospital-acquired infections lead to functional disability and emotional stress to patients (Ian 2014, Ponce-de-Leon 1991). Different bacteria, viruses, fungi and parasites may cause such infections and these microorganisms are acquired by cross-infection from one person to another in the hospital or by endogenous infection caused by the patient’s own flora. Some organisms may be acquired from environment through substances recently contaminated from another human source. Before the introduction of antibiotics, and basic hygienic practices in hospital settings, most hospital infections were due to microorganisms not present in the normal flora of the patients and pathogens of external origin. (WHO: A practical guide 2002). Progress in the antibiotic treatment of bacterial infections has considerably reduced mortality from many infectious diseases. Hospital acquired infections today are caused mostly by microorganisms common in the general population (e.g. Enterobacteriaceae, Enterococci, Proteus and Staphylococcus aureus). These organisms are transmitted through discharged patients and visitors to the community (Ian 2014, Ponce-de-Leon 1991). In this regard, nosocomial infections need to be taken seriously and diagnosed for proper treatment as they pose great danger
  40. 40. 10 if ignored. Recently, Centers for Disease Control estimated that the burden reflected by hospital-acquired bacterial infections on patients and the healthcare system exceeded 30 billion dollars each year. These incidences account for the significance in mortality and morbidity rates in ICUs and more than 30% of the death rate after being hospitalized (Giske et al 2008). Inspite of treatment, such nosocomial infections increase the medical cost up to $156,000 for patients with hospital acquired infection staying longer than uninfected patients. Figure 1.3 Different sources that cause hospital acquired infections (Adapted from Prevention of hospital-acquired Infections, WHO report 2012) 1.1.2 Multidrug resistance is a Major Threat and Challenge The major challenge in disease management is the resistance developed by the pathogens for antibiotics. Multi-drug-resistance increases the morbidity and mortality (Jyoti et al 2014). Emergence of such superbugs is purely a huge man-made problem stemming out of the following factors:
  41. 41. 11 1. Indiscriminate use of antibiotics Unnecessary use of antibiotics, self-medication and non- completion of the course as health improves lead to bacterial resistance and ineffectiveness of antibiotics. Frequent use of antibiotics can harm vital organs like liver and kidney and cause other serious side effects too. 2. Horizontal gene transfer and acquisition of MDR by pathogens For the past few decades the spectrum and frequency of antibiotic- resistant infections have increased. It is attributed to mutational changes and acquisition of resistance-encoding genetic material transferred from other bacteria. This is also related to the overuse of antibiotics in human health care and in animal feeds, a combination of microbial characteristics, selective pressure of antimicrobial use, social and technical changes that enhance the transmission of resistant organisms. Hospitals play a major role in selection of multi-drug-resistance organisms by their widespread use of antimicrobials in the ICU and for immuno-compromised patients (Senka & Vladimir 2003). Methicillin-resistant Staphylococci, Vancomycin resistant Enterococci and extended-spectrum betalactamase (ESBL) producing gram negative Bacilli are identified as major problem in nosocomial infections due to horizontal gene transfer (Erika 2011). 3. Lack of new antibiotics A WHO report states that the antibiotics pipeline is drying up while resistance to existing drugs is increasing day-by-day. Two major reasons for such a situation are non-development of new formula drugs and modifications of existing ones leading to poor commercial returns as they are used only during infections (Braine 2011).
  42. 42. 12 Nosocomial infections acquired in hospital settings occur worldwide and affect both resource-poor and developed countries. They are a significant burden for both patient and public health and one among the major causes of death leading to increased morbidity among hospitalized patients (Saranraj and Stella 2001). Every year, organisms resistant to even most potent antibiotics are identified, attracting great public concern worldwide. Since the discovery of penicillin, antibiotics were considered the “magic bullets” in curing infectious diseases. They have been misused and abused in clinical treatment due to inappropriate prescription to patients through misdiagnosis. Premature cessations of therapy not only fail to eradicate the pathogens, but also trigger resistance in the surviving bacteria. Moreover, antibiotics are sold without prescription over the counter especially in developing countries. Another major factor that causes drug resistance is the large-scale use of antibiotics in animal farming which are later consumed by human and accumulated in food chain (Report by the IMS Institute for Healthcare Informatics 2013). Major clinical challenges in both humans and animals are the MDR phenotypes. Consequently, microbes have developed cross resistance to a series of functionally and structurally unrelated drugs. Most of the life threatening pathogens for humans are zoonotic. In India the outbreak of H1N1 virus (swine flu) in 2009 killed more than 500 people and other zoonotic diseases like plague, leptospirosis are often a threat to human lives. Zoonotic diseases such as anthrax, Hepatitis E, Rabies are also very dangerous and difficult to handle when it develops multi-drug-resistance (WHO 2014). Not only underdeveloped or developing countries like India suffer from such out breaks but so do developed countries. The ampicillin (Ap), streptomycin (Sm), sulfamethoxazole (Su), tetracycline (Tc), and trimethoprim (Tp) (Ap– Sm–Su–Tc–Tp) pattern is increasingly reported among MDR E. coli and S. enterica strains isolated from food producing animals. The O104:H4 strain of
  43. 43. 13 E. coli outbreak is well known for displaying resistance to an extended spectrum of β–lactams. It was also resistant to (Ap–Sm–Su–Tc–Tp) making it difficult to locate the genes responsible for encoding the resistance phenotype (Steven et al 2013). Antibiotic resistance was identified in a miniscule portion only in Pseudomonas aeruginosa that have intrinsic and constitutive high drug tolerance (Leclercq & Courvalin 1991, Hancock 1998). Strains have attained elevated drug tolerance due to the usage of antibiotics which serve as an environmental selective pressure. The horizontal transfer of genetic materials enables the wide spread of resistance (Alonso et al 2001). The resistant genes can be transferred either by cell-to-cell conjugation, phage-mediated transduction or by naked DNA transformation. The prevalence of MDR increases the mortality and morbidity of bacterial infection, making the treatment more difficult (Ochman et al 2000). In 2010, Centre of Disease Control (CDC) has reported that bacterial infection resulted in approximately 30,000 deaths each year in the United States (Aminov 2010). MDR strains have been found towards all available antibiotics, presenting one of the biggest threats to public health. 1.2 INADEQUACY OF CLASSICAL AND CURRENT DIAGNOSTIC METHODS AND LACK OF SCREENING AND SURVEILLANCE METHODS FOR PREVENTIVE HEALTHCARE The classical method of detecting and identifying bacteria is based on culturing, enumeration and isolation of presumptive colonies for further identification analysis. In some cases, the sample needs to be homogenized, concentrated or pre-enriched prior to analysis. Bacterial cells can become injured or viable-but-nonculturable (VNC) due to the sub-lethal stressors, such as osmotic shock, acid, heat and cold which makes the analysis difficult
