Antimicrobial Activities of Nitrofurantoin and Some Novel Nitrofuran

Antim icFolDial activities of nitrofiiFantoin and some novel nitFofiiFan componnds

X lie s i tm itte i io T lie University of Lon ty Catlierine A nn Skarpe £ Jegree of D octor of Pkilosopky

Microbiology Section, Department of Pbarmaceutics, TLe School of Pharmacy, Brunswick Square, London

1 ProQuest Number: 10104255

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ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Dedicated to my parents M^argaret and. A llan Sliarpe Declaration and Acknowledgments

The work presented in this thesis was carried out in the Microbiology Section of the Department of Pharmaceutics and in the Department of Pharmaceutical and Biological Chemistry at The School of Pharmacy, University of London between November 1994 and November 1997. During this period ten days were spent at laboratories of the Genetic and Reproductive Toxicology Department, Medicine Safety Evaluation Division of GlaxoWellcome in Ware, Hertfordshire.

I would like to thank my supervisors Dr. R. J. Pinney and Dr. R. A. Watt for their substantial support and encouragement throughout this endeavour.

I should also like to thank: Marianne and Marion for their technical help and suggestions. Wilf for achieving such marvellous nuclear magnetic resonance spectra from the most contrary of compounds. Members of the GlaxoWellcome laboratories, especially Rob, Julia, Dave, Steve, Brian and Dr. Gatehouse, for providing facilities and guidance for the Salmonella and SOS/umu mutagenicity testing. Dr. Kazuei Igarashi of the Faculty of Pharmaceutical Sciences, Chiba University, Japan for the gift of Escherichia coli polyamine transport-deficient mutants. Proctor & Gamble Pharmaceuticals Inc., Norwich, NY, USA for their gift of semicarbazide acetic acid, used for the synthesis of a novel nitrofuran compound. The School of Pharmacy, University of London for their financial support.

DECLARATION AND ACKNOWLEDGMENTS The mechanism of action of nitrofurantoin against the Escherichia coli K12 strain AB1157 was investigated. Like quinoione antibacterials, nitrofurans damage DNA and induce SOS error-prone DNA repair. However, unlike the quinolones, which exhibit three separate mechanisms of action depending on whether RNA or protein synthesis or cell division is functioning, the activity of nitrofurantoin was shown not to be dependent on protein or RNA synthesis or on cell division. The resistance of Escherichia co//AB1157, which is wild-type with respect to DNA repair, and some DNA repair-deficient mutants to nitrofurantoin decreased in the order TK702 umuC > wild-type AB1157 > AB2494 lexA > AB2470 recB > AB2463 recA > JG138 pci A > TK501 uvrB umuC > JC3890 uvrB > AB1886 uvrA. Plasmid R46, which confers mutagenic error-prone DNA repair on its host, increased the minimum inhibitory concentration (MIC) of nitrofurantoin against uvrB- deficient strains but did not affect the MICs of the wild-type or other DNA repair- deficient strains. R46 conferred some protection against bactericidal concentrations of nitrofurantoin to AB1157 and the umuC, uvrA, uvrB and uvrB umuC mutants but not to the lexA, recA, recB or po/A-deficient strains. Other mutator plasmids pGW12, pGW16, pKM101, pYD1, R16, R124, R144, R391, R446b, R621a and R805a, from a variety of incompatibility groups, were also tested. None affected the MIC of nitrofurantoin against E. coli strain AB1157. However, pKM101, R446b, R621a and R805a conferred some protection whereas pGW12, pGW16, R144 and R391 sensitized AB1157 to a bactericidal concentration of nitrofurantoin. E. coli AB1157 showed low spontaneous mutation to nitrofurantoin resistance. Mutation frequencies were not increased in cells exposed to an inhibitory concentration of nitrofurantoin. R46 was the only plasmid of a variety of incompatibility groups to be eliminated from E. coli AB1157 by nitrofurantoin. Nine novel nitrofuran compounds were synthesized. The general method involved condensation of a polyamine (putrescine, spermidine or spermine) with a nitrofuran and isolation of the required products by extraction and other techniques. The products were characterized by UV spectroscopy, ^H nuclear magnetic resonance and mass spectroscopy. The inhibitory activities of the novel nitrofuran compounds were determined on a range of Gram-positive and Gram-negative

ABSTRACT 4 bacteria and fungi. Bis-5-nitrofurfurylidenylputrescine (compound I), bis-5- nitrofurfurylidenylspermine (compound II) and bis-5-nitrofurfurylidenylspermidine (compound III) showed excellent broad spectrum antibacterial activity and good antifungal activity. Bis-5-nitrofurfurylidenylethylspermidine (compound IX ) also showed good antifungal activity and 5-nitrofurfurylidenylglucosamine (compound VI) good antipseudomonal activity, neither of which are exhibited by nitrofurantoin. Compounds I, II, III and 4-(N-5-nitrofurfurylidenyl)aminobenzoyl-2-acetylaminoethylamide (compound IV ) were screened for mutagenicity and genotoxicity using the Ames Salmonella mutagenicity assay and the SOSIumu genotoxicity test. Nitrofurantoin was not mutagenic against S. typhimuhum strain TA98, which detects frameshift mutations, but was highly mutagenic against strain TA100, which detects base-pair substitutions. All four novel nitrofuran compounds tested were less mutagenic than nitrofurantoin in strain TA100. Similarly, all four novel nitrofuran compounds were shown to be considerably less genotoxic than nitrofurantoin in the SOSIumu test. The activities of nitrofurantoin and the four novel nitrofuran compounds I - IV were also compared in wild-type and polyamine transport- deficient mutants of E. coli K12. The novel compounds were active against transport-deficient mutants suggesting they would enter the cell via uptake mechanisms other than those determined by polyamine transport pathways.

ABSTRACT a

Page Line Corrigendum

27 8 (Smith, 1980) should read (Smith, 1984) 50 2 (Lewin, 1994) should read (Lewin, 1994; Wang, 1985) 50 3/4 Insert “such as topoisomerases II and IV ” to read “Type 2 topoisomerases, such as topoisomerases II and IV, produce a transient...”

50 8 Insert “Topoisomerase IV is necessary for chromosome segregation (Kato at a!., 1990; Kato at a/., 1992)” to read “...(Srivenugopal at a/., 1984). Topoisomerase IV is necessary for chromosome segregation (Kato at a/., 1990; Kato at a!., 1992). Type 1...” 58 15 (Ferrrante ef a/., 1995) should read (Ferrante ef a/., 1995) 54 18 Replace (Lewin, 1994) with (Hardy, 1988; Jacob ef a/., 1963)

8 6 8 Insert “using strains of E. coli 343/113 thy arg lys (Materials & Apparatus Table 2.1.1)” to read “Plasmids were transferred by conjugation in nutrient broth using R^ strains of E. coli 343/113 thy arg lys (Materials & Apparatus Table 2.1.1).”

8 8 9 Replace “Bactericidal” with “Bacteriostatic” to read “Bacteriostatic activity was determined from the zones of inhibition.” 112 N/A Figure 4.1.19. legend should read Survival of E. coli AB1886 uvrA and uvrA(R46) in 20xMIC of nitrofurantoin. # = uvrA] O = uvrA(R46) 125 32 Replace “Quinolones target DNA as their site of action (Goss at a/., 1964)...” with “Quinolones bind to DNA;topoisomerase II (DNA gyrase) complexes blocking DNA replication (Chen at ai, 1996)...”

CORRIGENDA 169 last Replace “thanthose” with “than those” 170 13 Replace “polyamine-deficient” with “polyamine uptake- deficient” 178 27 Replace “stevens” with “Stevens” 184 25/26 After “...exposed to clinically-attainable concentrations of nitrofurantoin.” insert “Nitrofurantoin-induced mutants may have been observed had the concentration of nitrofurantoin to which the cells were exposed been less lethal. Mutants may also have been observed had the cells been given a chance to recover and express resistance in nitrofurantoin- free medium after exposure to nitrofurantoin and before plating onto nutrient agar containing nitrofurantoin.” 194 26 Replace “antimicrobialsmight” with “antimicrobials might”

Figures Error bars on all figures show standard error of the mean (SEM)

R e fe re n c e s

Chen, C.-R., Malik, M., Snyder, M. & Driica, K. (1996) DNA gyrase and topoisomerase IV on the bacterial chromosome: quinolone-induced DNA cleavage. Journal of Molecular Biology 258:627-637

Jacob, P., Brenner, S. & Cuzin, F (1963) On the regulation of DNA replication in bacteria. Cold Spring Harbor Symposium on Quantitative Biology 2B\ 329- 348

Kato, J., Nishimura, Y., Imamura, R., Niki, H., Hiraga,8 . & Suzuki, H. (1990) New topoisomerase essential for chromosome segregation in E. coll. Cell 63: 393-404

Kato, J., Suzuki, H. & Ikeda, H. (1992) Purification and characterization of DNA topoisomerase IV in Escherichia coli. Joumal of Biological Chemistry 2S7: 25676-25684

Wang, J. 0. (1985) DNA topoisomerases. Annual Review of Biochemistry 54: 665-697

Appeniis A (pages 232-235) makes up part of this corrigenda

CORRIGENDA Page

Declaration and Acknowledgements 3 Abstract 4 Contents 6 Abbreviations and Symbols 14 List of Figures 18 List of Tables 24 List of Chemical Schemes 26 Aims of This Thesis 27

PART ONE INTRODUCTION 28 1. Nitrofurans 29 1.1. Metabolism of nitrofurans 34 1.2 Action of nitrofurans 36 2. Polyamines 38 2.1. Polyamine biosynthesis 40 2.1.1 Arginine decarboxylase 40 2.1.2. Agmatine ureohydrolase 40 2.1.3. Ornithine decarboxylase 40 2.1.4. Spermidine synthetase 41 2.1.5. S-Adenosylmethionine synthetase 41 2.1.6. S-Adenosylmethionine decarboxylase 41 2.1.7. Lysine decarboxylase 41 2.2. Polyamine regulation 42 2.3. Polyamine metabolism 43 2.3.1. Acetyltransferases 43 2.3.2. Amine oxidases 43 2.3.3. Glutathionylspermidine and trypanothione synthetase 43 2.4. Polyamine transport 44 2.4.1. The putrescine/spermidine transport system 44

CONTENTS 6 2.4.2. The low activity putrescine transport gene 44 2.4.3. The putrescine-only transport system 44 2.5. Functions of polyamines 45 2.5.1. Interactions with DNA 45 2.5.2. Interactions with RNA 46 2.5.3. Interactions with ribosomes 46 2.5.4. Effects on growth 47 2.5.5. Involvement in macromolecular synthesis 47 2.5.6. Interactions with membranes 49 2.5.7. Interactions with proteins 49 3. DNA mutation 50 3.1 Causes of DNA mutation 51 3.1.1. Tautomerism 51 3.1.2. Spontaneous deamination 51 3.1.3. A base-analogue 52 3.1.4. Chemical deamination 52 3.1.5. Chemically-induced tautomerism 52 3.1.6. Alkylating agents 52 3.1.7. Intercalating agents 53 3.1.8. Ultra-violet irradiation 53 3.1.9. Ionizing radiation 53 3.2 DNA repair by Escherichia coli 54 3.2.1. Photoreactivation 54 3.2.2. Nucleotide excision repair 54 3.2.3. Base excision repair 55 3.2.4. The adaptive response 56 3.2.5. Mismatch repair 59 3.2.6. Recombination repair 60 3.2.7. Error-prone repair 63 3.2.8. The 8 0 S regulon 63 4. Plasmids 64

CONTENTS PART TWO MATERIALS AND APPARATUS 67 1. Micro-organisms 68 2. Plasmids 71 3. Media 72 4. Antimicrobial agents 75 5. Nutritional requirements 75 6. Chemicals 76 7. Materials for mutagenicity studies 8. Apparatus 80

PART THREE EXPERIMENTAL METHODS 83 1. Culture suspensions 84 1.1. Overnight stationary phase MB 84 1.2. Overnight stationary phase PBS 84 1.3. Log phase NB 84 1.4. Log phase PBS 84 2. Concentrated culture suspensions 84

3. Viable counting 85 3.1. Drop-plate viability 85 3.2. Spread-plate viability 85 4. Post-UV survival 85 5. Plasmid transfers 86

6 . Determination of minimum inhibitory concentration 87 6.1. Preparation of antibiotic/antimicrobial nutrient agar 87 6.2. Inoculation of plates 87 6.2.1. Multipoint inoculations 87 6.2.2. Loop inoculations 87 7. Agar diffusion tests 87 7.1. Seeded agar 88 7.2. Zones of inhibition 88

8 . Determination of bacterial sensitivity in liquid media 88

9. Mutation to nitrofurantoin resistance 8 8

CONTENTS 8 9.1. Spontaneous mutation on solid media 0 8

9.2. Mutation in liquid media 8 9 10. Replica plating 89 11. Plasmid elimination 89 11.1. Lethal concentration 90 11.2. Sublethal concentration 90 12. Ames Salmonella plate incorporation mutagenicity assay 90 12.1. Preliminary toxicity tests 90 12.2. Microbial mutagenicity tests 91 13. SOS/umu test 91 14. Manufacture of novel nitrofuran compounds 92 14.1. Condensation of nitrofuran and amine in ethanol 92 14.2. Condensation of nitrofuran and amine in acetonitrile 93 14.3. Condensation of nitrofuran, 4-aminobenzoic acid and amine 93 14.4. Condensation of 5-nitrofurfuraldehyde diacetate and amine 95 in ethanol 14.5. Exchange reaction of 5-nitrofuraldoxime and amine via N- 96 (heptylidene)-amine in isopropanol/water 14.6. Exchange reaction of 5-nitrofuraldoxime and amine via N- 96 (heptylidene)-amine in isopropanol/water 14.7. Reaction of 5-nitrofurfuraldehyde and semicarbazide acetic 97 acid 14.8. Reaction progress 99 14.9. Method of acétylation 99

PART FOUR RESULTS 100 1. Sensitivities to nitrofurantoin of Escherichia coli DNA repair- i oi deficient strains and their plasmid R46-containing derivatives 1.1. Introduction 101

1.2. Plasmid transfers 101

1.3. Post-UV survival ofEscherichia coli and the effect of R46 102 1.4. Minimum inhibitory concentrations of nitrofurantoin and the 106

CONTENTS 9 effect of R46 1.5. Bactericidal profile of nitrofurantoin 107 1.6. Sensitivities of DNA repair-deficient strains to 20xMIC of 109 nitrofurantoin 1.7. Summary 113 2. Protective or sensitizing effects, to lethal activity of 115 nitrofurantoin, of a variety of plasmids from different incompatibility groups that either protect against or sensitize to UV light 2.1. Introduction 115 2.2. Plasmid transfers 115 2.3. Effects of plasmids on post-UV survival ofEscherichia coli 116 AB1157 2.4. The effects of plasmids on the minimum inhibitory 116 concentration of nitrofurantoin for Escherichia co//AB1157 2.5. Survival of Escherichia coli AB1157, containing a range of 120 different plasmids, in a bactericidal concentration of nitrofurantoin 2.6. Summary 124 3. Antibacterial action of nitrofurantoin against Escherichia coU 125 when protein synthesis, RNA synthesis or cell division are inhibited 3.1. Introduction 125 3.2. Antibacterial activity of nitrofurantoin in different liquid 126 media and drug combinations 3.3. Summary 129 4. Plasmid stability to nitrofurantoin inEscherichia coii AB1157 130 4.1. Introduction 130 4.2. Elimination of plasmids from cells exposed to a lethal 130 concentration of nitrofurantoin 4.3. Elimination of plasmids from cells exposed to a sub- 132

inhibitory concentration of nitrofurantoin 4.4. Summary 132

CONTENTS ÏÔ 5. Mutation of Escherichia coli to nitrofurantoin resistance 134 5.1. Introduction 134 5.2. Confirmation of the R46 mutator effect 134 5.3. Spontaneous mutation of Escherichia coli to nitrofurantoin 137 resistance 139 5.4. Mutation to nitrofurantoin resistance in nitrofurantoin- exposed cultures of Escherichia coli 140 5.5. Summary 140 6. Preparation of novel nitrofuran compounds 140

6 .1. Preparation of bis-5-nitrofurfurylidenylputrescine (I) 141 6.2. Preparation of bis-5-nitrofurfurylidenylspermine (II) 142 6.3. Preparation of bis-5-nitrofurfurylidenylspermidine (III) 142 6.4. Preparation of 4-(N-5-nitrofurfurylidene)aminobenzoyl-2- acetylaminoetylamide (IV) 143 6.5. Preparation of bis-5-nitrofurfurylidenylmethyllysine (V) 143

6 .6 . Preparation of bis-5-nitrofurfurylidenylglucosamine (VI) 144 6.7. Preparation of acetyl ated bis-5-nitrofurfurylidenylglucosamine (VII) 145

6 .8 . Preparation of N-acetyl-N-(5-nitrofurfurylethenylidenyl)- ethylenediamine (VIII) 145 6.9. Preparation of bis-5-nitrofurfurylidenylethylspermidine (IX) 145 6.10. Summary 146 7. Antimicrobial activities of novel nitrofuran compounds 146 7.1. Introduction 146 7.2. Minimum inhibitory concentrations 149 7.3. Agar diffusion 154 7.4. Summary 156 8. Mutagenicity of novel nitrofuran compounds 156 8.1. Introduction 157 8.2. Ames Sa/mone//a plate-incorporation assay 157 8.2.1. Preliminary toxicity tests 157 8.2.2. Microbial mutagenicity tests 155 8.3. SOS/umu test 159

CONTENTS ÏT 8.4. Summary -|7 0 9. Activities of novel nitrofuran compounds against polyamine

transport deficient mutants of Escherichia coii 170 9.1. Introduction 171 9.2. Antibacterial activity of the novel nitrofuran compounds in liquid medium 174 9.3. Summary

175 PART FIVE DISCUSSION 176 1. Sensitivities to UV and nitrofurantoin of Escherichia coli DNA repair-deficient strains and the effect of R46 1 78 2. Effect of a variety of plasmids on the survival of Escherichia

coli AB1157 in a lethal concentration of nitrofurantoin 1 79 3. Antibacterial action of nitrofurantoin against Escherichia coli when protein synthesis, RNA synthesis or cell division are inhibited 181 4. Plasmid stability to nitrofurantoin inEscherichia coli AB1157 182 5. Mutation of Escherichia coli to nitrofurantoin resistance 184 6. Antimicrobial activities of novel nitrofuran compounds 188 7. Mutagenicity of novel nitrofuran compounds 190 8. Activities of novel nitrofuran compounds against polyamine transport deficient mutants of Escherichia coli

PART SIX SUMMARY AND CONCLUSIONS 192

REFERENCES 195

CONTENTS 12 PAPERS

Abstracts of the following publications are included as part of this thesis

Sharpe, C. A. & Pinney, R. J. (1995) Resistance of R plasmid-containing strains of

Escherichia coli to nitrofurantoin. Journal of Pharmacy and Pharmacology 47:

1062

Watt, R A., O'Reilly, Y. M., Williams, B. J., Sharpe, C. A. & Pinney, R. J. (1997) Synthesis and antimicrobial activity of nitrofuran-polyamine conjugates.