  44. 44. 14 (Kell et al 1998). Biochemical tests depend on the unique biochemistry of microbes. Classical biochemical tests like Indole, methyl red, Voges- Proskauer and citrate (IMViC); Triple sugar iron test (TSI) are used routinely in clinical practice. However, chromogenic and fluorogenic media are now being developed by virtue of specific enzymes on the microbe converting the given substrate to coloured or fluorescent products. These methods are both tedious and time-consuming requiring a series of tests with the incubation of the microorganisms for 2-3 days. Another approach in wide use are the enzyme/substrate methods like enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) based upon either chromogenic or fluorogenic substrate methods (Siddhesh et al 2012). Antibody (Ab)-based techniques, which takes the advantage of specific binding affinities of antibodies to specific antigens, can either be developed in the laboratory or purchased commercially. The antibodies can be specific for a single strain of bacteria, or can potentially be produced for a single species (E. coli). Once antibodies are produced, their specificity is tested for by mounting onto a support system like nylon supports, polystyrene waveguides, cantilevers and glass slides (Valerie 2014). Still all these techniques have their own disadvantages viz. development of specific antibody, laboriousness, high cost instrumentation, and lacking skilled personnel (Rachel & Stephen 2005). The limitations of these methods have led to the research focusing on development of rapid and accurate techniques to identify pathogens. 1.2.1 Limitations of Emerging Modern Methods Microarray technique combines the potential of simultaneous identification and speciation of bacteria. The rapid identification of bacteria in clinical samples is important for patient management and antimicrobial
  45. 45. 15 therapy (Georg et al 2004). In this method the bacterial samples are discriminated on a single slide. For quick detection and identification of bacteria using species-specific oligonucleotide probes designed for specific regions of various targeted genes DNA-based microarray approach is becoming popular. The high-throughput nature of microarray experiments impose numerous limitations, which apply to simple issues such as sample acquisition and data mining, to more controversial issues that relate to the methods of biostatistical analysis required to analyze the enormous quantities of data obtained (Abdullah et al 2006). The limiting step for commercialisation and further development of microarrays is the complexity and the time required to design and test discriminatory genetic regions that separate one species from another. This lack of discriminatory information also limits other molecular identification methods, including sequencing (Dennise et al 2002). Methods involving identification of surface proteins or whole-cell or its genetic material are gaining interest now. These include immunoassay techniques and molecule-specific probes, such as lipid or protein attachment- based approaches. Because of the design of the immunoassay, sample contaminants that might interfere with the antigen-antibody reaction can produce false positive results. On the other hand, nucleic acid detection methods target specific nucleic acid sequences of bacteria. These include Polymerase Chain Reaction (PCR), Quantitative PCR (qPCR), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), and Nucleic Acid Sequence Based Amplification (NASBA). These methods identify specific sequences from a complex mixture of DNA and therefore are useful for determining the presence and quantity of pathogen-specific or other unique sequences within a sample (Mark & Joyce 2005). qPCR facilitates a rapid detection of low amounts of bacterial DNA accelerating therapeutic decisions
  46. 46. 16 and enabling an earlier antibiotic treatment. Molecular recognition approaches have the potential for being more rapid, more sensitive and adaptable to a wider class of pathogens (Rachel & Stephen 2005). However, all these techniques have a setback due to the following disadvantages listed in the Table 1.2. Table 1.2 Advantages and disadvantages of molecular methods used for bacterial identification Methods Advantages Disadvantages PCR (Single) Provides sensitive detection of single gene or bacteria PCR conditions must be optimized. Multiplex PCR Reduces cost and allows rapid detection of multiple bacteria Primer design is critical and primers may interfere with each other leaving some genes and bacteria undetected. Real-time PCR Shortens detection time, detects and quantifies bacteria, high sensitivity, specificity and reproducibility Requires expensive equipment, reagents and operations by skilled technicians. Reverse- transcriptional PCR Can detect only viable cells of pathogens Skill required to handle unstable RNA for pathogen detection Nested PCR Has improved sensitivity and specificity than conventional PCR Greater expense than regular PCR as twice as much enzyme and reagents are used DNA sequencing Has high discriminatory power and reproducibility Complex method, time consuming and relatively expensive (Adapted from Frederick et al 2013)
  47. 47. 17 1.3 NEED FOR NEW APPROACHES TO DEVELOP NEXT GENERATION TOOL WITH MODERN KNOWLEDGE Despite the advances in technology and medicine, infectious diseases remain a major cause of death and socioeconomic disruption for millions of people. Many bacteria are responsible for causing infectious diseases in animals and humans. Among these, bacteria like E. coli (UPEC), Shigella and Salmonella are common. Bacteria like Proteus spp. and Pseudomonas are associated with hospital-acquired infections and these are also multi-drug-resistant. Obviously these are quite dangerous and there is an increasing demand to keep them away from communities. Existing protocols for field detection and identification of such bacteria are unavailable or ineffective for surveillance. Hence it is imperative to develop next generation tool with modern knowledge. 1.3.1 Intra and Extracellular Targets for Non-Invasive and Non-Destructive Detection Methods Biomarkers are critically important tools for detection, prognosis, treatment and monitoring (Pothur et al 2002). Biomarkers are biological molecules that are indicators of physiological state and also of change during a disease process (Pradeep et al 2011). The value of a biomarker lies in its ability to provide an early indication of the disease and to monitor disease progression (Judith et al 2007). Recent studies have accumulated scientific evidences suggesting that certain surface-associated and extracellular components produced by bacteria can be used as biomarkers assisting in their identification. These bacterial components would be able to directly interact with the host cells including bacteriocins, exopolysaccharides, surface-associated and extracellular proteins. Extracellular proteins include proteins that are actively
  48. 48. 18 transported to the bacterial surroundings through the cytoplasmic membrane, as well as those that are simply shed from the bacterial surface. Compared to other bacterial components, the interactive ability of extracellular proteins has been less extensively studied (Borja et al 2010). Bacterial Volatile Organic Compounds (BVOCs) have been considered as sensitive and specific biomarkers for bacterial detection in human samples and culture media. The possibility of using VOC markers as one of the largest groups of bacterial metabolites would open a new frontier for developing more efficient techniques in the diagnosis of bacterial infections (Mohsen et al 2014). Table1.3 provides the list of common diseases and/or infections with their characteristic odours. Table 1.3 Diseases and their odours S.No Disease Odour Source 1. Anaerobic infection Rotten apples Skin / sweat 2. Bacterial vaginosis Amine-like Vaginal discharge 3. Bacterial infection Foul Sputum 4. Bladder infection Ammoniacal Urine 5. Cystic fibrosis Foul Infant stool 6. Diabetes mellitus Acetone-like Breath 7. Diphtheria Sweet Sweat 8. Phenylketonuria Musty / horsey Infant skin 9. Pseudomonas infection Grape Skin / sweat 10. Rotavirus gastroenteritis Foul Stool 11. Tuberculosis Stale beer Skin 12. Typhoid Baked brown bread Skin (Adapted from Pavlou & Turner 2000)
  49. 49. 19 1.3.2 Volatile Organic Compounds (VOCs) as Extracellular Targets Volatile Organic Compounds (VOCs) play an important role in structuring and characterizing life. These kinds of compounds are produced by animals, bacteria, humans and plants and also provide diverse functions in both natural and artificial systems. The volatility of VOCs in the environment gives them unique characteristics making studies of such compounds challenging (Chidananda et al 2015). The production of volatiles has been recognized since millennia and has been exploited as aroma or flavour components in the production of cheese, wine and other fermenting food. Repellent odours from rotting material are produced by bacteria, indicating a chemical communication between different species (Stefan & Jeroen 2007). VOCs have relative molecular masses ranging between 30 and 300 g/mole and heavier molecules are not considered VOCs because they generally have a vapour pressure that is too low at room temperature (Alphus & Manuela 2009). Molecules with one or two polar functional groups are the most volatile ones than those with more functional groups. Non-polar molecules are generally more volatile than polar ones as the volatility is determined by their molecular weight and their intermolecular interaction. (Sichu 2009). Hence, a compound with a low molecular weight, a carbon backbone, a high vapour pressure, (greater than 0.27 KPa) and a boiling point between 50-260 ºC existing as gas under standard temperature and pressure are classified as VOCs (Turner et al 2006). Bacterial Volatile Organic Compounds (BVOCs) are produced from the primary and secondary metabolism of the organisms. The BVOCs are produced as a by-product of primary metabolism involving the breakdown of food in the environment to extract nutrients needed for the maintenance of cell structures. However, the BVOCs are produced by the microbes due to the
  50. 50. 20 environmental stress during growth through secondary metabolism (Kai et al 2009, Hughes & Sperandio 2008). Information on bacterial BVOCs produced through its primary and secondary metabolisms are limited though there are many reports on VOCs released. Bacteria release a number of characteristic VOCs like aldehydes (benzaldehyde, acetaldehyde, formaldehyde, 2-methylbutanal, 3- methylbutanal, Decanal), ketones (2- tridecanone, 2-heptanone, 2-nonanone, Acetophenone, 2-undecanone, acetone), alcohols (2-pentadecanol, propanol, 1-decanol, ethanol, 1-butanol, 1-pentanol), acids (Crotonic acid, phenyl acetic acid) and compounds like hydrogen sulphide, methyl mercaptan, dimethyl sulphide, ethyl butanoate, isoprene, trimethyl amine, n-propyl acetate, dimethyl disulphide, ammonia, trimethyl amine as chemical messengers or secondary metabolites, (Lieuwe et al 2013) under defined growth conditions. These are attractive targets for developing into non-invasive diagnostic markers. In ancient times, physicians relied heavily on their senses to diagnose the infections before sophisticated analytical techniques were available. Colour, smell and taste were used to detect biological markers. VOCs are one such metabolite released from microorganisms as protection against antagonists or as signalling molecules that can be exploited for their specific detection (Nicholson & Lindon 2008). Different pathogens possess similar VOCs and therefore, the volatile profiles under defined growth conditions should be compared in order to identify the unique compound serving as an effective tool for identification. Hence, an alternate method for identifying pathogenic bacteria can be based on such characteristic metabolites generated by these organisms using specific biochemical pathways.
  51. 51. 21 1.4 CURRENT METHODS FOR DETECTION OF VOLATILE ORGANIC COMPOUNDS (VOCs) Volatile organic compounds are currently detected using a variety of methods including colorimetric sensor array, using fluorescent reagents, Gas Chromatography and Mass Spectroscopy (GC-MS), biosensors and E- nose. The description of each method is given below. 1.4.1 Colorimetric Sensor Array The colorimetric sensor array represents a new approach to array- based chemical sensing (Michael et al 2006). Such approach has emerged as a potential tool for the detection of chemically diverse analytes. Similar to the mammalian olfactory system, these arrays produce composite responses unique to an odourant based on cross-responsive sensor elements. In such sensor design architecture, one receptor responds to many analytes and many receptors respond to any given analyte (Christopher et al 2010). A distinct pattern of responses produced by the array provides the possibility of a characteristic fingerprint for each analyte. The different indicators that are available for detection on the array are shown in the Figure 1.4. Based on a broad range of chemical-sensing interactions, rather than on weak nonspecific van der Waals forces, the disposable array exhibits both excellent sensitivity and selectivity to a broad range of organic compounds. The array is well-suited for the detection of biogenically important analytes such as acids, amines and thiols. The arrays are basically nonresponsive to changes in humidity, which avoids the problem of interference due to changes in humidity during environmental sample analyses (Chen Zhang & Suslick 2005).