Journal of Pharmacy and Pharmacology 47: 1111

Sharpe, C. A. & Pinney, R. J. (1997) Antibacterial action of nitrofurantoin against

Escherichia coli when protein synthesis, RNA synthesis or cell division are

inhibited. Journal of Pharmacy and Pharmacology 49 (Suppl. 4): 65

Sharpe, C. A., Watt, R. A. & Pinney, R. J. (1997) Antimicrobial activities of novel

nitrofuran-polyamine conjugates. Abstracts of the 37th ICAAC Annual Meeting of the American Society for Microbiology : 151

CONTENTS 13 aii

A bbreviations Aldrich-Chemical Co Aldrich-Chemical Company Ltd., Dorset, England Amersham Amersham, Bury, England Ap ampicillin ADP adenosine diphosphate ara arabinose arg arginine ATCC American Type Culture Collection ATP adenosine triphosphate ATPase adenosine triphosphatase Beecham Research Beecham Research Labs, Welwyn Garden City, England BDH Chemicals BDH Chemicals Ltd., Poole, England bio biotin (vitamin HO BOC British Oxygen Company, England Burroughs Wellcome Burroughs Wellcome and Co., The Wellcome Foundation Ltd., London, England C confluent growth cfu colony forming units CIC critical inhibitory concentration Cm chloramphenicol X degrees centigrade Da daltons Difco Laboratories Difco Laboratories, Detroit, USA DM Davis-Mingioli minimal salts solution DMA Davis-Mingioli minimal salts selecting agar DMSO dimethyl sulphoxide DNA deoxyribonucleic acid d/s double-stranded (DNA) D/S double strength Evans Evans, Horsham, Sussex, England Fisher Scientific Fisher Scientific UK Ltd., Loughborough, England

ABBREVIATIONS AND SYMBOLS 14 Fisons Fisons Scientific Equipment, Loughborough, England FlowFusor® FL FlowFusor® FL (Manufacturing) Ltd., Fresenius (Manufacturing) Ltd Healthcare Group, England Fluka Chemical Fluka Chemical AG, Buchs F plasmid fertility plasmid of Escherichia coli g grammes gal galactose glu glucose Gm gentamicin Hfr high frequency of recombination plasmid

Hg mercuric ions his histidine hr hour(s) Inc incompatibility group ISO Iso-sensitest agar J joules k kilo (1 0 ^) Km kanamycin Koch-Lights Labs Koch-Light Labs. Ltd., Haverhill, Suffolk, England I litre lac lactose Lancaster Chemical Lancaster Chemical Research, Morecombe, England Lederle Laboratories Lederle Laboratories, Gosport, Hampshire, England leu leucine lys lysine

m prefix - milli ( 1 0 '^) suffix - metre

M prefix - mega ( 1 0 ®) suffix - molar (moles per litre) MAC MacConkey agar mal maltose May & Baker Ltd. May & Baker Ltd., Eastbourne, England met methinine MIC minimum inhibitory concentration Millipore Millipore, Bedford, USA min minutes

ABBREVIATIONS AND SYMBOLS 15 n nano (1 0 ’®) N neat, undiluted (culture suspension) NA nutrient agar N/A not applicable nad nicotinamide adenine dinucleotide NADP nucleotidyl adenosine diphosphate NB nutrient broth NCTC National Collection of Type Cultures Nicholas Laboratories Nicholas Laboratories Ltd., Slough, England Nf nitrofurantoin Nm 0/N overnight (culture suspension) ONPG o-nitrophenyl-P-D-galactopyranoside p protection phenotype PBS phosphate buffered (normal) saline Pm paramomycin pro proline Proctor & Gamble Proctor & Gamble Pharmaceuticals, Norwich psi pounds per square inch R resistance phenotype Rf rifampicin rha rhamnose R plasmid resistance plasmid rpm revolutions per minute s sensitivity phenotype SAB Sabouraud agar or broth SC semiconfluent growth SDS sodium dodecyl sulphate Sigma Chemical Co Sigma Chemical Company Ltd., USA Sm streptomycin s/s single-stranded (DNA)

SSB single strand binding protein Su sulphonamides (sulphadiazine unless stated

ABBREVIATIONS AND SYMBOLS 16 Te tetracycline TGA tryptone glucose media thi thiamine (vitamin thr threonine thy thymine Tm trimethoprim trp tryptophan Unipath Ltd. Oxoid Ltd. / Unipath Ltd., Basingstoke, England UV ultraviolet irradiation (254 nm) w/v weight per volume xyl xylose

SYMBOLS

M micro (1 0 '®) % per cent registered trade mark

ABBREVIATIONS AND SYMBOLS 17 List of Figures

Page

Figure 1.1.1. Structure of heterocyclic furan ring 29 Figure 1.1.2. 2-Furfuraldehyde 29 Figure 1.1.3. 2-Furfuryl alcohol 29 Figure 1.1.4. 2-Furfuroic acid 29 Figure 1.1.5. Nitrated derivative 29 Figure 1.1.6. Hydantoin ring 33 Figure 1.1.7. Oxazolidinone ring 33 Figure 1.1.8. Example of a vinylogous nitrofuran 33 Figure 1.1.9. Example of a di-(nitrofuran) 34 Figure 1.1.10. Reduction of nitrofurans 35 Figure 1.2.1. Polyamine biosynthesis and metabolism in 39 Escherichia coli Figure 1.3.1. Spontaneous deamination of cytosine to uracil 52 Figure 3.14.1. Spermine analogue of nitrofurantoin 97 Figure 3.14.2. Spermidine analogue of nitrofurantoin 97 Figure 3.14.3. Nitrofurantoin with aminoacetaldehyde spacer 97 Figure 4.1.1. Post-UV survival of Escherichia coli AB1157 and the 103 DNA repair-deficient strains Figure 4.1.2. Post-UV survival of E. coli AB2494 lexA and 104 /exA(R46) Figure 4.1.3. Post-UV survival of E. coli AB2463 recA and 104 recA(R46) Figure 4.1.4. Post-UV survival of E. coli JG138 polA and po/A(R46) 104 Figure 4.1.5. Post-UV survival of E co//AB1157 and AB1157(R46) 104 Figure 4.1.6. Post-UV survival of E. coli TK702 umuC and 105 umuC{RA6) Figure 4.1.7. Post-UV survival of E. coli AB2470 recB and 105 recB( R46) Figure 4.1.8. Post-UV survival of E. coli AB1886 uvrA and 105

LIST OF FIGURES 18 uvrA(R46) Figure 4.1.9. Post-UV survival of E. coli JC3890 uvrB and 105 uvrB(R46) Figure 4.1.10. Post-UV survival of E. co//TK501 uvrB umuC and 106 uvrB umuC(R46) Figure 4.1.11. Bactericidal activity of nitrofurantoin on E. coli 108 AB1157

Figure 4.1.12. Survival of Escherichia coli AB1157 and DNA repair- 11 o deficient strains in 20xMIC of nitrofurantoin Figure 4.1.13. Survival of E. coli AB2494 lexA and /exA(R46) in m 20xMIC of nitrofurantoin Figure 4.1.14. Survival of E. coli AB2463 recA and recA(R46) in m

2 0 xMIC of nitrofurantoin Figure 4.1.15. Survival of E. coli AB2470 recB and recB(R46) in m 20xMIC of nitrofurantoin

Figure 4.1.16. Survival of E. coli JG138 polA and po/A(R46) in 1 20xMIC of nitrofurantoin

Figure 4.1.17. Survival of E. coli AB1157 and AB1157(R46) in 1 1 2 20xMIC of nitrofurantoin

Figure 4.1.18. Survival of E. coli TK702 umuC and umuC{R46) in 11 2 20xMIC of nitrofurantoin

Figure 4.1.19. Survival of E. coli AB1886 uvrA and uvrA(R46) in 11 2

2 0 xMIC of nitrofurantoin

Figure 4.1.20. Survival of E. coli JC3890 uvrB and uvrB(R46) in 1 1 2 20xMIC of nitrofurantoin

Figure 4.1.21. Survival of E. coli TK501 uvrB umuC and uvrB 1 1 3 umuC(R4S) in 20xMIC of nitrofurantoin

Figures 4.2.1. - 4.2.4. Post-UV survival of E. coli AB1157(R") and 1 1 7 AB1157(R+) 4.2.1. R16 4.2.2. R46 4.2.3. R124 4.2.4. R144

Figures 4.2.5. - 4.2.8. Post-UV survival of E. coli AB1157(R~) and 1 1 8 AB1157(R+) 4.2.5. R391 4.2.6. R446b 4.2.7. R621 a 4.2.8. R805a

LIST OF FIGURES 19 Figures 4.3.7. - 4.3.8. Effect of chloramphenicol or rifampicin when 129 present with nitrofurantoin in nutrient broth (NB) or phosphate buffered saline (PBS) from the start of incubation 4.3.7. NB 4.3.8. PBS Figures 4.5.1. -4.5.2. Survival and mutation frequencies of E. coli 138 AB1157 and AB1157(R46) in 0.0 or 20 pg mM of nitrofurantoin (I) 4.5.1. Survival 4.5.2. Mutation frequencies

Figures 4.5.3. - 4.5.4. Survival and mutation frequencies of E. coli 139 AB1157 and AB1157(R46) in 0.0 or 20 pg ml'^ of nitrofurantoin (II) 4.5.3. Survival 4.5.4. Mutation frequencies Figure 4.6.1. Bis-5-nitrofurfurylidenylputrescine 140 Figure 4.6.2. Bis-5-nitrofurfurylidenylspermine 140 Figure 4.6.3. Bis-5-nitrofurfurylidenylspermidine 141 Figure 4.6.4. 4-(N-5-nitrofurfurylidenyl)aminobenzoyl-2-acetyl- 142 aminoethylamide

Figure 4.6.5. Bis-5-nitrofurfurylidenylmethyllysine 1 4 2 Figure 4.6.6. 5-Nitrofurfurylidenylglucosamine 143

Figure 4.6.7. Acetyl ated 5-nitrofurfurylidenylglucosamine 1 4 4 Figure 4.6.8. N-Acetyl-N-(5-nitrofurfurylethenylidenyl)ethylene- 144 diamine

Figure 4.6.9. Bis-5-nitrofurfurylidenylethylspermidine 1 4 5

Figure 4.7.1. Structures of novel nitrofuran compounds I - IX, 1 4 7 parent compounds X - XII, nitrofurantoin (XIII) and the polyamines, putrescine, spermidine and spermine (XIV - XVI)

Figures 4.7.2. - 4.7.4. Antimicrobial activities of novel nitrofuran 1 5 0 compounds I, II and IV

4.7.2. Compound I 4.7.3. Compound II 4.7.4. Compound IV

LIST OF FIGURES 21 Figures 4.7.5. - 4.7.7. Antimicrobial activities of novel nitrofuran 151 compounds V and V II and 5-nitro furfuraldehyde (X) 4.7.5. Compound V I 4.7.6. Compound V II 4.7.7. Compound X Figures 4.7.8. - 4.7.10. Antimicrobial activities of 5-nitrofuranacrolein 152 (XI), 5-nitrofuraldoxime (X II) and nitrofurantoin XIII 4.7.8. Compound X I 4.7.9. Compound x n 4.7.10. Compound XIII

Figures 4.7.11 . Antimicrobial activities of novel nitrofuran compound 153 III which was only active against S. cerevisiae and compounds V, V III and IX which were only tested against E. coli, S. aureus and S. cerevisiae Figure 4.8.1. Structures of novel nittrofuran compounds I - IV 156 Figure 4.8.2. Dose-mutagenicity relationships for nitrofurantoin 159 using S. typhimurium strains TA98 and TA100 in the presence and absence of 89-mix Figures 4.8.3. - 4.8.4. Dose-mutagenicity relationships for novel i so nitrofuran compounds I, n, m and IV using S. typhimurium TA98 in the presence and absence of 89-mix 4.8.3. In presence of 89-mix 4.8.4. In absence of 89-mix Figures 4.8.5. - 4.8.6. Dose-mutagenicity relationships for novel 161 nitrofuran compounds I, n, m and IV using S. typhimurium TA100 in the presence and absence of 89-mix 4.8.5. In presence of 89-mix 4.8.6. In absence of 89-mix Figure 4.8.7. Dose-mutagenicity relationships for nitrofurantoin 162 using S. typhimurium TA98NR in the presence and absence of 89-mix Figure 4.8.8. Dose-mutagenicity relationships for compounds I 162 and IV using S. typhimurium TA98NR in the presence and absence of 89-mix

LIST OF FIGURES 22 Tiri»le 4.5.1. Mutation frequencies to nalidixic acid resistance 135 (5xMIC)

Table 4.5.2. Spontaneous mutation frequencies of E. coli AB1157 135 and AB1157(R46) to nitrofurantoin resistance determined on solid media

Table 4.5.3. Number of E. coli AB1157 and AB1157(R46) 136 colonies maintaining resistance to nitrofurantoin

Table 4.7.1 Minimum inhibitory concentrations of the novel 148 nitrofuran compounds, their parent compounds, nitrofurantoin and the polyamines, putrescine, spermidine and spermine

Table 4.7.2. Critical inhibitory concentrations of the nine novel 154 nitrofuran compounds, their parent compounds, nitrofurantoin and the polyamines, putrescine, spermidine and spermine

Table 4.8.1 Minimum inhibitory concentrations of compounds I, 158 II, III, IV and nitrofurantoin against S. typhimurium TA98 using the plate-incorporation method Table 4.8.2. Minimum concentrations of compounds I, II, III, IV 159 and nitrofurantoin which cause mutagenesis Table 4.8.3. Mutagenic potencies of the novel nitrofuran 164 compounds and nitrofurantoin

Table 4.8.4. Lowest concentration at which p-galactosidase 166 activity is doubled by the novel nitrofuran compounds I, II, III, IV and nitrofurantoin in S. typhimurium TA1535(pSK1002)

LIST OF TABLES 25 Aim s oi Tikis TIkesis

Like the quinolones of twenty years ago, nitrofurans are used in human medicine solely for the treatment of urinary tract or gastro-intestinal infections. They are rapidly metabolised in liver and body tissues, which reduces their systemic effect, but 30-50% of an oral dose is eliminated unchanged in the urine. They are considered too toxic for systemic use, giving rise to hypertension, pulmonary toxicity and inhibition of mitochondrial respiration. They are also proven mutagens, although carcinogenicity has not been demonstrated. The full potential of nitrofurans as antimicrobials has yet to be awakened (Smith, 1980). As nitrofuran activity is due to DNA adduct formation, a derivative which both increases the uptake of nitrofuran into the cell and targets the molecule to DNA, its site of action, might well improve activity. Holley et al. (1992) showed that conjugating nitroimidazoles to polyamine carriers facilitated entry of the former into Ehrlich ascites tumour cells (EATC) and targeted them to DNA. In addition, this increased uptake did not lead to increased toxicity. In an analogy with this work, polyamine derivatives of nitrofuran will be synthesized and tested to compare their antimicrobial activities and their mutagenic and genotoxic potentials with nitrofurantoin. Their activity in polyamine transport-deficient E. coli mutants will also be examined. The work will also study the sensitivities, to nitrofurantoin, of various DNA repair-deficient mutants and determine whether R plasmids, which confer a DNA repair capacity on their host, increase resistance to the DNA-damaging nitrofurans. Since many DNA-damaging drugs eliminate plasmids from host bacteria, this will also be tested for a range of clinically-isolated R plasmids.

AIMS OF THIS THESIS 27 PART ONE

INTRODUCTION 28 1. Nitrofurans Dodd & Stillman (1944) were the first to report the antibacterial activities of furan derivatives. Furans are characterized by a heterocyclic ring consisting of 4 carbon and 1 oxygen atoms (Figure 1.1.1).

4 3 Figure 1.1.1. Structure of heterocyclic furan ring

The majority of compounds studied by Dodd & Stillman (1944) were derivatives of either the basic furan structure shown above or the three simple furan compounds; 2-furfuraldehyde (Figure 1.1.2), 2-furfuryl alcohol (Figure 1.1.3) and 2- furfuroic acid (Figure 1.1.4). Twenty-five of the compounds were also nitrated at the Cg position on the furan ring (Figure 1.1.5).

CHgO H

Figure 1.1.2. 2-Furfuraldehyde Figure 1.1.3. 2-Furfuryl alcohol

COOH

Figure 1.1.4. 2-Furfuroic acid Figure 1.1.5. Nitrated derivative

Seventeen nitrated compounds were compared with their non-nitrated parent. Only three of the non-nitrated compounds showed any bacteriostatic action, these being acid esters of 2-furfuryl alcohol. However, the addition of a nitro group in the Cg position conferred bacteriostatic properties on the fourteen previously non-active compounds and increased the bacteriostatic properties of the three slightly active compounds. Seven other nitrated derivatives also showed bacteriostatic action with only one of the twenty-five nitrated compounds being inactive. The group present

INTRODUCTION 29 at the Cg position of the furan ring was also found to influence the antimicrobial effectiveness of the compound, furfuroic acid derivatives being the least active (Dodd & Stillman, 1944). During the period 1944 to 1960 many hundreds of similar compounds were synthesized and tested for antimicrobial activity (Chamberlain, 1976; Miura & Reckendorf, 1967) including compounds in current use or which have been used in human or veterinary medicine (Table 1.1.1).