  52. 52. 22 Figure 1.4 Colorimetric sensor array using metalloporphyrins, metal nanoparticles and acid-base indicators showing different coloured spots when reacted with VOC (Adapted from Sung 2009). 1.4.2 Fluorescent Method for VOC Detection New technologies are being developed using conjunction of high- sensitivity fluorescence based detection to reduce the time required for the assay (Bhaskara et al 2012). Fluorescence-based assays are widely used in high-throughput screening due to their ease of operation, diverse selection of fluorophores, high sensitivity and various display readout modes. As a result, fluorescence-based assays have been applied to monitor a broad range of activities in life-science research such as air analyses, distribution of molecules, organelles or cells, enzymatic activities, molecular dynamics and interactions, and signal transduction (Frank 2008). Detection is achieved through fluorophore-tagged growth substrates included in the media that are added to samples. Upon growth, specific bacterial enzymatic activity cleaves the fluorophore from the substrates, causing fluorescence or increase in fluorescence. This fluorescence can then be detected by a number of
  53. 53. 23 instruments. It is a simple assay that is economical and time saving (Rachel & Stephen 2005). Both colorimetric or fluorimetric method provides cost effective, non-invasive and high throughput diagnostic assays. 1.4.3 Gas Chromatography and Mass Spectroscopy (GC-MS) Traditional analytical methods for VOC detection usually combines Gas Chromatography (GC) coupled most often with Mass Spectrometry (GC-MS) or a certain detection approach such as flame ionization detection (GC/FID), photoionization (GC/PID) (Petr 1984) and Electron Ionisation mode (EI). Sometimes other approaches such as membrane-inlet mass spectrometry or isolation followed by NMR spectroscopy are used (Thorn et al 2011). Though several general mass spectral libraries such as the Wiley and the NIST are available, more specialized, critically evaluated libraries are sometimes more useful for volatile compounds. These libraries are of immense use, as the closest hit within the library might uncritically be taken as positive identification. The inclusion of additional data, especially gas chromatographic retention indices, is critical for structure elucidation. GC/MS has excellent detection sensitivity and specificity, and are thus the best suited for VOC trace detection and identification but real-time direct detection could pose a challenge (Sichu 2014). Even though GC-MS analyses have enabled comprehensive studies, these tools have not emerged as routine instruments for clinical diagnosis due to high operating costs, laborious and time-consuming sample-preparation methods and requirements for significant training and expertise for effective operation and data interpretation. The limited applicability of traditional methods and analytical instruments in clinical diagnoses has prompted the need to develop simpler, cheaper, non-invasive and more user-friendly diagnostic assays for routine clinical applications. Major techniques recently involved in VOCs based
  54. 54. 24 detection of infectious diseases their advantages and disadvantages are given in the Table 1.4. Table 1.4 Advantages and disadvantages of some of the methods currently used for VOC analysis in clinical aspect Method Advantage Disadvantage Gas chromatography with mass spectroscopy 1. Identification of unknown VOCs and profiling possible 2. Sensitivity in the ppb range 3. Reproducible 1. Cannot detect non‐ volatile, polar and thermally labile compounds 2. Requires lengthy sample preparation (hydrolysis/ derivatization) Ion mobility spectrometry (IMS) 1. No pre-concentration needed 2. Sensitivity in the ppm range 3. Mobile system 4. Low cost 5. Suitable for clinical use 1. Low resolving power, 2. Lack of positive identification 3. Instability of response (due to humidity and other matrix interferences) 4. The sensitivity of the IMS is reduced due to the low pressure operation of the ionization region and drift tube. 5. Real-time measurements not Possible Selected ion flow tube mass spectrometry 1. Measures VOCs in real time 2. Potential for online testing 3. VOC measurement in headspace (serum/urine) 4. Sensitivity in the ppb range VOC chemical identification and profiling not possible Uses carrier gas, less sensitive than PTR-MS
  55. 55. 25 Table 1.4 (Continued) Method Advantage Disadvantage Proton transfer spectrometry 1. No pre-concentration needed 2. Real-time measurements and online monitoring 3. Sensitivity in the ppb range Large and costly instrument mass interferences, library of compounds still to be created. Various chemical sensor matrix platforms/e-noses 1. Easy to use 2. Portable 3. Sensitivity in the ppb range May need chemometric processing, suffers from cross-sensitivities (Adapted from Shneh et al 2013) 1.4.4 Biosensors A variety of chemical sensors, including biosensors and E-noses have demonstrated the feasibility of VOC detection. Chemical sensors detect odour molecules based on the reaction between the odour molecules and the target sensing materials on the sensor surface. This reaction triggers a certain change in mass, volume or other physical properties which is then converted to an electronic signal by a transducer. There are different types of transducers for chemical sensors like optical, electrochemical, heat-sensitive and mass-sensitive. The most common chemical sensors includes surface acoustic wave sensor, quartz crystal microbalance sensor, metal oxide semiconductor sensor, and polymer composite-based sensor biosensors. They are currently drawing interest as it comes with reliable results in much shorter detection time (Vijayata et al 2014).
  56. 56. 26 1.4.5 E-nose E-noses have drawn much attention since it is the most promising approach so far for high sensitivity and mimicking the biological nose respectively for sensing. The electronic-nose detects volatile compounds with an array of semi-conducting polymer sensors that enables the user to map aroma pattern in a graphical or digital format. It comprises of an array of chemical sensors with different selectivity, a signal-preprocessing unit and a pattern recognition system. The interaction between volatile organic compounds with an array of sensors generates a characteristic fingerprint which can be recognized by comparing it with previously recorded patterns in the recognition system (Simeng et al 2013). Electronic noses can be used for detecting bacterial pathogens, either in vitro or in vivo, or as a potential tool for the identification of patients with diseases. They employ conductivity sensors like Metal oxide semiconductors (MOS), Intrinsically conductive polymer chemiresistors (ICP) and Conductive Polymer composite chemiresistors (CP); Electrostatic Potential sensors like Metal oxide semiconductor field effect transistors (MOSFET) and Gas Sensitive Field Effect Transistor sensors (GASFET); Acoustic Resonance Sensors like Thickness-shear mode / Quartz Crystal Microbalance / Bulk Acoustic Wave (TSM / QCM / BAW) and Surface Acoustic Wave (SAW) and Optical Vapour sensors like Polymer-deposited Optical sensors (DPO) and self-encoded bead (SEB) (Simeng et al 2013). Though biosensors/ E-nose can process in a single run, the chance of capturing and identifying a small amount of pathogens present in samples is difficult (Andre et al 2002). Different sampling methods have been used for the volatile compound detection in order to distinguish between normal and infected specimens and their detectable range (Ida et al 2006). The rapid screening of biological samples could allow faster and appropriate therapeutic
  57. 57. 27 treatment and would lead to decrease in mortality rate over classical cultivation and isolation methods. However, there is still much work to do before biosensors become a real alternative for pathogen detection (Olivier et al 2007). A recent review states that studies on VOC based identification of infectious diseases are limited when compared to other identification methods. The major sources for detection of VOCs related to infections are the respiratory tract, gastrointestinal tract, urinary tract and nasal cavity. The upcoming analytical technologies for detection and measurement of volatile organic compounds (VOCs) had shown advantages in clinical applications. Hence, the interest for their use in evaluating the diagnostic potential of VOCs for different diseases has increased. VOCs as specific biomarkers in clinical samples open up a new direction for developing rapid and potentially inexpensive disease screening tools. Most of the studies on volatile biomarkers have been carried out on exhaled-breath samples, although other clinical matrices, such as urine and faeces, have also been investigated (Kamila & Ian 2015). 1.5 REGULATION OF VOLATILE ORGANIC COMPOUND METABOLISM Bacteria and fungi are capable of producing a wide variety of biochemical compounds via primary and secondary metabolism. During primary metabolism, the organism breaks down food in the environment to extract nutrients needed for the maintenance of cell structures and, in the process, creates VOC's as by-products (Karen & Santo-Pietro 2006). In secondary metabolism, there is a competition for resources in a nutrient-poor environment thereby driving the production of VOC. Although the distinction between primary and secondary metabolism is not absolute, the secondary metabolism is known to start after active growth has ceased. Secondary
  58. 58. 28 metabolites have diverse chemical structures and are distinct products of particular groups of organisms and sometimes even strains (Vining 1990). The function of secondary metabolites in the organism is not clear, but the process seems to have different purposes owing to their remarkable variety and many different chemical structures (Bentley & Bennett 1988). Volatile aldehydes have been found to be produced by a variety of microorganisms. Acetaldehyde is formed through oxidative carboxylation of acetolactate, a by-product of the synthesis of leucine in yeasts (Berry 1988). Unsaturated fatty acids may be transformed to volatile aldehydes such as hexanal, heptanal and nonanal, and the precursors of 2-decenal, 2 undecenal and 2-heptenal are linoleic and linolenic acid (Korpi 2001). In certain studies investigating the emission of VOCs during microbial growth showed that the concentration of aldehydes decreased as though the microorganisms had consumed the aldehydes. The growth of microorganisms generates volatile metabolites, but the lack of knowledge about metabolic routes makes it generally unclear whether all compounds found in relation to microbial growth really are a metabolic product or whether microbial growth or moisture promote(s) emission of compounds from a substrate (Ezeonu et al 1994). Amino acid, such as alanine, valine, leucine, isoleucine, phenylalanine and aspartic acid, are also involved in aroma biosynthesis as direct precursors, and their metabolism is responsible for the production of a broad number of compounds, including acids, carbonyls, alcohols and esters. The information available to date on the biosynthesis of amino acid-derived volatiles is based on precursor feeding experiments with radio-labelled, stable-isotope-labeled, or unlabeled precursors (Muna et al 2013). Amino acids can undergo an initial deamination or transamination leading to the formation of the corresponding molecules alpha-keto acid.
  59. 59. 29 Subsequent decarboxylation followed by reductions, oxidations or esterifications give rise to aldehydes, acids, alcohols and esters (Reineccius 2006). A general pathway is shown schematically in Figure 1.5. Branched chain volatile alcohols, aldehydes and esters arise from the branched chain amino acids leucine, isoleucine and valine. The general scheme of biosynthesis is thought to proceed in a similar way as that in bacteria or yeast, where these pathways have been more extensively studied (Beck et al 2002, Tavaria et al 2002). Figure 1.5 Representative VOC metabolic pathway involving amino acids From the wide range of reported VOCs, a number of aldehydes and ketones were found to be predomina3.5 ntly produced by bacteria. Besides hydrazines, a multitude of different groups of derivatizing agents has been established for the analysis of carbonyl compounds. All of these comprise of a condensation reaction of the reagent with the analyte under formation of a colored and/or fluorescent derivative.
  60. 60. 30 Therefore, detection may be performed by photometry or fluorescence spectroscopy (Martin et al 2000). Though reports suggest a variety of dyes like 2,4-dinitrophenylhydrazine (DNPH), 1-Dimethylaminonaphthalene- 5-sulfonylhydrazide (Dansyl hydrazine, DNSH), nitroaromatic hydrazines, 2-diphenylacetyl-1, 3-indandione-1-hydrazone (DAIH), 4-nitrophenylhydrazine (pNPH), 1-methyl-1-(2,4-dinitrophenyl)hydrazine (MDNPH), Nitrobenzooxadiazole (NBD derivatives), a Dimethylaminosulfonyl group (DBD) or an aminosulfonyl (ABD) group, 2,4,6-trichlorophenylhydrazine (TCPH), Pentafluorophenylhydrazine (PFPH) and halogenated phenyl hydrazine reagents specific for carbonyl compound, DNSH has been found to be best suited owing to its lower level detection in atmospheric samples (Laurent et al 2004). The importance of derivatizing agents for the analysis of aldehydes and ketones becomes apparent from the literature search for respective analytical developments and applications. The chemical abstract database which covers literature from 1967 until today, lists more than 1500 articles which focus on derivatization techniques for the analysis of carbonyl compounds (Jan & Ki-Hyun 2015) Therefore, release of a number of carbonyl compounds as specific VOCs by bacteria and availability of a variety of reagents for their detection prompted us to focus on carbonyl compounds as specific biomarker in this study. 1.6 RATIONAL DESIGN OF MEDIA FOR ENHANCED VOLATILE ORGANIC COMPOUND PRODUCTION The biosynthesis of VOCs depends on the availability of carbon, nitrogen and sulfur as well as energy provided by primary metabolism. Therefore, the availability of these building blocks has a major impact on the
  61. 61. 31 concentration of any secondary metabolite, including VOCs, demonstrating the high degree of connectivity between primary and secondary metabolism. Biosynthesis of the wide array of different VOCs branches off from only a few primary metabolic pathways. Based on their biosynthetic origin, all VOCs are divided into several classes, including fatty acid derivatives and amino acid derivatives in addition to a few species-/genus-specific compounds not represented in those major classes (Stefan & Jeroen 2007). The medium composition has a great influence on both qualitative and quantitative production of volatile metabolites. In general, nutrient-rich media promote larger quantities of VOC than nutrient-poor media. The emission of VOC changes with the growth phase of the bacterial culture (Malik 1979). Additionally many factors affect volatile composition, including the genetic makeup, degree of maturity, environmental conditions such as pH of the medium, levels of CO2 or O2, moisture and temperature (Maria et al 2013). There are several pathways involved in volatile biosynthesis starting from lipids and amino acids. Once the basic skeletons are produced via these pathways, the diversity of volatiles are achieved via additional modification reactions such as acylation, methylation, oxidation/reduction and cyclic ring closure (John et al 2007). Thus, the medium composition / growth conditions can be manipulated in order to achieve an enhanced VOC release. 1.7 PROTEUS AS A MODEL STUDY ORGANISM In this work we have chosen Proteus, a notorious nosocomial pathogen as a model organism and have identified its VOC biomarker. The general introduction about the organism and its pathogenicity are described in detail.