Table 1.1.1. Nitrofurans used in human and/or veterinary medicine. (Based upon Budavari et a i, 1989; Debuf, 1991; Fraser ef a/., 1991; Martindale, 1996; Sittig, 1979; Therapeutic Drugs, 1991)

Nitrofuran compounds (Molecular mass) Use and date of introduction

Furaltadone Veterinary use: orally for C13H16N4O6 coccidiosis and (324.3) J \ À OoN O histomoniasis in poultry and intestinal infections; topically for mastitis. Norwich Pharmacal 1957

Furazolidone Veterinary use: orally for CgHyNaOs 7 V intestinal infections; intra (225.2) 0 ,N o uterine use for endometriosis. VU Norwich Pharmacal 1956

Furazolium 0 |- Antibacterial chloride 1964 (Netherlands) C9H8N3O3SCI (273.7)

Nidroxyzone O Anti-infective 7 V C8 H10N4O5 N, A Eaton Labs 1947 (242.2) 0 ,N 0 N NK CHgCHgOH

Nifuradene O Antibacterial C8 H8 N4O4 7 V Norwich Pharmacal 1956 (224.2) 0 ,N o VU continued overleaf

INTRODUCTION 30 Nifuraldazone Veterinary use as C7 H5N4O5 N. NH2 antibacterial for enteritis in (226.2) OoN o N calves. I H O Eaton Labs 1947

Nifuratel Orally and vaginally as

C10H11N3O5S antibacterial, antifungal and (285.3) antiprotozoal Polichimica 1963 CH2SCH3

Nifurfoline Antibacterial // V C 1 3 H 1 5 N 5 O 0 0,N' 'o' N' 'N 1964 (Spain) (337.3)

Nifuroquine Veterinary use for bovine C14H8 N2O6 mastitis. (300.2) 1967 (South Africa)

Nifuroxazide Intestinal antiseptic for // V C12H9N3O5 OgN 0 colitis and diarrhoea. (275.2) Robert et Carrière 1963 OH

Nifuroxime Topical antiprotozoal and C5H4N2O4 anti-infective. Also used in N (156.1) Â OH conjunction with 0 ,N ^ 0 furazolidone to treat vaginitis. Norwich Pharmacal 1943

Nifurpirinol Antibacterial in fish diseases C12H10N2O4 and for human use. (246.2) Dainippon 1967

CHLOH œntinued overleaf

INTRODUCTION 31 Nifurprazine n A Topical antibacterial CioHgN^Og ^ Boehringer Mannheim 1963 (232.2)

Nifursol fj—\ Veterinary use as NO, C12H7 N5O9 Q N o N (365.2) ' ° 1 H

NO2

Nifurtimox Antiprotozoal (Chaga’s C10H13N3O5S disease). (287.3) 1964 (Germany) SO. HX

Nifurtoinol A ^ Antibacterial

( 2 ® ° ' o,n ' ^ o^ " ' n ^ n - ch ,oh 1962 (Belgium) H

Nifurzide rj~\ m Anti-infective, antibacterial CizHsNAS (diarrhoea). (336.3) H ^ 1972 (Germany)

Nihydrazone r— ^ O Veterinary use as C7 H7 N3O4 J! isj 11 antibacterial and (197.2) OgN CH 3 antiprotozoal. ^ Eaton Labs 1947

Nitrofurantoin /— v O Orally for UTIs. CgHgN^Og / / \ \ N Â Eaton Labs 1952 (238.2) " n m

continued overleaf

INTRODUCTION 32 Nitrofurazone O Veterinary use in bovine CgHgN^O^ N. A mastitis and metritis and for (198.1) OgN N NHL prevention of intestinal I infections. Human use as a H topical antibacterial. Eaton Labs 1947

Nitrovin Veterinary use as Ci4Hi2NgOg OgN NÜ2 antibacterial and g (360.3) promoter. NH 1954 (Japan) HN NH;

AsO,H NPA acid 3' '2 Veterinary use for

C12H11ASN4O 7 coccidiosis. 0,N' ^ 0 (398.2) I I Norwich Pharmacal 1957 H H

Nitrofurans fall into one of four groups: azomethine, vinylogous, di- (nitrofuran) and others; i.e. nitrofurans which don't fall into the previous three categories. The azomethine group contains compounds which have heterocyclic rings joined to the nitrofuran (NF) at position C 2 via an azomethine bond (C=N). The majority of these heterocyclic rings are hydantoin (Figure 1.1.6) or oxazolidinone rings (Figure 1.1.7).

Figure 1.1.6. Hydantoin ring Figure 1.1.7. Oxazolidinone ring

Vinylogous nitrofurans have an additional vinyl group ( CH=CH-) between the nitrofuran ring and the azomethine group (Figure 1.1.8).

N R

Figure 1.1.8. Example of a vinylogous nitrofuran

INTRODUCTION 33 The di-(nitrofuran) œmpounds have two nitrofuran groups joined by various chains (Figure 1.1.9).

N-R I R'

Figure 1.1.9. Example of a di-(nitrofuran)

1.1. Metabolism of nitrofurans Nitrofurans themselves are not active antibacterials but are enzymatically reduced to highly reactive intermediates by endogenous nitroreductases which are found in a variety of bacteria including Escherichia coli. Figure 1.1.10 shows possible reduction schemes for nitrofurans. The intermediates produced and their activity depend upon the electronic properties of the compound being reduced and the reductase reducing it (Paulino-Blumenfeld eta!., 1992). Two pairs of genes conferring sensitivity to nitrofurazone or nitrofurantoin have been identified on the E. coli chromosome. They are nfsA and nfsB at 19 (Zenno et al., 1996) or 21 (Bachmann, 1990) and 11 (Bachmann, 1990) or 19 (Kumar & Jayaraman, 1991) min respectively and nfnA and nfnB at 80 and 13 min respectively (Bachmann, 1990). The former pair of genes are known to express nitroreductase I, one of the two nitroreductases which exist in E. coli and which are responsible for the reduction of nitrofurans. Nitroreductase I is a protein of approximately 50 kDa consisting of two components, la and Ib which are expressed by the nitrofurazone sensitivity genes nfsA and nfsB (Breeze & Obaseiki-Ebor, 1983b; McCalla et ai, 1978). However, there are two forms of component Ib, Ib^ and Ibg, with a third gene, tentatively called nfsC (Bryant et ai, 1981) possibly coding for the second form. The existence of three genes at 11,19 and 21 min on the E. coli chromosome, encoding the different components of nitroreductase I, would explain the contradictory data of Bachmann

(1990), Kumar & Jayaraman (1991) and Zenno et ai (1996). Nitroreductase I is equally capable of using either NADH or NADPH as cofactor (Knox et ai, 1995) and

INTRODUCTION 34 produces highly reactive, short-lived intermediates which are reduced to cyano derivatives (Peterson et ai, 1979). It is oxygen insensitive, being equally active in aerobic and anaerobic conditions. It initially reduces the nitro group by a 2e~ step, therefore not forming the nitrofuran radical anion (NO 2"*) which only requires 1 e~ (Spain, 1995).

OgN—( Jj— R Nitrofuran

O-N—(T > — R Nitrofuran ^ Superoxide 'J radical anion o^dation anion O J’

ON— —R Nitroso derivative

HOHN— ^—R Hydroxylamino derivative

HON - \ J r L Oxime Amine derivative derivative HN=C "9— R

N=C Nitrile (cyano) Nitrenium derivative

O. DNA Adduct N=C ' ^ R '— ' Inactive saturated nitrile (cyano) derivative

Figure 1.1.10. Reduction of nitrofurans. (Based upon Debnath et a i, 1993; Edwards, 1980; Peterson et s i, 1979)

INTRODUCTION 35 Nitroreductase n consists of at least two components (McCallaet a/., 1970), Ila and lib of 120 kDa and 760 kDa respectively (McCalla at al., 1975). It is inhibited by the presence of oxygen and therefore only active in anaerobic conditions (McCalla at al., 1978). The anion radical is an obligate intermediate of nitroreductase n reduction (Peterson at a!., 1979), as the first step involves transfer of 1e~ (Spain, 1995), and can be oxidized back to the parent or non-enzymatically decayed to the amine derivative (Figure 1.1.10). Nitrofuran resistant strains have been isolated, which are deficient in nitroreductase I (McCalla at a!., 1978; Peterson at a!., 1979). The level of resistance varied depending on which enzyme component was lacking and appeared to occur in four steps suggesting that three genes are responsible for nitroreductase I (Breeze & Obaseiki-Ebor, 1983c; Szybalski & Nelson, 1954). No nitrofuran resistant mutants have been found deficient in nitroreductase II, thus the latter enzyme is probably essential to the cell for reasons as yet unknown.

1.2. Action of nitrofurans From Figure 1.1.10 it can be seen that various types of intermediate and end- product are produced when nitrofurans are reduced by bacterial nitroreductases. Due to this range of metabolites, there exist considerable differences in the degree of damage produced by the various nitrofuran compounds and possibly in the manner in which they cause this damage. The DNA of nitrofuran-treated nitroreductase-deficient cells shows no difference to DNA of untreated cells (McCalla at a/., 1971), therefore, with few exceptions (Lu & McCalla, 1978; McOsker & Fitzpatrick, 1994), the drugs are activated by reduction (McCalla at a!., 1971) to produce metabolites. Repair of DNA and recovery of treated cells occurs fairly quickly after drug removal and the number of DNA strand breaks (McCalla at a!., 1975; Tu & McCalla, 1975) produced does not appear to correlate with lethality (McCalla at al., 1971) using density gradient sedimentation of the cells in alkaline and neutral sucrose gradients. Nitrofurans form different types of adduct which bind to DNA (Touati at al., 1989; Wentzell & McCalla, 1980). From studies in Vibrio cholaraa and Escherichia coli it is likely that the nitrofuran ring intercalates into the DNA molecule (Mukherjee & Chatterjee, 1992) and may then bridge the two strands, forming an interstrand

INTRODUCTION 36 cross-link (Chatterjee et al., 1987). Of those adducts whose structures have been characterised, one covalently binds to DNA and is removed by excision repair (Introduction 3.2) whilst another type is more persistent and produces daughter- strand gaps when the DNA is replicated (Lu et al., 1979; Wentzell & McCalla, 1980). This stimulates the synthesis of RecA and induces SOS error-prone repair (Introduction 3.2) (Bryant & McCalla, 1980; Chatterjee et al., 1983; Sengupta et al., 1990; Sturdikef a/., 1986). Cell division is suspended leading to filamentation (lida & Koike, 1977; Sengupta et al., 1990). The nature of adduct binding sites is still controversial; reduced nitrofurans bind to phage DNA at AT-rich sequences (Hadi et ai, 1989; Shahabuddin & Hadi, 1990) and to plasmid DNA at guanine bases in GC-rich sequences (Touati et ai, 1993). The inhibition of growth by nitrofuran compounds thus results from inhibition of nucleic acid synthesis (Endo et ai, 1963; McCalla, 1964). ATP levels also change but since they either increase or decrease, depending on the dose of nitrofuran (Lu & McCalla, 1978), inhibition of energy metabolism is not responsible for reduced nucleic acid synthesis. More specifically, reduced nitrofurans cause simultaneous synthesis and degradation (a rapid turnover) of RNA (Kato et ai, 1970) and inhibition of ribosomal RNA synthesis (Tu & McCalla, 1976). This degradation is not due to RNase as RNase activity is the same in both treated and untreated cells (Kato et ai, 1970). Reduced nitrofurans also bind to ribosomes but do not cause degradation of pre-existing subunits (Tu & McCalla, 1976). They bind extensively to proteins (McCalla et ai, 1970; McCalla et ai, 1975) and to specific ribosomal proteins in a dose-dependent manner (McOsker & Fitzpatrick, 1994). Polysome formation is reduced in nitrofuran-treated cells which could be due to lack of ribosomal RNA or to binding of metabolites to ribosomal proteins. At low concentrations reduced nitrofurans inhibit the initiation of translation of mRNA of specific inducible genes (Wagner et ai, 1977). DNA-dependent RNA polymerase activity is unaffected and therefore not responsible for the reduction in RNA synthesis (Tu & McCalla, 1976). The elongation of all proteins is inhibited at higher concentrations (Wagner et ai, 1977) due to chain termination. Nitrofurans have been shown to be mutagenic in various Ames tester strains of Salmonella typhimurium (Jurado et ai, 1994; Jurado & Pueyo, 1995; Koch et ai, 1994; Pal et ai, 1992; Sturdik et ai, 1986) but in over thirty years of clinical use

INTRODUCTION 37 there has been no report of human genotoxicity (Martindale, 1996). Nitrofurans exhibit antibacterial, antiprotozoal, antihelminthic, antihypertensive and antineoplastic characteristics (Paul & Paul, 1964). However, the toxicities of these compounds and their derivatives have restricted their development and use in clinical medicine (Kedderis & Miwa, 1988; Martindale, 1996). During their long period of use as antibacterial agents, resistance to nitrofuran compounds has remained low (Beunders, 1994; Bonten etal., 1992; Cheong etaL, 1995; London et ai, 1994; Murdoch et ai, 1995).

2. Polyamines Polyamines are polycationic (polybasic), aliphatic compounds containing two or more amine groups. Those occurring naturally include cadaverine

(NH2(CH2)5NH2), putrescine (NH 2(CH2)4NH2), spermidine (NH 2(CH2)3NH(CH2)4NH2) and spermine (NH 2(CH2)3NH(CH2)4NH(CH2)3NH2). They are widely distributed in nature, some being found in all living organisms. Many bacteria contain spermidine and putrescine whilst mammalian cells, yeasts, fungi and protozoa also contain spermine. Under steady-state conditions intracellular polyamine pools are maintained within a relatively constant range by the collective efforts of biosynthetic, catabolic and transport mechanisms and regulatory proteins (Figure 1.2.1).

The abbreviations for Figure 1.2.1 are as follows: 1 Arginine decarboxylase 2 Agmatine ureohydrolase 3 Ornithine decarboxylase 4 Spermidine synthetase 5 Acetyltransferases 6 Polyamine/amine oxidases 7 Methylthioadenosine phosphorylase 8 S-adenosylmethionine synthetase 9 S-adenosylmethionine decarboxylase 10 Glutathionylspermidine synthetase 11 Trypanothione synthetase 12 Lysine decarboxylase 13 Putrescine/spermidine transporter 14 Putrescine/ornithine antiporter 15 Putrescine transporter 3-APP 3-Acetamidopropanal dcSAM decarboxylated S-Adenosylmethionine Ex. Extracellular compartment In. Intracellular compartment

INTRODUCTION 38 Met Methionine MTA Methylthioadenosine 5-MTR-1 -P 5-Methy!thioribose-1 -phosphate Pi Inorganic phosphate PPi inorganic pyrophosphate SAM S-Adenosylmethionine

L-Arginine L-Lysine

CO ^ 1 CO 12

Glutamate Agmatine Cadaverine

CO. CO. Urea-^, ^ 9 L-Omithine ^ Putrescine dcSAM ------SAM A PPi + Pi A A c e ty h \ CoA 5^ /O Acetate 3-APP ^6 ATP Acetylputrescine Met

Acetylspermidine

Adenine Acetyl CoA Spermidine MTA > 5 -M T R-1-P A Acetyl-^ CoA 5^^^>Acetate

Glutathione + ATP Acetylspermidine

ADP + Pi Glutathionylspermidine

— Glutathione + ATP

^ A D P + Pi

Ex. In.

Figure 1.2.1. Polyamine biosynthesis and metabolism in Escherichia coli. (Based upon Bollinger ef a/., 1995; Morris & Pardee, 1966; Pegg & Williams-Ashman, 1987; Vogel, 1970)

INTRODUCTION 39 2.1. Polyamine biosynthesis There are two pathways of putrescine synthesis in E. coli (Morris & Pardee, 1966). The main pathway is by the decarboxylation of ornithine but, since this is dependent on the concentration and availability of ornithine, a second route is available (Morris & Koffron, 1969). Arginine is decarboxylated to agmatine which is converted to putrescine with the production of urea (Morris & Koffron, 1967). Putrescine is then combined with S-adenosylmethionine to produce spermidine (Tabor et ai. , 1958; Zappia et al. , 1980). 2.1.1. Arginine decarboxylase exists as two different enzymes, one is constitutively expressed and the other induced at low pH (Morris & Koffron, 1969). The constitutive enzyme is responsible for the synthesis of putrescine and spermidine and is the product of the speA gene (Moore & Boyle, 1991) at approximately 63 min (Bachmann, 1990; Satishchandran etal., 1990) on the E. coli chromosome. It is produced in small amounts and is unaffected by pH variation (Meng & Bennett, 1992) having an optimum at pH 8.4. It requires pyridoxal phosphate (2 molecules per molecule of enzyme) and Mg^"" for maximal activity and is specific for arginine. It can exist in monomeric, dimeric and tetrameric forms, the monomers exist as a mixture of 71 kDa and 75 kDa proteins (Tabor & Tabor, 1985). It is inhibited by two independent mechanisms; putrescine negative feedback and by one of a small group of proteins known as antizymes (Introduction 2.2). The inducible enzyme is the product of the adi gene (Meng & Bennett, 1992). As well as decarboxylating arginine it also accepts guanidine derivatives, canaverine and L-ornithine (Blethan et al., 1968). It is produced at low pH having an optimum of pH 5.2. It requires pyridoxal phosphate (10 molecules per molecule of enzyme) and Mg^'’ for activity. It consists of ten subunits, each a single polypeptide chain, which form five dimers of 165 kDa each (Boeker & Snell, 1968). These dimers arrange into a decamer of 820 kDa consisting of two pentameric rings which is the active form (Boeker et al., 1969). 2.1.2. Agmatine ureohydrolase is the product of the speB gene (Cunningham-Rundles & Maas, 1975) positioned at approximately 64 min on the E.

CO//chromosome (Bachmann, 1990; Satishchandranetal., 1990). The promoter of SpeB is induced by agmatine itself (Szumanski & Boyle, 1992). 2.1.3. Ornithine decarboxylase also exists as two different enzymes, one

INTRODUCTION 40 constitutive and the other induced. The constitutive enzyme is present in all strains at neutral pH, having an optimum at pH 8.1 (Applebaum et al., 1977) and is the product of the speC gene (Cunningham-Rundles & Maas, 1975) positioned at approximately 64 min on the E. co//chromosome (Bachmann, 1990; Satishchandran etal., 1990). It is dependent on pyridoxal phosphate. The enzyme occurs as a dimer of 82 kDa (Applebaum etal., 1977) and is specific for ornithine, with less than 0.1% activity for arginine or lysine. It is activated by the presence of GTP and other nucleotides and inhibited by putrescine and spermidine negative feedback (Applebaum etal., 1977) and by antizymes. At low pH (Applebaum etal., 1975) and under semianaerobic conditions an alternative ornithine decarboxylase is induced. It occurs as a 160 kDa dimer with an optimum pH of 7.0 and is specific for ornithine (Applebaum et al., 1975). It is the product of the speF gene at 16 min on the E. coli chromosome (Kashiwagi et al., 1991) and also requires pyridoxal phosphate for activity (Applebaum et al., 1975) but is not found in most strains of E.coli including K12 (Meng & Bennett, 1992). 2.1.4. Spermidine synthetase is the product of the speE gene (Tabor & Tabor, 1985) at 3 min on the E. coli chromosome (Bachmann, 1990). It has two subunits of approximately 73 kDa (Bowman et al., 1973) and is regulated by the amount of substrate present. 2.1.5. S-Adenosylmethionine synthetase is the product of the metK gene (positioned at 63 or 63.7 min on the E. coli chromosome) which requires the presence of ATP for activity (Satishchandran et al., 1990; Tabor & Tabor, 1984; 1985). 2.1.6. S-Adenosylmethionine decarboxylase is the product of the speD gene (Tabor & Tabor, 1985) at approximately 3 min on the E. coli chromosome (Bachmann, 1990). It is 113 kDa to 115 kDa (Markhamet al., 1982; Wickner et al., 1970) and consists of six identical subunits each of 15 kDa to 17 kDa with one covalently attached pyruvate group per subunit (Markham et al., 1982; Wickner et al., 1970). These pyruvate groups, together with divalent cations such as Mg^"", are essential for activity. 2.1.7. Lysine decarboxylase also exists in both constitutive and inducible forms. As only 3% to 5% of lysine decarboxylase activity is due to the constitutive enzyme (Goldemberg, 1980) little is known about it. The inducible enzyme is a

INTRODUCTION 41 membrane-associated protein which acts as a defence mechanism at low extracellular pH by producing cadaverine and carbon dioxide. The carbon dioxide escapes into the external environment increasing the pH (Neely et al., 1994). Activity is dependent on products of both the cadABC operon and cadR at approximately 94 min and 46 min respectively on the E. coli chromosome (Bachmann, 1990). The cadR gene product is a regulatory protein and the cadC gene product is associated with the cytoplasmic membrane, having an extracellular domain which detects low pH and an intracellular domain which acts as a promoter for the cadAB operon (Neely et al., 1994). The cadA gene product is the decarboxylase enzyme and requires pyridoxal phosphate for activity (Sabo etal., 1974). Its subunits are 80 kDa and form dimers of approximately 158 kDa. These arrange into a decamer as two stacked pentameric rings (Sabo et al., 1974). The cadB gene product is thought to be a lysine/cadaverine antiporter (Meng & Bennett, 1992).