  62. 62. 32 1.7.1 Proteus –General Introduction Kingdom : Bacteria Phylum : Proteobacteria Class : Gamma proteobacteria Order : Enterobacteriales Family : Enterobacteriaceae Genus : Proteus Species : P. mirabilis and P. vulgaris Proteus species are Gram-negative, facultatively anaerobic, rod shaped bacterium. It shows swarming motility, and urease activity. Proteus organisms are implicated as serious reason of infections in humans, along with Escherichia coli, Klebsiella, Enterobacter and Serratia species. Some of the species of Proteus causing urological diseases are P. mirabilis, P. rettgeri, P. vulgaris, P. norganii, P. penneri, P. hauseri and P. myxofaciens. However, P.mirabilis and P.vulgaris are more prevalent than other species. Proteus species are found in multiple environmental habitats including human intestinal tract as part of normal human intestinal flora and long term care facilities. In hospital settings, it is not unusual for gram-negative bacillus to colonize both the skin and oral mucosa of both patients and hospital personnel. P. mirabilis causes 90% of all 'Proteus' infections in humans and also can be considered a community-acquired infection (http://emedicine. medscape.com/article/226434-overview). Proteus vulgaris and Proteus penneri are isolated from individuals in long-term care facilities hospitals and from patients with underlying diseases or compromised immune systems. Patients with recurrent infections, with structural abnormalities of the urinary tract, those who have had urethral instrumentation, and those whose infections were acquired in the hospital have an increased frequency of infection caused by Proteus (Guentzel 1996).
  63. 63. 33 Proteus species undergoes dramatic morphological changes, from a single rod-shaped swimmer cell to an elongated multicellular swarmer cell, in response to growth on solid surfaces (Holt et al 1994).Most strains produce a powerful urease enzyme, which rapidly hydrolyzes urea to ammonia and carbon dioxide (Ryan et al 2004, Rauprich et al 1996, Matsuyama et al 2000). Urea → 2NH3+ CO2 Proteus species are the causative agent of a variety of opportunistic nosocomial infections including those of the respiratory tract, eye, ear, nose, skin, burns, throat, and wounds; it also may cause gastroenteritis. Proteus mirabilis causes serious kidney infections which can involve invasion of host urothelial cells. Reports suggest prevalence of 17% for P. mirabilis and 5% for P. vulgaris in the faeces of healthy persons. Urinary pathogens are thought to originate mainly from the gut and it is interesting that P. mirabilis is disproportionately more frequently isolated from patients with urinary-tract infections than P. vulgaris (Krikler 1953). 1.7.2 Pathogenesis and Diseases Caused by Proteus Infection depends on the interaction between the infecting organism and the host defense mechanisms. Various components of the membrane interplay with the host to determine virulence. Proteus species in addition, to the outer membrane contains a lipid bilayer, lipoproteins, polysaccharides and lipopolysaccharides. The first step in the infectious process is adherence of the microbe to the host tissue. Fimbriae facilitate adherence and thus enhance the capacity of the organism to produce disease. P. mirabilis like E. coli, and other gram-negative bacteria contain pili, which are tiny projections on the surface of the bacterium. Specific chemicals located on the tips of pili enable organisms to attach to selected host tissue sites (eg. urinary tract endothelium). The virulence factors produced by P. mirabilis are shown in
  64. 64. 34 Figure 1.6. The adhesion of Proteus species to uroepithelial cells initiates several events in the mucosal endothelial cells, including secretion of interleukin 6 and interleukin 8. Proteus organisms also induce epithelial cell desquamation (Christopher et al 2000). Figure 1.6 A schematic diagram showing proteins produced by P. mirabilis that are known or hypothesized to be virulence factors important in urinary tract infections (Adapted from Christopher et al 2000) Urease production, together with the presence of bacterial motility and fimbriae, may favor the production of upper urinary tract infections. When the pathogen enters the bloodstream, endotoxin, a component of gram- negative bacteria cell walls, apparently triggers a cascade of host inflammatory responses and leads to major detrimental effects. Thus the factors for pathogenesis include adherence to host mucosal surfaces, damage and invasion of host tissues, evasion of host immune systems, and iron acquisition. The ability of Proteus organisms to produce urease and to alkalinize the urine by hydrolyzing urea to ammonia makes it effective in producing an environment in which it can survive. The activity of a urease enzyme, causes polyvalent cations, such as Mg2+ and Ca2+ , to precipitate out of the urine and form struvite and carbonate hydroxyapatite crystals (Griffith et al 1976). The mineral structures also provide bacteria a habitat to hide from antibiotic treatment and the host immune cells (Li et al 2002).