2.2. Polyamine regulation Polyamines are involved in a wide variety of biological reactions and are a vital component of all cells. However, excessive polyamines are toxic (Kramer et al., 1993) and so need to be regulated. As polyamine concentrations increase so do the concentrations of some proteins. Three of these have been identified, two are basic and one is acidic (Heller etal., 1983). They inhibit both ornithine decarboxylase and arginine decarboxylase constitutive enzymes but not the inducible enzymes, lysine decarboxylase or S-adenosylmethionine decarboxylase (Heller et al., 1983; Panagiotidis etal., 1988; Panagiotidis & Canellakis, 1984). The two basic proteins called antizymes 1 and 2 are 11 kDa and 9 kDa respectively (Huang et al., 1984) and have amino acid sequences identical to the E. coli ribosomal proteins S20/L26 and L34 respectively (Panagiotidis & Canellakis, 1984) which also inhibit ornithine decarboxylase and arginine decarboxylase (Panagiotidis etal., 1988). The acidic antizyme is49 kDa (Huang etal., 1984). Antizymes inhibit polyamine synthesis by non-covalently binding to the enzymes forming an antizyme-decarboxylase complex. They are also believed to increase the degradation of the decarboxylase enzymes (Hayashi, et al., 1988). These proteins also mediate negative transcriptional regulation of the decarboxylase enzymes by interacting with the promoter regions

INTRODUCTION 42 of their genes (Panagiotidis et al., 1994). An activator of the decarboxylase enzymes has also been identified (Kyriakidisetal., 1978). This may be the anti-antizyme protein which binds antizyme releasing the decarboxylase from the complex (Tabor & Tabor, 1985). The ratio of the inhibitors and activator is dependent on the phase of growth of the cell, the extracellular polyamine concentration and decarboxylase activity (Kyriakidis et al., 1978).

2.3. Polyamine metabolism

Few polyamine metabolites have been found, though all polyamines are acetylated and excreted from the cell. They can also be oxidatively deaminated and transformed to an amino acid (Large, 1992) and eliminated. Spermidine is also metabolised, sequentially, to glutathionylspermidine and trypanothione (Tabor & Tabor, 1975). 2.3.1. Acetyltransferases. Spermidine acetyltransferase has been identified as the product of the speG gene at 35.6 min on the E. coli chromosome (Fukuchi et al., 1994) and is constitutively expressed in all phases of growth (Carper et al., 1991). It is a 95 kDa protein consisting of four identical subunits (Fukuchiet al., 1994). Spermidine acétylation produces N^-acetylspermidine and N®-acetylspermidine in relatively equal amounts. However, spermidine acetyltransferase is not very active in bacteria and therefore few acetylated metabolites are actually found (Carper et al., 1991; Fukuchi et al., 1994). Despite this, monoacetylated putrescine and the two isomeric forms of monoacetylated spermidine have been recovered from growing cultures of E. coli (Dubin & Rosenthal, 1960). 2.3.2. Amine oxidases oxidatively deaminate amine groups. Cleavage can occur at a primary (terminal) amine site or at a secondary amine site (Mondovi et al., 1988). Little is known of the (poly)amine oxidases as they are not very active in E. co//(Fukuchi etal., 1994). 2.3.3. Glutathionylspermidine and trypanothione synthetases catalyse the reaction of spermidine with glutathione to produce, sequentially, glutathionyl spermidine and trypanothione (Bollinger etal., 1995). These metabolites are only found under anaerobic and acidic conditions such as exist in the stationary phase, even though the enzymes are present at all stages of growth. This is believed to be

INTRODUCTION 43 due to a rapid turnover of the metabolites during logarithmic growth (Tabor & Tabor, 1975).

2.4. Polyamine transport Three transport systems have been identified in E. coli to date. One transports putrescine and spermidine and the other two transport putrescine alone. One of the latter has low activity. All of the polyamine transport systems require the presence of a proton motive force (Kashiwagi et al. 1990; Munro et al., 1974). 2.4.1. The putrescine/spermidine transport system is produced from the potABCD operon at 15 min on the E. co//chromosome (Furuchi etal., 1991). The 43 kDa potA gene product provides an inner membrane-associated, ATR-dependent nucleotide-binding domain. The 31 kDa potB and 29 kDa potC genes produce a transmembrane protein having six segments with hydrophilic links. The 39 kDa potD gene product provides a substrate-binding domain in the periplasm which binds putrescine weakly and spermidine strongly. Putrescine transport is therefore inhibited in the presence of spermidine. All four proteins are necessary for the transport system to work, as well as the presence of ATP and a membrane potential (Kashiwagi etal., 1993). 2.4.2. The low activity putrescine transport gene is encoded by potE which is found in an operon with the inducible ornithine decarboxylase enzyme, speF at 16 min (Kashiwagi et al., 1991). The potE gene product provides one protein consisting of twelve transmembrane segments with hydrophilic links and appears to be a putrescine/ornithine exchange system (Kashiwagi etal., 1992). It is active when the environment is acidic and its main function is the excretion of putrescine. However, this protein can also transport putrescine into the cell, with a very low activity, in an energy-dependent manner without the exchange of ornithine (Kashiwagi et al., 1992). 2.4.3. The putrescine-only transport system is analogous to the putrescine/spermidine transport system. It is encoded by the potFGHI operon at 19 min on the E. coli chromosome (Pistocchi et al., 1993). The 38 kDa potF gene product provides a putrescine-specific binding domain in the periplasm. The 45 kDa potG gene product provides a membrane-associated nucleotide-binding site. The 35 kDa potH and 31 kDa potl gene products provide a transmembrane domain of

INTRODUCTION 44 six segments with hydrophilic links of variable length. All four domains are required for transport activity. The amino acid homologies of the components of this transport system and the putrescine/spermidine transport system are: PotA and PotG 42% similar, PotB and PotH 37% similar, PotC and Potl 36% similar and PotD and PotF 35% similar (Pistocchietal., 1993).

2.5. Functions of polyamines Under physiological conditions polyamines are likely to exist as polycations

with spermine existing as NH 3'^(CH2)3NH2XCH2)4NH2'^(CH js', spermidine as

NH3"‘(CH2)3NH2''(CH2)4NH3'‘ and putrescine as NH3''(CH2)4NH3'’. The charges are not necessarily localised charges, such as those present on inorganic ions, but may be distributed over the length of the molecule. Due to the presence of charge, polyamines interact with DNA, RNA, ribosomes and proteins. The majority of these interactions have been shown using mammalian cells, however, the specific molecular functions of polyamines remain elusive. In E. coli the majority of spermidine (90%) appears to be bound to RNA whilst only 50% of putrescine is bound to RNA and 40% is free (Miyamoto at al., 1993), thus the different polyamines have different distribution profiles in E. coll. The following are a few examples of how polyamines are believed to interact with and affect bacteria. 2.5.1. Interactions with DNA. The interactions of polyamines with DNA appear to be both specific and non-specific. Recent studies have been unable to confirm the binding preferences of polyamines (Schmid & Behr, 1991). One model considering DNA as a linear distribution of negative charge and spermine as having point concentrations of positive charge, though ignoring the structures of both, suggests that spermine interacts with DNA in the major groove by interactions between proton acceptors on the DNA and proton donors on the polyamine. Each amine in spermine forms hydrogen bonds with two sites on the DNA molecule involving either the oxygen of a phosphate group or the N^ of a guanine base. Binding to these specific sites produces a 25° bend in the helix axis thus stabilizing the complex (Feuerstein at al., 1986). This compares to an earlier model which considered the structures of both DNA and spermine but ignored base-specific interactions. In this example the spermine bridged the minor groove of the helix and the positively charged amine residues interacted with the negatively charged

INTRODUCTION 45 phosphate residues on opposite DNA strands (Liquori etal., 1967). In vitro studies have shown that non-specific interactions between DNA and spermine occur when DNA forms a condensate in the presence of spermine, thus protecting the DNA from shearing. As spermine binds to DNA, the DNA charge- density is reduced. A reduction of 90% appears to be the threshold at which DNA condenses in a relatively fast reaction, the spermine molecules appearing mobile along the DNA double-helix (Porschke, 1984). The presence of spermidine reduces the rate of unfolding of DNA and stabilizes DNA against thermally-induced unfolding. The presence of spermidine also protects against thermal dénaturation. These effects are thought to result from cooperative cross-linking interactions between two regions of the DNA double helix (Flink & Pettijohn, 1975). 2.5.2. Interactions with RNA. As with DNA, studies of RNA and polyamines are also conflicting with regard to the nature, specificity and number of cation binding sites. In vitro crystallographic studies showed that spermine binds at very specific sites on the yeast tRNA^''® molecule, in a ratio of 2:1. One spermine molecule forms hydrogen bonds at phosphate oxygens on opposite sides of the major groove of the double helix extending from the D stem into the anticodon stem. This produces a 25° bend in the helix, similar to that produced in DNA, and then stabilizes both the secondary and tertiary structures of tRNA. Such stabilization may facilitate positioning of the tRNA in the ribosomal aminoacyl site. The other spermine molecule is found in the variable loop (Quigley at al., 1978). Spermidine also binds to yeast tRNA^^® but, using equilibrium binding analyses, it was suggested that electrostatic forces were responsible for binding. It also appeared that two binding sites were available at low polyamine concentrations but only one at higher concentrations. There appeared to be no cooperative binding or conformational change in the tRNA (McMahon & Erdmann, 1982). More recently Frydman etal. (1992) have suggested that binding is probably due to both electrostatic forces and hydrogen bond formation. There may even be three binding sites at low polyamineitRNA ratios, with NHg"^ binding more efficiently than NHs"^, as spermine binds more strongly than spermidine. 2.5.3. Interactions with ribosomes. Polyamines can replace Mg^"^ at the cation binding sites of both 80S and 50S ribosomal subunits ofE. coli. This has no

INTRODUCTION 46 effect on phenylalanine polymerization, as long as remains above a critical level (Kimes & Morris, 1973; Weiss & Morris, 1973). It is therefore possible that polyamines may serve an essential role in preserving the structural and functional integrity of ribosomes (Weiss et al., 1973). Cell lysates prepared from polyamine-starved E. coli polyamine-requiring mutants were found to contain fewer 70S ribosomes and 308 subunits and more 508 subunits than lysates prepared from putrescine-supplemented cells. This suggested that polyamines affect the equilibrium of ribosomes and ribosomal subunits and may be required for efficient assembly of the 308 subunit and/or stabilization of the ribosome (Algranati etal., 1975). 2.5.4. Effects on growth. Polyamine-requiring mutants of E. coli require putrescine- or spermidine-supplementation to achieve optimal growth (Morris & Jorstad, 1973). A polyamine-starved E. coli speA speB speC speD mutant grew at approximately one-third the rate of polyamine-supplemented cells indicating that E. coli does not have an absolute requirement for polyamines (Hafner et al., 1979). However, introduction of the rpsL mutation into an E. coli speA speB speC polyamine-requiring mutant converted it from a partial requirement for polyamines to an absolute requirement. The rpsL gene encodes the 812 protein of the 308 ribosomal subunit which is involved in the initiation reaction, binding mRNA to the ribosome (Lewin, 1994). The change in polyamine requirement indicates that polyamines are involved in both ribosomal structure and function (Tabor et al., 1981).

Homologues of the structures NH 2(CH2)nNH(CH2 )3NH2, where n = 5, 6 , 7 or

8 , were compared to spermidine (n = 4) for their effect on growth enhancement in an E. coli auxotroph. All were taken up to an intracellular level equivalent to that of spermidine but increasing the chain length decreased the ability to stimulate growth, with the homologues where n=7 or n = 8 being essentially inactive. 8 imilar effects were seen in their ability to restore the rates of protein and mRNA chain elongation (Jorstadetal., 1980). 2.5.5. Involvement in macromolecular synthesis. Polyamine deficiency reduces the rate of DNA replication fork movement in E. coli (Geiger & Morris, 1978). The chain growth was halved when compared to that of putrescine- or spermidine-supplemented cells (Geiger & Morris, 1980) whilst the initiation of DNA

INTRODUCTION 47 of phage T7, which is relatively unaffected by polyamine starvation, in an E. coli polyamine-requiring mutant. Tabor & Tabor (1982) found that in a T7 mutant, carrying an amber mutation, polyamines were required for efficient translation even in the presence of amber suppressor genes. Thus it is likely that polyamines are required for efficient translation. 2.5.6. Interactions with membranes. Osmotically unstable E. coli protoplasts can be prevented from lysing by raising the osmotic pressure of their environment with, for example, sucrose. However, lysis is also prevented by the addition of spermidine or spermine (Tabor, 1962) which do not raise the environmental osmotic pressure and must therefore act on the protoplast itself, stabilizing it against lysis (Tabor & Tabor, 1964). As spermine could not be washed out of the protoplast, it was assumed that the spermine bound to components in the cell membrane (Tabor & Tabor, 1964) decreasing the repulsive forces of neighbouring phosphate groups (Tabor, 1962). Putrescine was not found to have a protective action (Tabor, 1962) but more recently it has been found in E. coli outer membrane together with spermidine and cadaverine (Koski & Vaara, 1991). It is possible that the polyamines form complexes with lipopolysaccharides thus stabilizing the membrane against osmotic lysis. There appears to be no structural specificity required for stabilization of membranes. Homologues of

NH2(CH2)3NH(CH2)nNH(CH2)3NH2 where n = 2, 3, 5 or 6 were all as effective as spermine (n = 4) in stabilizing E. coli protoplasts against osmotic lysis (Stevens, 1967). 2.5.7. Interactions with proteins. E. coli topoisomerases are involved in catenation (interlocking), or decatenation, of closed-circular DNA. High local concentrations of DNA are required for catenation, which means that the DNA has to be aggregated. Aggregation occurs when about 90% of the DNA charge is neutralized. Neutralization requires polyvalent cations (Krasnow & Cozzarelli, 1982). Topoisomerases regulate the supercoiling and therefore density of DNA. Bacterial gene expression and the replication and recombination of DNA is dependent on appropriate degrees of supercoiling (Lewin, 1994). Two types of topoisomerase have been identified in E. coli. Type 1 topoisomerases produce a transient single-strand (s/s) break in DNA and relax negatively supercoiled DNA

INTRODUCTION 49 without the need for ATP hydrolysis. Topoisomerases I and III are of this type with topoisomerase I acting on double strand (d/s) and s/s DNA (Lewin, 1994) whereas topoisomerase III is inhibited by s/s DNA (Srivenugopal et al., 1984). Type 2 topoisomerases produce a transient d/s break in DNA and require ATP. Topoisomerase n (DNA gyrase) introduces negative supercoils into DNA with ATP hydrolysis (Gellert at a/., 1976) and catalyses the relaxation of negative supercoils in the absence of ATP (Lewin, 1994). Topoisomerase II' relaxes both positive and negative supercoils (Srivenugopal etal., 1984). Type 1 topoisomerases, I and III, are inhibited by the presence of spermidine (Srivenugopal et al., 1984; Srivenugopal & Morris, 1985), compared to topoisomerase II which is stimulated by spermidine (Gellert et al., 1976). The inhibition is proportional to the decline in topoisomerase binding to DNA (Krasnow & Cozzarelli, 1982). However, reasons for the stimulation of DNA gyrase have yet to be clarified. Another protein affected by polyamines is single-strand binding protein (SSB). This enzyme exhibits a number of different binding modes in vitro which affect the number of nucleotides occluded by the SSB tetramer and the type and degree of cooperative complexes that are formed with s/s DNA. Polyamines were shown to bind cooperatively to SSB and induce transition of the binding mode so that more subunits of the tetramer could interact with more nucleotides (Wei et al., 1992).

3. DNA mutation Mutation of DNA results in a change of base sequence. Mutations in which the DNA sequence is changed at a single position are called point mutations. These include base-pair substitutions and frameshift mutations (Dale, 1994; Lewin, 1977). A base-pair substitution (nucleotide replacement) occurs when one base-pair is replaced by another base-pair. If the purine-pyrimidine axis is maintained after substitution, a purine replaces a purine or a pyrimidine replaces a pyrimidine, the change is called a transition mutation. However, if the purine-pyrimidine axis is altered, a purine replaces a pyrimidine or vice versa, the change is known as a transversion mutation. The consequence of substitution is dependent upon the change, if any, in amino acid residue for which the nucleotide ultimately partly codes. If the amino acid has not changed then the mutation is said to be neutral or

INTRODUCTION 50 silent (Friedberg etal., 1995). If the amino acid has changed the mutation could be silent or a missense or a nonsense mutation. A silent mutation is said to occur when there is no detectable effect on the function of the protein despite a change in its amino acid sequence. Missense mutations alter proteins in one of several ways; a partial loss of function, a gain of function, an alteration of function or a change in stability with respect to temperature or proteolytic degradation. A nonsense mutation results in a complete loss of protein function due to premature termination of protein synthesis (Dale, 1994; Friedberg etal., 1995). A frameshift mutation is a shift of the reading frame due to insertion or deletion of any number of nucleotides not divisible by three. This usually results ultimately in a nonsense mutation being produced downstream of the nucleotide insertion or deletion (Dale, 1994).

3.1. Causes of DNA mutation Many physical and chemical agents in the environment interact with DNA or interfere with its replication resulting in mutagenic or lethal effects (Dale, 1994; Davis etal., 1990). DNA damage can also arise spontaneously due to tautomerism or deamination of bases. 3.1.1. Tautomerism is exhibited by each of the bases in DNA and alters their hydrogen-bonding, and therefore base-pairing, capabilities. Adenine and cytosine can exist in amino (NHg, normal) and imino (NH, tautomeric) forms. Thymine and guanine can exist in keto (C=0, normal) and enol (COM, tautomeric) forms (Friedberg etal., 1995). The tautomeric forms are thermodynamically unfavourable with only one molecule in 10"* to 10® molecules existing at any one time (Dale, 1994). Even so, they can be introduced during DNA replication to form base-pairs other than adenine-thymine and guanine-cytosine; this results in transition mutations (Marti etal., 1988). 3.1.2. Spontaneous deamination of the exocyclic amino group to a keto group (example in Figure 1.3.1) can occur for several of the bases. Cytosine, adenine, guanine and 5-methylcytosine are deaminated to uracil, hypoxanthine, xanthine and thymine respectively (Friedberg etal., 1995; Stryer, 1995). The first three of these products of deamination are not normal constituents of DNA and are removed by specific DNA glycosylases (Marti et al., 1988; Saparbaev & Laval,

INTRODUCTION 5? 1994). However, thymine is a normal DNA base and mismatched GT base-pairs are ‘corrected’ to GC or AT (Hart! etal., 1988).

NHg O

NH 1 ------" jl 1 H' 'N^O H N'^O I I H H

Cytosine Uracil

Figure 1.3.1. Spontaneous deamination of cytosine to uracil

3.1.3. A base-anatogue is a compound similar to one of the four DNA bases which can be incorporated into DNA during replication (Marti at al., 1988). One example is 5-bromouracil (BU) which can substitute for thymine (Dale, 1994). Since BU can also exist in a tautomeric (enol) form which base pairs with guanine, incorporation of the incorrect tautomer may result in AT to GC transition mutations (Freifelder, 1987). 3.1.4. Chemical deamination of bases occurs on exposure to nitrous acid which oxidatively deaminates amino groups to keto groups, giving rise to transition mutations (Dale, 1994). 3.1.5. Chemically-induced tautomerism can also occur by exposure of a cytosine base to hydroxylamine. This increases the frequency of the imino form resulting in GC to AT transition mutations (Dale, 1994). 3.1.6. Alkylating agents are electrophilic compounds with affinity for nucleophilic centres in organic macromolecules which introduce alkyl groups onto nucleotides. Monofunctional alkylating agents have one active site and bifunctional alkylating agents have two active sites. They interact with many sites on DNA bases. Cytosine may be alkylated at nitrogens in positions 3 or 4 or at oxygen in position 2; thymine at nitrogen in position 3 or at oxygens in positions 2 or 4; adenine at nitrogens in positions 1, 3. 6 or 7; guanine at nitrogens in positions 1, 2,

3 or 7 or at oxygen in position 6 (Friedberg at al., 1995). Alkylation of oxygen in the phosphodiester linkage of the DNA backbone also occurs producing an alkylphosphotriester (Friedberg at al., 1995). The addition of

INTRODUCTION 52 alkyl groups may be directly mutagenic causing mispairing and leading to transition mutations. It may also be indirectly mutagenic leading to depurination. The loss of a purine is not itself mutagenic but if replication occurs before repair of the depurinated site, replication of the new strand ceases at the gap and can only continue by incorporation of a random base into the new strand by SOS error-prone repair (Dale, 1994). Bifunctional alkylating agents can also cause interstrand cross links between opposite DNA strands. These prevent strand separation and block DMA replication and transcription (Lodish et ai, 1995). 3.1.7. Intercalating agents contain a planar-ring structure which inserts

(intercalates) into the DNA double helix between adjacent bases (Dale, 1994). The bases become misaligned resulting in the addition or deletion of a nucleotide during replication causing a frameshift mutation. 3.1.8. Ultra-violet (UV) irradiation causes lethal and mutagenic effects by covalently linking adjacent pyrimidines on the same DNA strand (intrastrand links) or cross-linking DNA to proteins or breaking the DNA strand (Friedberg et ai, 1995). Pyrimidine dimers distort the DNA helix; they are non-coding lesions which cannot be replicated. The most common isomers are cyclobutane rings and pyrimidine (6,4) pyrimidone photoproducts. Several types of isomer can be repaired by reversal of the damage (photoreactivation) or by excision. If replication occurs before repair, a gap is produced in the new strand opposite the lesion, which can be filled in by recombination with a homologous strand (Dale, 1994; Davis et ai, 1990). Replication of DNA past a non-coding lesion without gap formation is prone to error and is therefore mutagenic (Introduction 3.2). 3.1.9. Ionizing radiation includes X-rays and the products of radioactive

decay (a and (3 particles and y-rays). It causes mutagenic and lethal effects by direct interaction of radiation energy with DNA or indirectly due to the interaction of reactive species on DNA. Reactive species arise from the splitting of water and other molecules into ionized molecules, free radicals and ion radicals such as HgOg,

O2"’ and «OH (Friedberg et ai, 1995). Many of these are mopped-up by enzyme- scavengers before they react and cause damage. However, not all can be detoxified. In these ways ionizing radiation may alter nucleotide bases, produce alkali-labile sites, break sugar-phosphate bonds or destroy the deoxyribose sugar

INTRODUCTION 53 of the DNA backbone, the latter lesion producing single-strand or double-strand DNA breaks (Friedberg ef a/., 1995; Lodish et al., 1995).

3.2. DNA repair by Escherichia coii To date, seven DNA repair mechanisms have been identified in Escherichia

CO//(Friedberg eta!., 1995). Repair mechanisms that occur prior to DNA replication or act directly on the altered site include photoreactivation, nucleotide excision repair, base excision repair and the adaptive response. Repair mechanisms occurring after replication include mismatch repair, recombination repair and error- prone repair. 3.2.1. Photoreactivation is the repair of pyrimidine dimers facilitated by enzymes encoded by the phrA and the phrB genes. The phrB gene maps at 16.2 min on the E. coli chromosome (Dorrell et a/., 1993) and encodes a 54 kDa photolyase protein of 471 amino acid residues (Park eta!., 1993) required for light-dependent photoreactivation. It also enhances light- independent excision repair (Yamamoto, 1992). Photolyase repairs cis-syn and trans-syn cyclobutane pyrimidine dimers, the latter with a lOVold lower affinity than the former (Kim et a/., 1993) but does not repair pyrimidine (6,4) pyrimidone photoproducts. The phrA gene codes for a 38 kDa protein which confers photorepair on E. coli phrB mutants, possibly having a role in the repair of (6,4) photoproducts (Dorrell eta!., 1993). This repair, unlike that due to phrB, is dependent on temperature and intensity of light fluence (Dorrell et a/., 1995). 3.2.2. Nucleotide excision repair repairs a wide variety of DNA lesions and is the major pathway for removing damage from DNA (Alberts et al., 1994). Damage is removed within an excised region of oligonucleotide by a multisubunit ATP- dependent nuclease (Sancar & Tang, 1993; Yasbin & Miehl-Lester, 1991). The process of excision repair uses the products of genes uvrA, uvrB, uvrC and uvrD. The UvrA protein has two ATP binding sites (Wang & Grossman, 1993), one of which, together with a cysteine residue at position 763 in the carboxy-terminal (Wang et al., 1994), is required for the non-specific binding of two molecules of UvrA (UvrAg) to an undamaged DNA site (Thiagalingam & Grossman, 1991 ; 1993). Next, one unit of UvrB protein binds to the UvrAg-DNA complex in the major

INTRODUCTION 54 AP endonucleases have been divided into four classes (Sancar & Sancar, 1988) depending on whether the phosphodiester bond is hydrolysed 3' or 5' to the abasic deoxyribose. One example is the class I iron-sulphur containing (Kuo et al., 1992) nth gene product, endonuclease III, which removes several altered bases with its glycosylase activity (Hatahet at al., 1994) and then hydrolyses the phosphodiester bond 3' to the AP site. Another example is the class II nfo gene product, endonuclease IV, which removes deoxyribose phosphate fragments from 3' termini (Sandigursky & Franklin, 1993). Once the deoxyribose phosphate bond has been incised the residue has to be removed so that repair can continue. Some of the AP endonucleases exhibit deoxyribophosphatase activity as does the recJ gene product (Dianov at al., 1994). The resultant gap is then repaired by insertion of the appropriate nucleotide by DNA polymerase I and the nick sealed by ligase. 3.2.4. The adaptive response is exhibited by cells exposed to sublethal concentrations of a substance which induce resistance to the mutagenic and lethal effects of subsequent exposure to a lethal dose. Two types of adaptive response have been identified, one repairs damage caused by alkylating agents and the other counteracts oxidative stress. Two constitutively expressed enzymes repair endogenous (MacKay at al., 1994) or exogenous alkylation (Vidal at al., 1995). The ogt gene at 29 min on the

E. CO//chromosome encodes a 19 kDa alkyltransferase (Shevell at ai, 1990). This transfers the alkyl group attached to either 0®-alkylguanine or O'^-alkylthymine onto one of its own cysteine residues at position 139 within the sequence Pro-Cys-His- Arg (lhara at al., 1994). Transferral is irreversible and is therefore a suicide reaction. The tag gene at 79.3 min on the E coli chromosome (Seeberg, 1993) encodes the 19 kDa N^-methyladenine DNA glycosylase I which also removes N^- methylguanine residues (Bjelland at al., 1993). Excision of these lesions leaves an apurinic site which is repaired by DNA polymerase I and ligase. Very low levels (Vaughan at al., 1993) of another alkyltransferase are also found in unadapted cells. This is encoded by the ada gene at 47.2 min on the E. coli chromosome (Seeberg, 1993). The 39 kDa ada gene product consists of 354 amino acids divided into two domains and a hinge region (Saget at al., 1995). Both domains contain a cysteine residue capable of accepting alkyl groups from alkylated

INTRODUCTION 56 Differences are seen in the activities of these régulons between log and stationary phase cells (Bishai et al., 1994; Liochev & Fridovich, 1992). Two of the régulons, oxyR and soxRS, are adaptively inducible by oxidative stress from environmental sources (Crawford & Davies, 1994). The oxyR regulon protects against HgOg with as many as thirty gene products being induced (Storz & Tartaglia, 1992). OxyR itself is redox sensitive (Schellhorn, 1995) and is therefore susceptible to oxidation and reduction, with a cysteine residue at position 199 being critical for redox sensitivity (Kullik at a/., 1995b). In both its oxidized and reduced states, OxyR undergoes a conformational change which increases its amino-terminal DNA binding affinity (Kullik at al., 1995a). However, only oxidized OxyR functions as a transcriptional activator (Tartaglia at al., 1992), binding to promoter sequences of OxyR-dependent genes (Tao at ai, 1993; 1995). Examples of the genes induced by OxyR are katG, which encodes hydroperoxidase I (Mukhopadhyay & Schellhorn, 1994), the ahpCF operon, which encodes alkylhydroperoxidase reductase (Ferrrante at al., 1995), oxyS (Rosner & Storz, 1994) and dps, which encode non-specific DNA binding proteins (Altuvia at ai, 1994). The soxRS regulon protects against superoxide generating agents (Nunoshiba & Demple, 1993) and nitric oxide (Nunoshiba at ai, 1993a) by the induction of as many as fourteen enzymes (Fawcett & Wolf, 1994). SoxR is a dimer containing two 2Fe-2S clusters (Hidalgo at ai, 1995), one per polypeptide chain (Wu at ai, 1995). These clusters are oxidized to 2Fe-2S^'' (Wu at ai, 1995) which enables SoxR to bind specifically to the soxS promoter sequence (Nunoshiba at ai, 1992) and activate transcription of this gene (Hidalgo & Demple, 1994). The level of SoxS is thus increased by a relatively small amount of SoxR (Wu & Weiss, 1992). SoxS binds to the promoter sequences of other genes of the regulon, facilitating subsequent binding of RNA polymerase and inducing transcription (Li & Demple, 1994). Genes encoding a variety of enzymes are induced by SoxS including zwf,

which encodes glucose- 6 -phosphate dehydrogenase (Fawcett & Wolf, 1995), sodA, encoding a manganese-containing superoxidedismutase (Privalle at ai, 1993), fumC, encoding a fumarase (Park & Gunsalus, 1995) and the n/b-encoded endonuclease IV repair enzyme (Nunoshiba at ai, 1995). SoxS also negatively regulates soxS transcription by binding to its own promoter sequence (Nunoshiba

INTRODUCTION 58 et al., 1993b). 3.2.5. Mismatch repair removes and repairs replicative mismatches that have been missed by the editing (proof-reading) exonuclease activity of DNA polymerase. It also recognises and removes frameshift mutations that have left unpaired bases in newly-synthesized daughter strands (Yasbin & Miehl-Lester, 1991 ). There are two types of mismatch repair, long patch and very short patch, and although their precise mechanisms are still unclear, a variety of genes have been identified as being involved. Long patch mismatch repair (LPMR) is also known as methyl- or dam- directed post-replication repair (Yasbin & Miehl-Lester, 1991). The dam gene product, deoxyadenine methylase, methylates adenine residues in 5-GATC-3' sequences. LPMR identifies the recently replicated strand because of its undermethylation (Yasbin & Miehl-Lester, 1991) with the muthi gene product providing strand specificity by recognizing an unmethylated GATC sequence near the mismatch and cleaving the sugar-phosphate backbone 5' to it (Lieb, 1987). The mutU (uvrD) gene product (helicase II) and the ssb gene product (single-strand DNA binding protein) then combine to unwind the DNA helix and the mutL gene product, together with a specific exonuclease, removes the damage-containing DNA oligonucleotide (Stryer, 1995). Exonuclease I activity is required if GATC cleavage occurred 3' to the mismatch and exonuclease V II or RecJ exonuclease activity is required if GATC cleavage occurred 5' to the mismatch (Cooper at al., 1993). The length of the removed DNA segment can be several hundred to several thousand nucleotides (Lieb, 1987). The gap is then resynthesized by DNA polymerase III and the nick sealed by ligase (Davis at ai, 1990; Yasbin & Miehl-Lester, 1991). Very short patch mismatch repair (VSPMR) removes and repairs oligonucleotides consisting of between ten and twenty-five nucleotides (Yasbin & Miehl-Lester, 1991). The méthylation of adenine residues in GATC sequences does not interfere with VSPMR (Lieb, 1987). It corrects GT mismatches found in sequences which are normal substrates for the dcm gene product, such as CCWGG and CCWG where W = A or T. Dcm, deoxycytosine methylase, methylates the internal C residue within these sequences (Sohail at al., 1990). Such GT mismatches arise from spontaneous deamination of 5-methylcytosine to thymine (Lieb, 1987). Repair is always in favour of the G-containing strand (Sohail at al.,

INTRODUCTION M 1990) causing T to C transitions which deplete the genome of T residues and increase the number of C residues (Bhagwat & McClelland, 1992). It is possible that this mechanism maintains sequences having important roles, for example chi (Lieb & Rehmat, 1995). The dcm product is not itself essential for VSPMR but another gene, vsr, which overlaps the 3' end of dcm by seven codons and is transcribed from the dcm promoter, is essential for VSPMR (Sohail et al., 1990). Other genes involved are mutL, mutS, pciA and fig (Sohail et al., 1990). 3.2.6. Recombination repair has been categorized into three pathways in E. coli (Smith, 1988) with over twenty-five gene products involved (Table 1.3.1). RecBGD recombination is the major pathway in wild-type cells. RecF recombination occurs inrecB- or recC-deficient strains in the presence of sbcB and sbcC or sbcD suppressor mutations. RecE recombination also occurs inrecB- or recC-deficient strains, but in the presence of the sbcA suppressor mutation (Kowalczykowskiet al., 1994). However, the type of recombination is not only dependent upon the genotype of the cell but also upon the structure of the substrate (Mendonca et al., 1995). Substrates include chromosomal, plasmid and bacteriophage DNA, with recombination events playing a role in conjugation, transduction, transformation and repair of DNA damage. There are two types of recombination repair, daughter- strand gap repair and double-strand break repair. Daughter-strand gap repair is required when synthesis of a DNA daughter strand is halted during replication opposite a non-coding lesion on the parent strand. Synthesis is re-initiated on the far side of the damage leaving a gap in the newly-synthesized DNA (Freifelder, 1987). The resulting s/s segment attracts RecA and single-strand binding protein. Homologous pairing with d/s DNA and RecA- promoted strand exchange are then initiated (Umezu & Kolodner, 1994; Umezu et al., 1993) and SOS regulated genes (Introduction 3.2.8) induced (Hegde et al., 1995). The 3'-hydroxyl end of the daughter-strand gap invades the intact homologous d/s DNA (Smith, 1988) which causes displacement of the complementary strand and its degradation. A Holliday junction (Holliday, 1964) is formed and the products of the derepressed ruvA and ruvB genes promote branch migration and dissociation of RecA (Adamset al., 1994). The Holliday junction intermediate is resolved either by RuvC cleavage (Bennett & West, 1995a) or by RecG-promoted reverse branch migration (Whitby & Lloyd, 1995). The gap left in

INTRODUCTION 60 Table 1.3.1. Recombination repair genes and products

Gene & Functions of product and references product

This is the recombination hotspot (5'-GCTGGTGG-3'). It converts RecBCD exonuclease activity to recombinase activity by altering chi (x) RecBCD so that RecD is ejected, therefore RecD is not required for recombination activity. (Horiuchi & Fujimura, 1995; Kuzminov et al., 1994; Myers at al., 1995)

RecA binds cooperatively to s/s and d/s DNA, 3 bases or base pairs per subunit. Polymerizes with s/s and d/s DNA in the presence of ATP to form right-handed, nucleoprotein filaments. Promotes homologous recA pairing and renaturation and catalyses strand-exchange reactions of 58 min DNA Binds and hydrolyses nucleoside triphosphates (DNA-dependent NTPase). Activated by s/s DNA to a coprotease which causes autodigestion of target proteins. (West, 1992; Wittung at al., 1994)

One of each subunit combines to produce RecBCD which is ATP- dependent DNA exonuclease V, a helicase and an ATP-stimulated s/s recBCD DNA endonuclease specific for the chi sequence. It binds to d/s DNA 61 min ends, the RecB subunit binding to the 3' end and the RecG and RecD subunits binding to the 5' end. (Ganesan & Smith, 1993; Korangy & Julin, 1994; Smith, 1988; Taylor & Smith, 1995)

RecF binds non-cooperatively to DNA containing s/s gaps, in the recF presence of ATP. RecO binds s/s and d/s DNA promoting renaturation 83 min and is functionally similar to RecA. RecF OR functions as a complex in recO stimulating RecA filament-DNA junction formation. (Griffin & Kolodner 55 min 1990; Hegde ef a/., 1996; Luisi-DeLuca, 1995; Madiraju & Clark, 1992 recR Tseng at al., 1994; Umezu & Kolodner, 1994; Umezu at al., 1993 11 min Webb at al., 1995)

3' to 5' ATP-dependent helicase specific for short lengths of d/s DNA and Holliday junctions. DNA-dependent ATPase binds to Holliday junctions and dissociates them in the presence of ATP and Mg^"". Catalyses branch migrations and reverse branch migrations of Holliday recG junctions. Functional similarities to the three ruv genes providing an 82 min alternative in rwv-deficient strains. Involved in all three pathways of recombination and in the repair of both s/s gaps and d/s breaks. (Hong atal., 1995; Lloyd & Buckman, 1991; Lloyd & Sharpies, 1993a; 1993b; Sharpies atal., 1994b; Whitby & Lloyd; 1995; Whitby atal., 1994)

RecJ is a 5' to 3' s/s DNA exonuclease involved in branch migration of Holliday junctions by RecA. Degrades the displaced strand facilitating recJ strand transfer. Cooperates with RecQ in double-strand break repair 62 min at crossovers. (Corrette-Bennett & Lovett, 1995; Kusano atal., 1994; Lovett & Kolodner, 1989) continued overleaf

INTRODUCTION 61 RecQ is a s/s DNA-dependent ATPase and ATP-dependent 3' to 5' recQ DNA helicase. Cooperates with RecJ in branch migration of Holliday 86 min junctions in double-strand break repair. (Kusano et al., 1994; Mendonca eta!., 1995; Umezu eta!., 1990)

rus Works with RecG to resolve Holliday junction intermediates by 12.5 junction-specific cleavage. Suppressor of the ruv genes. (Mandai et al., min 1993; Sharpies et al., 1994a)