  65. 65. 35 An infection occurs when microorganisms, usually bacteria, from the digestive tract, cling to the opening of the urethra and begin to multiply. An infection limited to the urethra is called urethritis. From there, bacteria often move on to the bladder, causing a bladder infection called cystitis. If the infection is not treated promptly, bacteria may then go up the ureters to infect the kidneys (Mobley 1987). This infection is called pyelonephritis shown in Figure 1.7. Presumably, males are less prone to ascending UTIs than females because of their longer urethrae. Since the urinary tract is open to the external environment, it is easy for pathogens to gain entry and establish infection. Due to the production of urease by this organism, infection with P.mirabilis not only develops into cystitis and acute pyelonephritis but also causes stone formation in the bladder and kidneys. Urolithiasis is a hallmark of infection with this organism (Griffith 1976). Figure 1.7 A schematic diagram of the urinary tract showing urethra, bladder, ureters & kidneys and the indicating (red spots) are the diseases that are associated with Proteus. The virulence factors listed under each infection contribute to their pathogenicity (Adapted from Caroline et al 2000).
  66. 66. 36 1.7.3 Proteus as a Nosocomial Organism Proteus mirabilis is the second most common cause of urinary tract infection and is also an important cause of nosocomial infections. Bacteriuria occurs in 10% -15% of hospitalized patients with indwelling catheters. The risk of infection is 3% -5% per day of catheterization. In contrast, individuals with multiple prior infections of UTI, multiple antibiotic treatments, urinary tract obstruction, or infection developing after instrumentation frequently become infected with Proteus bacteria. Proteus mirabilis is susceptible to nearly all antimicrobials except tetracycline. It is sensitive to ampicillin, broad-spectrum penicillins such as ticarcillin, piperacillin, first-, second-, and third generation cephalosporins, imipenem and aztreonam; Proteus vulgaris and Proteus penneri are sensitive to trimethoprim and sulfamethoxazole, quinolone, imipenem and fourth generation cephalosporins. P. mirabilis, is believed to be the most common cause of infection-related kidney stones, one of the most serious complications of unresolved or recurrent bacteriuria (Ali et al 1998). Multi-drug-resistance strains of P. mirabilis generally produce extended-spectrum lactamasesor the AmpC type cephalosporinase and rarely carbapenemases and their prevalence in some settings is relatively high. Proteus species were found to have high antimicrobial resistance against tetracycline, chloramphenicol. It is susceptible to some antibiotics like chloramphenicol, vancomycin, and amoxicillin (Gus & Michael 2014). However, regular drug administration to these strains increases the multi-drug resistance property. Coliforms and Proteus species rarely cause extra-intestinal disease unless host defenses are compromised. Disruption of the normal intestinal flora by antibiotic therapy may allow resistant nosocomial strains to colonize or overgrow. Nosocomial strains progressively colonize the intestine and
  67. 67. 37 pharynx with increasing length of hospital stay, resulting in an increased risk of infection. These infections are often difficult to treat because of high levels of antibiotic resistance among bacteria in the hospital environment. The bacteria responsible for many common outpatient infections have also developed resistant strains, which are creating new obstacles to effective treatment (Butler et al 2001). Prevention of infections, particularly those that are hospital acquired, is difficult and perhaps impossible. Sewage treatment, water purification, proper hygiene, and other control methods for enteric pathogens will reduce the incidence of such enteropathogens. However, these control measures are rarely available in less developed regions of the world. Doctors, staff and other workers in hospitals can do much to reduce nosocomial infections through identification and control of predisposing factors, education and training of hospital personnel, and limited microbial surveillance (Emily & Trish 2011). Since field deployable rapid detection methods are not available for Proteus, developing effective non-invasive detection method using Volatile Organic Compounds (VOC) released by them has been conceived for next generation diagnostics and surveillance. We have developed a technique that has tremendous potential in non-invasive diagnosis and remote identification. 1.8 OVERVIEW OF THE THESIS The analysis of Volatile Organic Compounds (VOCs) in biological specimens has attracted a considerable amount of clinical interest over the past two decades. It is well known that a number of infectious or metabolic diseases could liberate specific odour characteristics of the disease stage, which can be noticeable in the sweat, breath, urine or the stools (Bekir 2004).
  68. 68. 38 Any disorder in the normal function of the body results in the liberation of complex volatile mixtures through the same media. Urinary Tract Infection (UTI), a disease which is dangerous and unrecognized forerunner of kidney disorders is addressed in this thesis. The potential of diagnostic power of VOC is not much prominent because the odour that is emanating from pathogenic bacteria may be tough to be detected and discriminated. The forthcoming chapters analyses the conventional techniques available for identification of bacteria by volatiles. It provides a potentially non-invasive means of diagnosis and monitoring of pathological processes through simple fluorescent assay named ProteAl. The first chapter of the thesis deals with the basic information on infectious diseases their mortality rate and the availability of conventional and modern methods for their identification. The chapter then elaborates on the use of extracellular target (VOC) for bacterial identification, the current analytical methods available, their limitations and alternate methods that can be employed. The next aspect of the chapter focuses on the metabolic pathways that are well established in bacteria for the production of various VOCs. The last aspect of the chapter describes why Proteus, has been taken as case study in this thesis. The methodology and the resources used in the study in order to execute the objectives are dealt in the second chapter. The study in general employed the common biochemical, microbiological and molecular biology reagents, solvents and techniques. The details of all the analytical instruments involved are also described. A few methods that were slightly modified for specific application are also elaborated in this chapter. The results obtained from the experiments carried out in the study are described in the third chapter. The first section of this chapter provides the
  69. 69. 39 results pertaining to the literature survey done to catalogue characteristic bacterial VOCs, extraction of VOCs from Proteus. The second section explains the results obtained from GC-MS, FT-IR analyses. The third section describes the development of colorimetric and fluorimetric assays for bacterial volatile aldehyde detection. The final section deals with the identification of metabolic pathway for 2-methylbutanal production in Proteus. The enhancement of 2-methylbutanal production by manipulating the growth medium with an amino acid isoleucine is revealed in this section. The variation in gene expression due to isoleucine supplementation is also focused in this chapter. The fourth chapter discusses the important findings of this study relating it to the existing methods. The first section explains the need for new thoughts for developing diagnostic assays, the significance of the method developed and their need. The next section elaborates on the 2-methylbutanal pathway and the significance of the supplemented media. The last section explains how the current findings are novel and its applications. The future scope of the study is explained with a conceptual diagram in the final section of this chapter. 1.9 OBJECTIVES The emergence and necessity for constant surveillance of UTI pathogens prompted us to develop an appropriate non-invasive instrumentation methodology. Since nondestructive and remote identifications are preferred for early diagnostics and surveillance, identification of such volatile compounds offered a promising approach. Considering the current clinical/diagnostic requirement the following objectives have been framed:  Investigation of characteristic Volatile Organic Compound of various organisms under defined growth conditions.