RuvA forms a tetramer and facilitates the binding of RuvB to DNA. RuvB, an ATPase, forms a dodecamer, present as two hexameric rings, when ATP, Mg^"^ and d/s DNA are present. RuvAB, an ATPase- dependent d/s DNA 5' to 3' helicase, specifically recognizes Holliday junctions and catalyses branch migration and dissociation of RecA ruvAB nucleoprotein. The RuvA subunits, bound to DNA, are sandwiched operon between the two RuvB hexameric rings, altering the Holliday junction 41 min structure. Maximal effects are seen when RuvA, RuvB, ATP, DNA and Mg^^ are all present. 5' to 3' strand displacement. (Adams & West, 1995; Adams et al., 1994; Hiom & West, 1995; Iwasaki et al., 1992; Mitchell & West, 1994; Muller etal., 1993a; 1993b; Parsons & West, 1993; Parsonset al., 1992; 1995; Shiba et al., 1993; Stasiak, etal., 1994; Tsaneva & West, 1994; Tsaneva etal., 1993)

RuvC is an endonuclease, cleaving 3' to phosphate groups leaving 5' phosphate and 3' hydroxyl free ends, in a specific homologous ruvC sequence of the Holliday junction core. Forms a complex with Holliday 41 min junctions without the need for divalent ions. (Bennett & West, 1995a; Bennett & West, 1995b; Benson & West, 1994; Dunderdale et al., 1994; Shah etal., 1994; Takahagi etal., 1994)

sbcA 30 min Mutations in these genes restore recombination to recB- or recC- sbcB deficient strains. SbcB is 3' to 5' s/s DNA exonuclease I and a 44 min deoxyribophosphodiesterase which removes 3' hydroxylated ends. sbcCD (Kowalczykowskietal., 1994; Kushner ef a/., 1971) 9 min

one strand of the homologous d/s DNA is resynthesized by DNA polymerase I using its other strand as a template. The non-coding lesion is excised and repaired using the now intact daughter strand as template. The nicks are sealed by DNA ligase. Double-strand break repair requires RecBCD exonuclease V. This binds to d/s DNA ends (Taylor & Smith, 1995), unwinding and rewinding the DNA (Ganesan & Smith, 1993), using ATP (Korangy & Julin, 1994), until it reaches a correctly

oriented chi sequence (Horiuchi & Fujimura, 1995). It cuts at chi, removing a s/s DNA segment and the exonuclease is then converted into a recombinase (Horiuchi

INTRODUCTION 62 regulation, including lexA and recA themselves (Brent & Ptashne, 1981; Little & Harper, 1979; Little et al., 1981), have promoter/repressor regions (Little at a!., 1981) to which LexA binds, repressing expression of these genes (Kenyon & Walker, 1980). When a non-coding lesion is present on the parental strand, replication is blocked and the synthesis/editing system of the DNA polymerase stalls it at the replicative fork. This leads to a region of s/s DNA and generation of small DNA fragments and free bases (Little etal., 1980) which act as signalling molecules (Freifelder, 1987). These activate RecA to exhibit coprotease activity which causes LexA to autodigest (Little, 1984) and allows expression of SOS-controlled repair genes. Once damage is repaired and replication continues, the level of signalling molecules decreases. RecA is no longer activated, allowing uncleaved LexA to re repress genes of the SOS regulon. Over twenty genes are under SOS regulation, including recN, ruvAB, sulA, umuDC, uvrA, uvrB and uvrC (Fogliano & Schendel, 1981; Kenyon & Walker, 1980; 1981; Kitagawa etal., 1985).

4. Plasmids Plasmids are naturally-occurring DNA molecules present in many bacterial species. They are in addition to, and are distinct from, the bacterial chromosome, being non-essential for bacterial growth (Lewin, 1994). They are capable of independent replication, relying on the DNA-replication systems of their host for reproduction but controlling initiation of their own replication (Marti et al., 1988). Several types of plasmid are found in E. coll. The first plasmid to be discovered was called F and confers conjugal ability on E. coli K12 strains. Colicinogenic (Col) plasmids are generally small (molecular mass 10®), non-conjugative, multicopy (more than one per chromosome) plasmids which encode proteins that kill closely related bacterial strains. R plasmids are large (molecular mass up to 200 x 10®) ubiquitous plasmids which carry genes conferring resistance to antibiotics and heavy metals. Resistance is conferred in one of four ways by plasmid-encoded enzymes. These enzymes can breakdown or modify the antibiotic molecule, modify the antibiotic target site, reduce cellular accumulation of antibiotics or provide drug- resistant alternatives to the host enzymes which are inhibited by the antibiotic. Some colicinogenic plasmids and a wide variety of R plasmids confer resistance to ultraviolet (UV) light (MacPhee 1972; 1973a; Pinney, 1980; Siccardi,

INTRODUCTION 64 1969). This property is dependent on plasmid-encoded analogues of the chromosomal umuDC genes (Venturini & Monti-Bragadin, 1978; Walker, 1977). Thus, these plasmids increase error-prone repair and suppress the non-mutability of umu-deficient cells (Lodwick et al., 1990). They enhance the susceptibility of cells to UV- and chemically-induced mutagenesis (Friedberg at a/., 1995). Chromosomal umu-Wke genes are also widely distributed in Enterobacteriaceae (Sedgwick at al., 1991) producing proteins with varied mutagenic activities (Nohmi at al., 1992; Sedgwick at al., 1991). The mutagenic activities of plasmid-encoded Umu-like proteins also vary. Six analogous opérons have been identified on chromosomal and plasmid DNA that affect mutagenesis: umuDC, umuDCsj, samAB, impCAB, mucAB, and rumAB (Bagg atal., 1981; Ho atal., 1993; Lodwick at al., 1990; Nohmi atal., 1991; Perry & Walker, 1982). The two umuDC opérons are chromosomally encoded; umuDC on E. coli (Bagg atal., 1981) and umuDCsj on Salmonalla typhimurium (Nohmi atal., 1991). The samAB operon is present on a cryptic plasmid of S. typhimurium (Nohmi at al., 1991) and increases the poor mutability of S. typhimurium (Lodwick at al., 1990). The impCAB operon was found on plasmid TP110 isolated from S. typhimurium (Lodwick atal., 1990). ImpAB proteins are analogous to UmuDC (Glazebrook at al., 1986), however, the impC gene has no equivalent in other mutator opérons and is not absolutely required for UV protection and mutator effects (Glazebrook at al., 1986; Lodwick at al., 1990). It is unknown whether impC has been acquired or the other opérons have lost an equivalent gene. However, ten plasmids isolated from the ‘pre-antibiotic’ era (between 1917 and 1934) all harbour sequences similar to impCAB (Sedgwick at al., 1989). Another mutator operon, the mucAB operon, is also analogous toumuDC with 52% homology at the nucleotide level (Perry at al., 1985). UmuD and MucA proteins are 41% homologous and UmuC and MucB proteins are 55% homologous (Perry at al., 1985). As with the umuDC operon, mucAB has a LexA binding region (Elledge & Walker, 1983) and MucA, like UmuD, is processed in a RecA-dependent fashion (Hauser at al., 1992; McNally at al., 1990; Shiba at al., 1990). However, MucA also appears to be active when not processed by RecA (Shiba at al., 1990) as plasmids containing this operon can restore mutability to racA- and /exA-deficient strains (Hauser at al., 1992) unlike UmuDC. Kulaeva at al. (1995) characterized the rumAB operon found on plasmid

INTRODUCTION 65 R391 which was originally isolated from a Proteus species (Pembroke et al., 1986). It too has a LexA binding region and ambivalent RecA dependence. The majority of plasmids which have been found to increase mutagenesis in response to UV and chemicals have also been found to protect their host from UV- and chemically-induced DNA damage (Venturini & Monti-Bragadin, 1978; Walker, 1977). Protection is recA- and lexA- dependent (Venturini & Monti-Bragadin, 1978; Waleh & Stocker, 1979) bringing plasmid-mediated error-prone DNA repair under 8 0 8 control (Walker, 1977). The level of protection differs with the plasmid used and the mutator genes it carries (Upton & Pinney, 1983). Mutator effects also differ, depending on the type of damage, with plasmid-mediated activity inducing different base substitutions or frameshift mutations, therefore different levels of mutagenesis, according to the type of chemical or radiation-induced damage (Doyle & Strike, 1995; Watanabe et a/., 1994). Some plasmids do not protect their host from UV- induced DNA damage, despite increasing mutagenesis, but sensitize the cell (Siccardi, 1969; Pinney, 1980) therefore decreasing post-UV survival (Little etal., 1991; Upton & Pinney, 1983). This is possibly due to differences in control of the plasmid-encoded umu-like genes and how their products interact with the analogous chromosomal products.

INTRODUCTION 66 PART

AND

MATERIALS AND APPARATUS 67 1. Micro-organisms All micro-organisms used in this thesis are listed in Tables 2.1.1, 2.1.2, 2.1.3 and 2.1.4. All strains, except the fungi, were maintained in long term storage as nutrient broth cultures, frozen at -196°C in liquid nitrogen (BOC). Strains in use were maintained on nutrient agar slopes for three months or on plates for up to two weeks at 4°C. Plasmid presence was monitored by checking for resistance to appropriate antibiotics.

Table 2.1.1. Escherichia coli K12 DNA repair-proficient and -deficient strains

Escherichia Source, collection number Genotype coli K12 strain and reference

AB1157 thi-1 thr-1 proA2 leu-6 his-4 R. J. Pinney DP406 argE3 galK2 lacY1 ara-14 Howard-Flanders etal., 1964 mal xyl-5 mtl-1 rps1-31 tsx- 33 supE44 X~ S n f

AB1157(R16) AB1157(R446b) This thesis AB1157(R46) AB1157(R621a) AB1157(R124) AB1157(R805a) AB1157(R144) AB1157(pKM101) AB1157(R391)

AB1157(pGW12) R. J. Pinney DP1155 Walker, 1978

AB1157(pGW16) R. J. Pinney DP1156 Walker, 1978

AB1157(pYD1) J. Ambler JA63 Ambler ef a/., 1993

AB1886 As AB1157 but uvrAd Sm^ R. J. Pinney DP1140 Howard-Flanders etal., 1966

AB1886(R46) This thesis

AB2463 As AB1157 but recA13 Sm® R. J. Pinney DP1131 Howard-Flanders & Theriot, 1966

AB2463(R46) This thesis

continued overleaf

MATERIALS AND APPARATUS AB2470 As AB1157 but recB21 Sm® R. J. Pinney DP1133 Howard-Flanders, 1968

AB2470(R46) This thesis

AB2494 As AB1157 but lex A3 metB1 R. J. Pinney DP1132 thi leu* arg* Sm® Howard-Flanders, 1968

AB2494(R46) This thesis

J62-2 pro glu trp his R f R J. Pinney DP985 Meynell & Datta, 1966

J62-2(R805a) This thesis

JC3890 As AB1157 but DE30Y R. J. Pinney DP1138 (deletion bio-chIA) Sm® Kato & Shinoura, 1977

JC3890(R46) This thesis

JG138 polA1 thy rha lac R. J. Pinney DP1125 Monk etal., 1971

JG138(R46) This thesis

TK501 As AB1157 but umuC36 R. J. Pinney DP1105 DE301 thi bio Sm® Kato & Shinoura, 1977

TK501(R46) This thesis

TK501(R805a) R. J. Pinney DP1115

TK702 umuC pro his thi glu lac R. J. Pinney DP1117 Kato & Shinoura, 1977

TK702(R46) This thesis

343/113 thy arg nad gal thi lys R. J. Pinney containing one of the following plasmids Pinney, 1980 R46 (DP967) R446b (DP974) R144 (DP975) R391 (DP965) R16(DP969) R621a(DP976) R124(DP962) pKMIOI (DP972)

MATERIALS AND APPARATUS 69 Table 2.1.2 Escherichia coli Kl 2 polyamine transport-deficient strains

Escherichia Source, collection number Genotype coli Kl 2 strain and reference

MA261 speB spec serA thr leu thi K. Igarashi, Chiba University Cunningham-Rundles & Maas, 1975

KK313 MA261 pot A K. Igarashi, Chiba University Kashi wag i etal., 1990

KK313 MA261 pot A potF K. Igarashi, Chiba University potF::Km Pistocchi etal., 1993

NH1596 MA261 pot A potG K. Igarashi, Chiba University Kashi wag i etal., 1990

Table 2.1.3. Salmonella typhimurium LT2 strains used for mutagenicity testing. GlaxoWellcome = GlaxoWellcome Genetic and Reproductive Toxicology Department

Strain Genotype Source and reference

TA98 hisD3052 rfa chID bio uvrB GlaxoWellcome (pKMIOI) Maron & Ames, 1984

TA98NR hisD3052 rfa chID bio uvrB GlaxoWellcome (pKMIOI) Speck etal., 1981; Nitroreductase deficient Rosenkranz & Speck, 1975; 1976

TA100 hisG46 rfa chID bio uvrB GlaxoWellcome (pKMIOI) Maron & Ames, 1984

TA1535 hisG46 rfa chID bio uvrB GlaxoWellcome (PSK1002) Oda etal., 1985

The hisG46 mutation detects base-pair substitution events (Ames, 1971), the hisD3052 mutation detects frameshift reversions (Isono & Yourno, 1974) and the rfa (deep rough) mutation results in defective lipopolysaccharide thereby increasing cell permeability to bulky hydrophobic chemicals (Ames at al., 1973).

MATERIALS AND APPARATUS 70 Table 2.1.4. Other bacteria and fungi

Source and Strain Reference collection number

Bacillus subtilis R. J. Pinney NCTC 8236 JTS9

Candida albicans Laboratory strain N/A

Escherichia coli KL16 R. J. Pinney Bachmann, 1972 JTS 640

Klebsiella aerogenes R. J. Pinney NCTC 418 JTS 65

Pseudomonas aeruginosa R. J. Pinney NCIB 8626 DP 1244 ATCC 9027

Saccharomyces cerevisiae Laboratory strain N/A

Salmonella typhimurium LT2 R. J. Pinney Lilleengen, 1948 DP 421

Staphylococcus aureus R. J. Pinney NCTC 6571 DP 1210

2. Plasmids The plasmids used in this thesis are listed in Table 2.2.1 together with the antibiotics to which they confer resistance.

Table 2.2.1. Plasmids used in this thesis.

Plasmid Inc Resistance and Reference other information

pGW12 N Ap Walker, 1978

pGW16 N Ap UV^ at high UV dose Little etal., 1991 and UV^ at low UV dose Walker, 1978

pKMIOI N Ap Su U V Mortelmans & Stocker, 1979

pYDI Unknown Tm UV^ Ambler ef a/., 1993

continued overleaf

MATERIALS AND APPARATUS 71 R16 B Ap Cm Sm Su Hg Tc UV^ Jacob etal., 1977 Upton & Pinney, 1983

R46 N Ap Sm Su Tc UV^ Jacob etal., 1977 Upton & Pinney, 1983

R124 FIV Tc UV^ Jacob etal., 1977 Upton & Pinney, 1983

R144 la Km Tc UV^ Jacob etal., 1977 Upton & Pinney, 1983

R391 Km Nm Hg UV^ Ambler ef a/., 1993 Jacob etal., 1977 Upton & Pinney, 1983

R446b M Sm Tc UV^ Jacob etal., 1977 Upton & Pinney, 1983

R621a lY Tc UV^ Jacob etal., 1977 Upton & Pinney, 1983

R805a IC Km Nm Pm UV^ Jacob etal., 1977 Upton & Pinney, 1983

3. Media Media were sterilized by autoclaving. Liquid media were sterilized by autoclaving at 121 °C for 15 minutes, with the exception of nutrient broth used for growing overnight cultures which was autoclaved at 115°C (10 psi) for 20 minutes. Solid media were also autoclaved at 115°C (10 psi) for 20 minutes and cooled to 55°C in a water bath before pouring. An exception was DMA agar which was used whilst hot. Agar plates were made by pouring approximately 20 ml quantities of the cooled, sterile agar into sterile, disposable petri dishes laid on a marble slabL Once solidified, and if for immediate use, the lids were removed and the plates inverted and dried at 45°C for 45 minutes. The plates were cooled to 37°C before use. If not for immediate use, the plates were inverted, stored at 4°C and dried on the day required. Liquid media were stored at room temperature. 3.1. Nutrient broth was prepared by dissolving 25 g of Oxoid N®2 nutrient broth powder [CM67, Unipath Ltd.]^ in one litre of distilled water according to the

^ Apparatus and disposable materials are listed at the end of this section ^ Company and order code for materials in square parentheses

MATERIALS AND APPARATUS 72 manufacturer’s directions. 4.5 ml and 9.9 ml quantities were then dispensed into clean, glass bottles and sterilized. 3.2. Double-strength nutrient broth was prepared as for nutrient broth but dissolved in 500 ml of distilled water. Appropriate quantities were dispensed into clean, glass bottles and sterilized. 3.3. Nutrient broth with thymine was prepared by adding thymine solution to nutrient broth at a final concentration of 60 pg ml'\ 4.5 ml and 9.9 ml quantities were then dispensed into clean, glass bottles and sterilized. 3.4. Sabouraud liquid medium was prepared by dissolving 30 g of Oxoid Sabouraud liquid medium powder [CM147, Unipath Ltd.] in one litre of distilled water according to the manufacturer’s directions. 4.5 ml and 9.9 ml quantities were then dispensed into clean, glass bottles and sterilized. 3.5. Davis-Mingioii minimai saits solutionwas prepared as described by Davis and Mingioli (1950). The following salts [BDH Chemicals] were sequentially

dissolved in 2 litres of distilled water.

c//-Potassium hydrogen orthophosphate (K2HPO4) [29619] 14.0 g

Potassium dihydrogen orthophosphate (KH2PO4) [10203] 6.0 g

fr/-Sodium citrate (Na 3C6H507 -2 H2 0 ) [10242] 0.94 g

Magnesium sulphate heptahydrate (MgS 0 4 -7 H2 0 ) [10151] 0.2 g

Ammonium sulphate (NH4)2S0 4 [10033] 2.0 g 4.5 ml and 9.9 ml quantities were then dispensed into clean, glass bottles and sterilized. 3.6. Doubie-strength Davis-Mingioii minimal salts solutionwas prepared as above but using twice the quantity of salts. 50 ml volumes were dispensed into clean, 150 ml glass bottles and sterilized. 3.7. Phosphate buffered (normai) saline was prepared as described by Howard (1991). 0.9 g of sodium chloride [30123, BDH Chemicals] was dissolved in 100 ml of 0.025M phosphate buffer at pH 7.4. 0.5 M Phosphate buffer = 81 ml of Solution A + 19 ml of Solution B adjusted to pH7.4 with 1M sodium hydroxide

Solution A = of/-Sodium hydrogen orthophosphate dihydrate (Na 2HP0 4 -2 H2 0 ) in distilled water to give a solution of 0.5M [30157, BDH Chemicals]

Solution B = Sodium dihydrogen orthophosphate dihydrate (NaH 2P0 4 -2 H2 0 ) in distilled water to give a solution of 0.5M [30132, BDH Chemicals]

MATERIALS AND APPARATUS 73 0.025M Phosphate buffer = 5 ml of + 95 ml of 0.5M phosphate buffer deionized water 4.5 ml and 9.9 ml quantities were then dispensed into clean, glass bottles and sterilized. 3.8. Double-strength phosphate buffered (normal) saline was prepared by dissolving 1.8 g of sodium chloride in 100 ml of double-strength phosphate buffer (10 ml of 0.5M phosphate buffer + 90 ml of deionized water). 5.0 ml quantities were then dispensed into clean, glass bottles and sterilized. 3.9. Nutrient agar was prepared by the addition of 1.5 % (w/v) Lab M agar N®1 [MC2, Amersham] to nutrient broth before sterilization. 3.10. Iso-Sensitest nutrient agar was prepared by suspending 3.4 g of Oxoid iso-sensitest agar [CM471, Unipath Ltd.] in 100 ml of distilled water according to the manufacturer's directions and then sterilized. 3.11. MacConkey agar was prepared by suspending 5.2 g of Oxoid MacConkey agar powder [CM7, Unipath Ltd.] in 100 ml of distilled water according to the manufacturer’s directions and sterilizing. 3.12. Sabouraud dextrose agar was prepared by suspending 6.5 g of Oxoid Sabouraud dextrose agar powder [CM41, Unipath Ltd.] in 100 ml of distilled water according to the manufacturer's directions and sterilizing. 3.13. Antibiotic/Antimicrobial-containing agar was prepared by aseptically adding an appropriate volume of sterile drug solution to molten (55°C) medium and mixing well before pouring. Nutrient, iso-sensitest and MacConkey agars were used and prepared as previously described. 3.14. Davis-Mingioii minimal saits selecting agar was prepared by aseptically adding, as required, 1.0 ml of sterile amino acid solutions (Materials and Apparatus 5), 6.0 ml of sterile thymine solution, 5.0 ml of sterile biotin solution, 0.7 ml of sterile glucose solution (as a carbon source) and an appropriate volume of drug in solution to 50 ml of double-strength Davis-Mingioii minimal salts solution. The volume was then made up to 70 ml with sterile, distilled water. This mixture was poured into a 150 ml glass bottle containing 30 ml of sterile, hot, molten agar (1.5 g of Lab M agar NM in 30 ml of distilled water) and the contents thoroughly mixed.

MATERIALS AND APPARATUS 74 4. Antimicrobial agents

P r o d u c t C o d e C o m p a n y Ampicillin sodium BP PL38/5061R Beecham Research (Penbritin®) Chloramphenicol 0 0378 Sigma Chemical Co Gentamicin sulphate PL0188/5906 Nicholas Laboratories (Genticin®) Kanamycin sulphate K4000 Sigma Chemical Co Nalidixic acid N8878 Sigma Chemical Co Nitrofurantoin N7878 Sigma Chemical Co Rifampicin R3501 Sigma Chemical Co Streptomycin sulphate BP PL0039/6002 Evans Sulphadiazine sodium PL12/5057R May & Baker Ltd. Tetracycline hydrochloride PL0095/5035 Lederle Laboratories (Achromycin®) Trimethoprim lactate T 2611 Burroughs Wellcome

All antimicrobials were stored in a desiccator at room temperature. They were weighed aseptically into sterile, plastic bottles using an analytical balance and dissolved in sterile, distilled water (SOW) to give the required concentration. Exceptions were rifampicin which was dissolved in DMSO to a concentration of 10 mg ml'^ and then diluted to the required concentration in SOW; nalidixic acid which was dissolved in 0.5M NaOH (0.02 ml per mg) and then diluted to the required concentration in SOW; nitrofurantoin which was suspended in SOW to which was added 0.5M NaOH (0.02 ml per mg) to give the required dissolved concentration and sulphadiazine which was available as a solution of concentration 250 mg mM (1 g in 4 ml).

5. Nutritional requirements

P r o d u c t C o d e C o m p a n y CONCENTRATION

L-Arginine A 5006 Sigma Chemical Co 2.5 mg mM d-Biotin B4501 Sigma Chemical Co 0.2 mg ml'^ D-Glucose 28450 BDH Chemicals 40% L-Histidine 37221 BDH Chemicals 4.0 mg mM L-Leucine 37121 BDH Chemicals 4.0 mg mM DL-Methionine M 9500 Sigma Chemical Co 2.5 mg mM

MATERIALS AND APPARATUS 75 L-Proline 37143 BDH Chemicals 4.0 mg ml'"' Thiamine 44005 BDH Chemicals 1.0 mg mM L-Threonine T 8625 Sigma Chemical Co 4.0 mg mM Thymine 00972C Koch-Light Labs 1.0 mg mM

Solutions of these compounds were prepared, with distilled water, at the concentrations shown above. 20 ml volumes were then dispensed into clean, glass bottles and the solutions sterilized as follows, d-biotin, L-proline, L-leucine and L- threonine by steaming at 100°C for 30 minutes; L-histidine was adjusted to pH 7.2 with 1M HCI prior to steaming at 100°C for 30 minutes; L-arginine by steaming at 100°C for 10 minutes; thymine and thiamine by autoclaving at 115°C for 20 minutes; glucose by autoclaving at 115°C for 10 minutes and DL-methionine by filtering. All solutions were stored at 4°C (except DL-methionine which was frozen at -20°C ) for a maximum of six months.

6. Chemicals

P r o d u c t C o d e C o m p a n y Acetic anhydride 27017 BDH Chemicals Acetonitrile 29220 BDH Chemicals N-Acetylethylenediamine 39,726-1 Aldrich-Chemical Co Aminoacetaldehyde A 4898 Sigma Chemical Co diethyl acetal 4-Aminobenzoic acid 27103 BDH Chemicals Chloroform 27710 BDH Chemicals Diethyl ether 28132 BDH Chemicals Dimethyl sulphoxide 10323 BDH Chemicals Ethanol 96% 28719 BDH Chemicals Ethyl acetate E7770 Sigma Chemical Co D(+)-Glucosamine HCI G 4875 Sigma Chemical Co Heptaldehyde H 212-0 Aldrich-Chemical Co

Hydrochloric acid 28507 BDH Chemicals Isopropanol 405-7 Sigma Chemical Co L-Lysine methyl ester 2HCI L0645 Sigma Chemical Co Methanol 29192 BDH Chemicals

MATERIALS AND APPARATUS 76 5-Nitrofuraldoxime 14,928-4 Aldrich-Chemical Co 5-Nitro-2-furanacrolein 22,656-4 Aldrich-Chemical Co 5-Nitro-2-furfuraldehyde 17,096-8 Aldrich-Chemical Co 5-Nitro-2-furfuraldehyde 01715 Lancaster Chemical diacetate Norspermidine 17006 Sigma Chemical Co Oxalyl chloride 05376 BDH Chemicals Putrescine 01,320-8 Sigma Chemical Co Pyridine 29707 BDH Chemicals Semicarbazide acetic acid Gift from Proctor & Gamble Pharm; Sodium sulphate 30223 BDH Chemicals Sodium hydroxide pellets 30167 BDH Chemicals pearl 10438 BDH Chemicals Spermidine S3828 Aldrich-Chemical Co Spermine S3256 Aldrich-Chemical Co Sulphuric acid 30325 BDH Chemicals Toluene 30453 BDH Chemicals

7. Materials for mutagenicity studies If not indicated in the text, chemicals were received from BDH Chemicals, Difco Laboratories, Fisher Scientific, Fisons, FlowFusor® FL (Manufacturing) Ltd. or Sigma Chemical Co and were of analytical grade. 7.1. Vogel-Bonner E agar [P0674A, Unipath Ltd.] ready-poured plates were used containing 0.4 g of MgSO^-THgO, 4.0 g of citric acid HgO, 20.0 g of KgHPO^ (anhydrous) and 7.0 g of NaHNH^PO^ ^HgO per 1000 ml of distilled water. These were stored at 4°C until required and checked for contamination and dried on the day of testing. 7.2. Top agar (soft agar overlay) was prepared by suspending/dissolving 1.5 g of agar and 1.25 g of sodium chloride in 240 ml of distilled water and autoclaving at 121 °C for 15 minutes. It was stored at room temperature for 3 months, melted immediately prior to use and cooled to 45-50°C. Relevant supplements were added as required. 7.3. Blotin/Hlstidine solution 2.89 mg of biotin and 2.48 mg of L-Histidine monohydrochloride monohydrate were dissolved in 10 ml of distilled water and

MATERIALS AND APPARATUS 77 sterilized by filtration through a Millex-HV 0.45 pm filter unit [Millipore]. 10 ml was added to every 250 ml of top agar (for Salmonella typhimurium Ames tests) to give final concentrations of 9.55 pg ml’"' histidine and 11.13 pg ml'^ biotin. 7.4. Nutrient broth was prepared by dissolving 25 g of Oxoid nutrient broth N®2 powder [CM67, Unipath Ltd.] in 1000 ml of distilled water. 10 ml volumes were dispensed and autoclaved at 121 °C for 15 minutes. It was stored at 4°C for 3 months. 7.5. Ampicillin (10 mg ml'’’) 1 ml was added to every 400 ml of sterilized nutrient broth to give a final concentration of 25 pg mM as a supplement for S. typhimuriumTA98, TA98NR and TA100 to ensure the strains retained the pKMIOI plasmid. 7.6. Salt solution 12.3 g of potassium chloride (KCI, 1.65M) and 8.14 g of magnesium chloride (MgClg GHgO, 0.4M) were dissolved in 100 ml of distilled water and dispensed into 10 ml volumes. The solution was autoclaved at 121 °C for 15 minutes. 200 pi was added to every 10 ml of S9-mix to give final molarities of 33mM KCI and 8mM MgClj. It was stored at 4°C for 3 months. 7.7. Glucose-6-phosphate 304 mg was dissolved in 1 ml of sterile distilled water to give a 1M solution and stored at -20°C in deep freeze until required. 50 pi was added to every 10 ml of S9-mix giving a final concentration of 1.52 mg ml'^ (5mM). 7.8. Sodium phosphate buffer was prepared by combining 405 ml of solution A, 95 ml of solution B and 500 ml of distilled water in a 2 litre flask to give a 0.2M solution.

Solution A = 71.64 g of NagHPO^ 1 2 H2O in 1000 ml of distilled water

Solution B = 6.24 g of NaH2P0 4 *2 H2 0 in 200 ml of distilled water 250 ml volumes were dispensed into bottles and autoclaved at 121 °C for 15 minutes. 5 ml was added to every 10 ml of S9-mix to give a final molarity of 0.1 M buffer pH7.4. It was stored at room temperature for 3 months. 7.9. B-buffer was prepared by dissolving the following salts, with shaking, in 500 ml of distilled water.

Na2HP0 4 ‘2 H2 0 10.09 g

NaH2P0 4 -H2 0 2.75 g KCI 0.375 g

MATERIALS AND APPARATUS 78 o-Nitrophenyl-3-D-galactopyranoside N 1252 Sigma Chemical Go Sodium azide S 2002 Sigma Chemical Co

8 . Apparatus Autoclaves Dent & Hellyer LabClave: 121 °C for 15 minutes Midas 32 Prior Clave: 121 °C for 15 minutes bench-top autoclave: 115°C (10 psi) for 20 minutes Balances Mettler AT400 balance, Mettler Instruments Ag., Zurich Mettler H20, Mettler Instruments Ag., Zurich Mettler P I210, Mettler Instruments Ag., Zurich Mettler PK2000, Mettler Instruments Ag., Zurich Oertling 21TD, Maidstone, Kent Oertling V20 (Vernier), Maidstone, Kent R 160 P, Sartorius GmbH, Gottingen, Germany Centrifuges Centaur 2 (WR2204), MSB Scientific Instruments High Speed 18, MSB Scientific Instruments Micro Centaur, MSB Scientific Instruments Disposables & Chemical glassware by Quickfit and SGL Ltd. Miscellaneous Bppendorf tubes Glass spreaders Glass test tubes with oxoid lids Petri dishes (90 mm, triple-vent) Greiner Labortechnik Ltd. Pasteur pipettes Plastic 250 ml sterile centrifuge bottles Soda glass 5 ml vials TC Microwell 96F (96 well plates), Nunclon™ A Surface, Nunc Brand Products, Nalge Nunc International, Denmark Universal bottles: irradiated plastic, Greiner Labortechnik Ltd. sterile glass, laboratory stock Driers/Ovens Drying oven, Baird & Tatlock (London) Ltd., Bngland Agar drying cupboard, Hearson Laboratory Bquipment, London Vacuum oven, Bdwards High Vacuum Ltd., Sussex Filters Swinnex filter assembly, Millipore Ltd. Microcellulose membrane filters (0.45 pm pore diameter).

MATERIALS AND APPARATUS 80 Millipore Ltd., Bedford, USA Millex-HV 0.45 pm sterile filter unit 01730 (low protein binding, non-pyrogenic, single-use only, Durapore® membrane), Millipore, Bedford, USA Heating appliances “Dri-Block” BT3, Grant Instruments (test tube heater) Electrothermal MC221 Electrothermal MY6402 Incubators Baird & Tatlock (London) Ltd. 024 Environmental Incubator Shaker, New Brunswick Scientific Co. Inc., Edison, USA Gallenkamp and Company Ltd. at 135 revolutions per minute Heraeus Instruments B6760, Heraeus Equipment Ltd., Essex LEEC Mkll Proportional Temperature Controller Inoculator Denley multipoint inoculator Liquid dispenser Jencons [Scientific] Ltd. Perimatic Dispenser Microscope Olympus CK2, Olympus Optical Company Ltd., Japan Ikegami CCD camera, Kaiso RA1 (mount and measure) Domino image analysis system and Ames-SAS program pH Buffer solutions at pH4.00 [B-5020] and pH7.00 [B-4770], Sigma Chemical Company. Ltd., USA Digital meter: PTI-15, Data Scientific Ltd. Colour fixed indicator sticks (pH-Fix 4.5 -10.0), Macherey Nagel Whatman universal papers (pH1-11) Pipettes “pipetman”, Gilson, France: 1000 pi, 200 pi and 20 pi Anachem multichannel pipetters: 8 channel, 12 channel Pipette filler, Pipetboy acu, Integra Biosciences Ltd., UK Plastic disposable pipette (10 ml). Falcon® Serological Pipet, Becton Dickinson Labware, NJ, USA Sterile syringe (20 ml), Plastipak®, Becton Dickinson, Ireland Sonicator “Bransonic 12" Branson Ultrasonic Cleaner B12, Branson Cleaning Equipment Company, Shelton, CT, USA Spectrophoto Jenway 6100, Jencons [Scientific] Limited, Bedfordshire, meters England Cuvettes (N® 67.742, plastic, 10x4x45 mm^), Sarstedt, Germany Ceres UV 900 HDi, Bio-Tek Instruments, Inc. UV equipment J-225 Blak-Ray UV meter. Ultraviolet Products Inc., San

MATERIALS AND APPARATUS 81 Gabriel, California 254 nm low-pressure mercury lamp Model 12 (1 J m'^ s’"'), Hanovia Lamps Ltd., Slough, Buckinghampshire Vortex “Rotamixer”, Hook & Tucker Ltd. Water-bath Static waterbath. Grant Instruments (Cambridge) Ltd. Büchi 461 waterbath and Rotavapor®, Switzerland Laboritoriums Technik Ag. Workstation Howarth Particulate Biohazard Workstation, Howarth Air Engineering Ltd., Bolton, UK Class III type containment cabinet, Envair, Lancashire, UK

MATERIALS AND APPARATUS 82 3. Viable counting Cultures were serially diluted in nutrient broth or DM by aseptically adding 0.5 ml of a culture suspension to 4.5 ml of sterile diluent to give a 10^ dilution or by aseptically adding 0.1 ml of a culture suspension to 9.9 ml of sterile diluent to give a 10'^ dilution. These suspensions were shaken to mix and used to make further dilutions. Viability was determined by one of two methods. 3.1. Drop-plate viability. 20 pi of each dilution was accurately pipetted onto the surface of pre-warmed solid medium using automatic pipettes with sterile tips. Plates were divided into segments which were inoculated with two or more drops, depending on the number of dilutions used. After the inocula had absorbed into the medium, the plates were inverted and incubated at the optimum temperature for the organism. The number of colonies was recorded and the number of organisms present in 1 ml of original culture calculated.

Number of organisms = Mean number of x Dilution x 1000 per ml of original culture colonies per 20 pi drop factor 20

3.2. Spread-plate viability. 0.1 ml of a known dilution was accurately pipetted onto pre-warmed solid medium and spread over the surface using a sterile, glass spreader. After the inocula had been totally absorbed into the medium, the plates were inverted and incubated at the optimum temperature for the organism. The number of colonies was recorded and the number of organisms present in 1 ml of original culture calculated.

Number of organisms = Mean number of x Dilution x 10 per ml of original culture colonies per 0.1 ml drop factor

4. Post-UV survival 0/N cultures were serially diluted in nutrient broth to give dilutions from N (neat, undiluted 0/N culture) to 10*®. Viability was determined on nutrient agar. Once the inocula had been absorbed into the medium, the lids were removed and the plates irradiated under a low-pressure mercury lamp emitting light at 254 nm. The lamp was set at a height that gave a UV fluence of 1 J m'^ s'\ determined using a UV meter. The actual dose of UV, measured in J m'^, was therefore equivalent to

EXPERIMENTAL METHODS 85 6. Determination of minimum inhibitory concentration

Minimum inhibitory concentrations (MICs) were determined on solid media containing different concentrations of the drug. 6.1. Preparation of antibiotic/antibacterial nutrient agar. Concentration ranges increased by 25% to 50% at each consecutive step using ratios of 1, 1.5, 2, 3, 4, 5 and 7.5. 3 ml quantities of appropriate concentrations of drug solution were aseptically prepared in sterile, glass bottles. 17 ml of sterile, cooled (55°C), molten nutrient agar (2.5 g of Oxoid N®2 nutrient broth powder and 1.5 g of Lab M agar N“1 in 85 ml of distilled water) was then aseptically added to each bottle. The contents were gently mixed and immediately poured into a sterile, disposable petri dish laid on a marble slab. Once solidified the lids were removed and the plates inverted and dried at 45°C for 45 minutes. The plates were cooled to 37°C before use. Control, drug-free nutrient agar was also prepared using 3 ml of the diluent. If the diluent was not SDW, then agar was also prepared containing 3 ml of SOW to show that the diluent was not inhibitory. 8.2. Inoculation of plates. The series of antibiotic agar plates was inoculated with N, 10'^ and 10'"* dilutions of 0/N cultures. Drug-free plates were inoculated first, followed by plates of increasing drug concentration. Viability of the 0/N cultures was determined on nutrient agar. After the inocula had absorbed into the medium, the plates were inverted and incubated at the optimum temperature for the organism. The minimum inhibitory concentration (MIC) value was recorded as the lowest concentration of drug to inhibit growth of isolated colonies (BSAC Working Party, 1991). 6.2.1. Multipoint inoculations. The multipoint inoculator allowed twenty-one samples, each of approximately 1 pi, to be tested on every plate, i.e. seven organisms could be tested, at three dilutions, on one plate. 6.2.2. Loop inoculations. An alternative method of inoculation was used when only a few plates were involved. Loopfuls (approximately 1-2 pi) of the dilutions of 0/N cultures were placed onto the surface of the plates.