  70. 70. 40  Characterization and elucidation of structure of the characteristic VOC of Proteus species using instrumental analysis.  Development of simple non-invasive, non-destructive and most sensitive assay for the detection of the specific VOC of Proteus.  Validation of the developed assay using known clinical isolates and environmental samples.  Metabolic study using molecular biology tools to understand specific VOC biosynthesis and its regulation for hyper production.  Rational design of growth media for enhanced VOC production in order to improve the sensitivity.
  71. 71. 41 CHAPTER 2 MATERIALS AND METHODS 2.1 MATERIALS USED IN THIS STUDY The Table 2.1 and 2.2 gives the details of various chemicals, buffers and primer sequences used in our study. 2.1.1 Chemicals Used The chemicals such as organic, inorganic, acids, indicators, reagents etc. used in the study are tabulated below. Table 2.1 List of reagents, dyes and kits S. No. Chemicals Suppliers 1. Acids Acetic acid SRL, India Boric acid Merck Butyric acid Merck Hydrochloric acid SRL India Phosphoric acid Merck Propionic acid Merck 2. Alcohols Butanol Merck Ethanol Hayman, UK Methanol SRL India
  72. 72. 42 Table 2.1 (Continued) S. No. Chemicals Suppliers 3. Aldehydes Benzaldehyde Alfa Aesar Decanal Alfa Aesar Hexanal Alfa Aesar Nonanal Alfa Aesar 2-methylbutanal Spectrochem 4. Amino acids Isoleucine Himedia Leucine Himedia DL-Phenylalanine Himedia Valine Himedia 5. Enzymes DNase New England Biolabs Proteinase K Sigma-Aldrich Taq DNA Polymerase New England Biolabs 6. Growth medium Agar Himedia Casein acid hydrolysate Himedia Casein enzyme hydrolysate (Tryptone) Himedia Cetrimide Agar Himedia Eosin Methylene Blue Agar Himedia Methyl Red and Voges Proskauer agar Himedia Nutrient broth Himedia Salmonella Shigella Agar Himedia Simmons’ Citrate Agar Himedia Triple sugar iron agar Himedia Tryptone soya broth Himedia Urease broth Himedia Yeast extract Himedia
  73. 73. 43 Table 2.1 (Continued) S. No. Chemicals Suppliers 7. Ketones Acetophenone Alfa Aesar 2-heptonone Alfa Aesar 2-nonanone Alfa Aesar 2-pentanone Alfa Aesar 2-tridecanone Alfa Aesar 2-undecanone Alfa Aesar 8. Molecular kits PCR Purification Kit QIAGEN cDNA reverse transcription kit Applied Biosystems 9. Molecular Reagents Agarose Lonza, USA Diethylpyrocarbonate (DEPC) Sigma deoxynucleoside triphosphates (dNTP’s) New England Biolabs Ethylenediaminetetra acetic acid (EDTA) SRL India Ethidium bromide SRL India Phenol Sigma Aldrich Sodium dodecyl sulphate (SDS) SRL India Tris base Merck 10. Molecular markers DNA Ladder (100bp) New England Biolabs DNA Ladder (1Kb) New England Biolabs 11. Reagents Barritt reagent A Himedia Barritt reagent B Himedia 2,4 dinitrophenyl hydrazine (DNPH) Sigma Aldrich 5-dimethylaminonaphthalene- 1-sulphonyl hydrazine (DNSH) Sigma Aldrich Kovac’s reagent Himedia Methyl red Merck
  74. 74. 44 Table 2.1 (Continued) S. No. Chemicals Suppliers 12. Salts Disodium phosphate Merck Ferric chloride Merck Sodium acetate SRL India Sodium chloride Merck Sodium hydroxide Merck 13. Solvents Acetonitrile Fischer Scientific Chloroform Fischer Scientific Dichloromethane SRL India Diethyl ether SRL India Dimethyl sulphoxide SRL India Ethyl acetate SRL India Hydrogen peroxide Merck n-hexane SRL India 14. Vitamin Thiamine pyrophosphate (TPP) Himedia 2.1.2 Buffers used in this study The buffers used in the study and their composition are tabulated in Table 2.2. Table 2. 2 List of buffers used and their composition Buffers Composition pH Tris Borate EDTA (TBE) Tris base; Boric acid, 0.5M EDTA 8.0 TNES buffer 0.01 M Tris, 0.4 mM Nacl, 0.1 M EDTA, 0.5% SDS 8.0
  75. 75. 45 2.1.3 Cheminformatic Analysis of Bacterial Volatile Organic Compound Initially, an extensive literature survey was done to catalogue VOCs released by known pathogenic bacteria including Actinobacillus, Bacillus, Citrobacter, Clostridium difficile, E. coli, Enterobacter, Enterococcus faecalis, Klebsiella, Mycobacterium tuberculosis, Neisseria meningitides, Proteus, Psuedomonas, Salmonella, Serratia marcescens, Shigella, Staphylococcus and Xanthomonas campestris. Each organism produces a variety of compounds under different growth conditions. A set of acids, alcohols, aldehydes, esters, hydrocarbons, ketones, nitrogen and sulphur containing compounds have been identified to be produced by the bacteria during their growth. Each of these compounds serves as a signature for the organism in different growth medium. The lists of compounds produced by each organism grown under different growth medium are tabulated in the result section (Table 3.1). 2.1.4 Bacterial Strains used in the Study The details of the standard reference strains and well characterized clinical isolates are given below. 2.1.4.1 Standard strains Standard strains of Shigella flexineri (MTCC-1457 (ATCC- 29508), MTCC-9543), Salmonella paratyphi (MTCC 3220), Salmonella enterica subspecies (MTCC 3231), Proteus mirabilis (MTCC-425 (ATCC7002)), Proteus vulgaris (MTCC-426 (ATCC6380)), E. coli (MTCC-

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