7. Agar diffusion tests Agar diffusion tests (zones of inhibition) were determined by boring wells into solid media and filling these with solutions of test compound.

EXPERIMENTAL METHODS 87 7.1. Seeded agar was prepared by adding 1 ml of 0/N culture to 99 ml of cooled (55°C) molten medium appropriate for the organism. This was gently mixed and 20 ml quantities were poured into sterile, disposable petri dishes laid on a marble slab. The plates were set with the lids ajar to reduce condensation and used on the day of manufacture. 7.2. Zones of inhibition. Wells were cut into the seeded medium of each plate with a flamed and cooled cork borer. Each well was then filled with a different concentration of the compound being tested. The plates were incubated at a temperature appropriate for the organism. Bactericidal activity was determined from the zones of inhibition. Three measurements of the diameter of each zone of inhibition were made and the average calculated. The critical inhibitory concentration (CIC) was then determined by plotting the square of the distance from the source of diffusion to the edge of the zone (x^) against the concentration of drug and reading the concentration at x^ = 0 from the regression of the plot (Linton, 1961).

8. Determination of bacterial sensitivity in liquid media 0.1 ml of culture suspension (0/N, 0/N PBS, Log, Log PBS) was inoculated into glass bottles containing known concentrations of the drug being tested in 9.9 ml of liquid medium. These incubation mixtures were mixed thoroughly and maintained at an appropriate temperature for the organism in a static water bath. Viability of the incubation mixtures was determined at regular time intervals on appropriate agar. Survival was calculated as a percentage of the number of viable cells in the incubation mixture.

% survival = Number of viable cells per ml at time t x 100 Number of viable cells per ml at time 0

9. Mutation to nitrofurantoin resistance Mutation frequencies of cultures to nitrofurantoin resistance was determined on solid and in liquid media. 9.1. Spontaneous mutation on solid media was determined by spreading 0.1 ml of a 100N concentrated sensitive culture onto agar containing nitrofurantoin at MIC, 2xMIC, 5xMIC and lOxMIC, in duplicate. Viability was determined on

EXPERIMENTAL METHODS 88 nutrient agar and the mutation frequency calculated.

Mutation = Mean number of nitrofurantoin-resistant mutants per ml of 10ON culture frequency Total viable count per ml of 100N culture

9.2. Mutation in liquid media was determined by exposing sensitive cultures to nitrofurantoin and then selecting resistant mutants on nitrofurantoin-containing nutrient agar. 1.5 ml of 0/N culture was diluted 1 in 100 into nutrient broth (spontaneous mutation) or nutrient broth containing nitrofurantoin at 200 pg mM (induced mutation) in a 500 ml conical flask. The flasks were maintained at 37°C in a static water-bath and sampled at hourly intervals. Total viability was determined on nutrient agar and the count of nitrofurantoin resistant mutants was determined on nutrient agar containing 200 pg mM of nitrofurantoin. For these latter plates, 15 ml samples were centrifuged at 4,000 rpm for 25 minutes and resuspended in 0.15 ml of DM giving a 100-fold concentrated suspension of the incubation mixture. This was then serially diluted and spread. Modifications to this method included shaking the flask in at orbital incubator at 37°C to increase the growth rate and increasing the time of exposure to nitrofurantoin.

Mutation = Mean number of nitrofurantoin-resistant mutants per ml frequency Total viable count per ml

10. Replica plating A 20 cm^ piece of sterile velvet was stretched over the surface of a circular faced aluminium block of such diameter to allow a 9 cm Petri dish to fit over it. The plate on which colonies to be tested were growing was pressed onto the velvet leaving samples of each colony on the fibres. A fresh plate was then pressed onto the velvet, the orientation of the plate being noted, to deposit samples of the colonies onto this new plate. Imprints of up to six plates containing different antibiotics could be made. The plates were incubated overnight at the appropriate temperature and compared to the original and to an antibiotic-free control.

11. Plasmid elimination Plasmid elimination can occur from cultures dying in lethal concentrations or

EXPERIMENTAL METHODS 89 growing in sub-inhibitory concentrations of an eliminating agent. 11.1. Lethal concentration. 0.1 ml of an 0/N culture was inoculated into glass bottles containing a lethal concentration of the drug being tested in 9.9 ml of nutrient broth. After 2 or 3 hours incubation in a static water-bath, together with a drug-free control, samples were diluted 10'^ and spread on MacConkey agar. The plates were incubated and replicated as described above and the frequency of elimination determined.

Frequency of = Number of colonies not growing on antibiotic agar x 100 elimination Number of colonies on nutrient agar

11.2. Sublethal concentration. 0.1 ml of a lO'"* dilution of an 0/N culture was inoculated into glass bottles containing sublethal concentrations of the drug being tested in 4.9 ml of nutrient broth. These were then placed in an orbital incubator at the appropriate temperature together with a drug-free control and grown to turbidity. A 10’® dilution of the turbid culture was spread onto duplicate MacConkey agar plates and incubated overnight incubation at the appropriate temperature. Replica plates were produced on nutrient agar and antibiotic agar, containing an antibiotic to which the plasmid confers resistance. The frequency of elimination was then calculated as shown above.

12. Ames Salmonella plate incorporation mutagenicity assay Solutions of the test compounds were prepared in appropriate solvents. To a 2.0 ml aliquot of molten top agar (soft agar overlay) containing a trace of histidine (9.55 pg ml'^) and excess biotin (11.13 pg ml'^) in a sterile, oxoid-capped, disposable glass tube maintained at 45°C in a Dri-Block, was added 100 pi of the tester strain, 100 pi of the test solution and 0.5 ml of 0.2M buffer. This was mixed by flicking the tube with a finger and poured onto a 20 ml Vogel-Bonner E minimal medium agar plate. Controls were prepared by replacing the test compound with solvent. Duplicate plates were also prepared with S9-mix instead of 0.2M buffer to determine whether liver enzymes activated or destroyed the test compound. 12.1. Preliminary toxicity tests were conducted as above using one plate per concentration of test compound. The background lawn was examined microscopically after overnight incubation at 37°C. The lowest concentration giving

EXPERIMENTAL METHODS 90 no background lawn was recorded (MIC). Preliminary toxicity tests were used to determine the maximum concentration of test compound for use in the microbial mutagenicity tests by establishing the bactericidal effects of the compounds on the tester strains. 12.2 Microbial mutagenicity tests were conducted as described above using three plates per concentration of test compound, six solvent (control) plates and two positive control (Table 3.12.1) plates. All were tested with and without 89- mix. Once the top agar had set (within one hour of pouring) the plates were inverted and incubated for 3 days at 37°C in the dark.

Table 3.12.1. Positive controls and concentrations used in the Ames/Salmonella mutagenicity assay

S. typhimurium strain + 89-mix - 89-mix

TA98 / TA98NR 3-methylcholanthene 2-nitrofluorene (10 pg/plate) (1 pg/plate)

TA100 cyclophosphamide sodium azide (5000 pg/plate) (2pg/plate)

The critérium for a positive (mutagenic) response in this assay and for these strains is a two-fold increase in revertants over the spontaneous reversion. 8 pontaneous mutation frequencies (untreated and solvent controls) should fall between 15 and 60 colonies per plate for TA98 and TA98NR (Kier et a/., 1986) and between 75 and 260 colonies per plate for TA100 (Kier at a/., 1986 with modifications by GlaxoWellcome Genetic and Reproductive Toxicology Department).

13. SOSIumu test 0.2 ml of 0/N culture was added to 25 ml of TGA broth and grown for up to 10 hours at 37°C in an orbital incubator at 230 rpm. When an optical density, at 600 nm, of 0.22 or greater had been reached, 2 ml of the culture was added to 100 ml of fresh TGA medium in a conical flask. This was grown for 2 hours at 37°C in an orbital incubator at 230 rpm until an optical density, at 600 nm, of 0.22 or greater was obtained. The test compounds were dissolved at appropriate concentrations

EXPERIMENTAL METHODS 91 and 10 pi quantities added to 240 [^\ of the bacterial tester strain suspension and 50 pi of S9-mix or 0.2M buffer in the well of a microtitre plate. The plates were incubated for 2 hours at 37°C in an orbital incubator at 230 rpm. A 30 pi sample from each well was then transferred into 270 pi of fresh TGA medium in a fresh microtitre plate. These plates were incubated for 2 hours at 37°C in an orbital incubator at 230 rpm to allow recovery and expression of the P-galactosidase gene. Optical density readings for each well were then measured at 600 nm. Another 30 pi sample of each suspension was then added to 120 pi of B buffer in fresh microtitre plates. 30 pi of ONPG solution was then added with mixing to start the enzyme reaction and the plates were incubated for half an hour at 28°C in an orbital incubator at 230 rpm. The reaction was terminated by the addition of 120 pi of 1M sodium carbonate solution to each well. Optical density readings were measured at 415 and 600 nm and the punits of P-galactosidase produced were calculated. The positive controls 2-aminoanthracene (with S9) and ICR 191 (without S9) were also used. A positive umu induction was a 2-fold increase in p-galactosidase activity over the control.

14. Manufacture of novel nitrofuran compounds Nitrofurans are commercially synthesized by direct condensation (Sittig, 1979). For example, nitrofurantoin can be synthesized by condensation of 5- nitrofurfuraldehyde and 1-aminohydantoin (Remington’s Pharmaceutical Sciences, 1990) usually in a 1:1 ratio where the number of moles of reactants are equivalent. This uncomplicated method of synthesis was tried initially. 14.1. Condensation of nitrofuran and amine in ethanol (Scheme A).

H - H g O I R— C C + R' ► R—C=N R' H H

H H - Z H g O I I 2 R— M — R"— N - ► R— C=N— R"—N=C— R H H H

Scheme A. Condensation reaction of 5-nitrofurfuraldehyde or 5-nitrofuranacrolein with amine. R = nitrofuran; R' = amine; R" = polyamine

EXPERIMENTAL METHODS 92 Molar equivalents of either 5-nitrofurfuraldehyde or 5-nitrofuranacrolein and amine were each dissolved in 96% deoxygenated ethanol. The nitrofuran solution was then added dropwise to the amine solution and the mixture stirred mechanically, in the presence of nitrogen and at room temperature, for one hour. After this time the solvent was evaporated under reduced pressure at 45°C and the solid triturated in water and 1M hydrochloric acid. The aqueous phase was made basic with sodium hydroxide and extracted with several portions of ethyl acetate or chloroform. The portions were combined, dried with NagSO^ for 30 minutes and filtered to remove the NagSO^. The solvent was removed by evaporation under reduced pressure at 45°C and the product dried in a vacuum oven or desiccator. While this method was successful for the manufacture of bis-5-nitrofurfurylidenylputrescine, bis-5-nitrofurfurylidenylspermidine, bis-5-nitrofurfurylidenylspermine, bis-5-nitrofurfurylethenylidenylspermidine and N-acetyl-N-(5-nitrofurfurylethenylidenyl)ethylenediamine, as determined from NMR, it gave tarry products of N-acetyl-N-(5-nitrofurfurylidenyl)ethylenediamine, bis-5-nitrofurfurylidenyl- norspermidine and bis-5-nitrofurfurylethenylidenylputrescine whose NMR spectra were not consistent with those expected. 14.2. Condensation of nitrofuran and amine in acetonitrile. This method was a modification of 14.1 with similar reaction conditions but using acetonitrile as the reaction solvent instead of ethanol in an attempt to reduce the tarry state of the product. The solid remaining after evaporation of the solvent was not triturated or extracted in any solvent. This method did not, however, reduce the tarriness of the product. 14.3. Condensation of nitrofuran, 4-aminobenzoic acid and amine (Scheme B). This condensation differed slightly in that benzoic acid was used to link the nitrofuran and the amine. Molar equivalents of 5-nitrofurfuraldehyde and 4- aminobenzoic acid were dissolved in 96% ethanol. The benzoic acid solution was added dropwise to the nitrofuran solution and refluxed for 3 hours. The resultant 5- nitrofurfurylidenyl-4-aminobenzoic acid was filtered under reduced pressure and the precipitate dried in a vacuum desiccator. A portion of this solid was then suspended in toluene and heated with half of a molar equivalent of oxalyl chloride. Pyridine and N-acetylethylenediamine were then added and the mixture stirred. The toluene layer was separated and the remainder extracted into chloroform. The solvent was

EXPERIMENTAL METHODS 93 evaporated and the product dried in a vacuum desiccator.

O^N O CHO + HgN // COOH

OgN H Cl 01 I I o = c - c = o

HO OH I I o = c - c = o

0 ,N ^C ""-C V“"

NH 2(CH 2) 2NHC0 CH.

HO!

C-NH(CH 2)2NHC0 CH O^N 0

Schem e B. Condensation of 5-nitrofurfuraldehyde, 4-aminobenzoic acid and N-acetylethylenediamine

Although the direct condensation of nitrofuran with amines is an established industrial method, for example for nitrofurazone (Sittig, 1979), it often produces highly coloured, tarry, insoluble products with low molecular weight alkyl amines, which are difficult to isolate. As both methods 14.1 and 14.2 resulted in tarry products, the gentler reaction of the diacetyl-protected 5-nitrofurfuraldehyde with the

EXPERIMENTAL METHODS 94 amine, in the presence of sulphuric acid, was employed. This is a variation of the method used in the preparation of nifuratel (Sittig, 1979). 14.4. Condensation of 5-nitrofurfuraldehyde diacetate and amine in ethanol (Scheme C). Molar equivalents of 5-nitrofurfuraldehyde diacetate and amine compounds were each dissolved, with heating if necessary, in 96% deoxygenated ethanol. The 5-nitrofurfuraldehyde diacetate solution was added

dropwise to the amine solution and mixed. 1 0 % sulphuric acid was added dropwise to catalyze the reaction and the mixture allowed to reflux gently for seven hours. Solvent levels were maintained as necessary. The solvent was evaporated under reduced pressure at 45°C and the product taken into water and made basic with sodium hydroxide. The aqueous phase was extracted with several portions of ethyl acetate which were combined, dried with NagSO^for 30 minutes and filtered to remove the NagSO^. The ethyl acetate was removed by evaporation under reduced pressure at 45°C and the product dried in a vacuum oven or desiccator. The aqueous phase itself was evaporated under reduced pressure at 50°C and the resultant suspension combined with toluene which was evaporated under reduced pressure at 45°C. The product was dried in a vacuum oven.

OCOCH, OCOCH. / / C—H 2 R— C— H \ OCOCH. OCOCH,

H H

:n — R' 2 R — +:N—R"—N' H H H

HgO 2 H P

H H H

R C = N — R' R N— R"— N = C — R

Scheme C. Condensation reaction of 5-nitrofurfuraldehyde diacetate with amine. R = nitrofuran; R' = amine; R" = polyamine

EXPERIMENTAL METHODS 95 This method proved successful in producing bis-5-nitrofurfurylidenylputrescine and bis-5-nitrofurfurylidenylmethyllysine, as determined by NMR, but not bis-5-nitrofurfurylidenylspermine and bis-5-nitrofurfurylidenylspermidine. Hence, an alternative method, using an exchange reaction of N-(heptylidene)amine with 5- nitrofuraldoxime, was tried. This is a variation of the principal method used to prepare nitrofurantoin (Sittig, 1979). 14.5. Exchange reaction of 5-nitrofuraldoxime and amine via N- (heptylidene)-amine in isopropanol/water (Scheme D). Molar equivalents of heptaldehyde (dissolved in isopropanol) and amine (dissolved in water acidified with concentrated hydrochloric acid) were stirred for five hours at room temperature. This mixture and concentrated sulphuric acid were then added to 5-nitrofuraldoxime and stirred over a steam bath for VA hours. The product was filtered, washed and dried in a vacuum oven or desiccator.

2 CH,(CH2);C ^ H H

H^N R' H jN R" NHj

HgO

CH3(CH,)5C=N — R' CH3(CH2)sCH=N — R"— N=CH(CH2)gCH3

R CH = NOH 2 R CH = NOH

CH3(CH2)5CHN0 H 2 CH3(CH2)gCHN0 H

R— CH = N — R' R— CH = N — R" — N = C H - R

Scheme D. Reaction of 5-nitrofuraldoxime and amine via N-(heptylidene)amine. R = nitrofuran: R' = amine; R" = polyamine

This method only produced bis-5-nitrofurfurylidenylspermine and not bis-5- nitrofurfurylidenylspermidine or N-acetyl-N-(5-nitrofurfurylidenyl)ethylenediamine, as determined by NMR. 14.6. Exchange reaction of 5-nitrofuraldoxime and amine via N- (heptylidene)-amine in isopropanol. Method 14.5 was modified so that both the heptaldehyde and the amine were dissolved in isopropanol which was evaporated before the addition of 5-nitrofuraldoxime. This did not, however, increase the

EXPERIMENTAL METHODS 96 success of this method of manufacture. As other synthetic targets of this project were spacer-linked and polyamine derivatives of nitrofurantoin, for example a spermine analogue (Figure 3.14.1), a spermidine analogue (Figure 3.14.2) or a derivative with an aminoacetaldehyde spacer (Figure 3.14.3), a method of directly incorporating a protected aminoacetaldehyde was attempted. This was a variation of the commercial synthesis of 1-aminohydantoin (Remington’s Pharmaceutical Sciences, 1990).

Q ^ X q >-CHN(CH2)3NH(CH2)4NH(CH2)3-N NH

o Figure 3.14.1. Spermine analogue of nitrofurantoin

Q ^Xq3>-CHN(CH2)3NH(CH2)4-N NH

o Figure 3.14.2. Spermidine analogue of nitrofurantoin

^ CHNCHjCHN-N^ ^NH OjN O Y o Figure 3.14.3. Nitrofurantoin with aminoacetaldehyde spacer

14.7. Reaction of 5-nitrofurfuraldehyde and semicarbazide acetic acid (Scheme E). Molar equivalents of aminoacetaldehyde diethyl acetal and acetic

anhydride, together with pyridine, were reacted together for at least 18 hours to produce acetylaminoacetaldehyde diethyl acetal. This and semicarbazide acetic acid were then refluxed gently to condense them and given prolonged reflux to cyclise the semicarbazide moiety. Once cyclised, gentle refluxing was maintained and the solution made basic before the addition of 5-nitrofurfuraldehyde. This

mixture was stirred for at least 1 hour and then solvent removed by evaporation

EXPERIMENTAL METHODS 97 under reduced pressure at 45°C. The product was dried in a vacuum oven or desiccator. Unfortunately, this method did not prove successful and requires modification for future work.

NH2CH2CH(0C.,HJ 2 5^2

+ Pyridine + Acetic anhydride for 18 hours CONH, I 2 CHXONHCHXH(OC^HJ^ + KNN 2 5^2 2 I CH^COOH + Sulphuric acid + Ethanol Gentle reflux CONH^ I 2 CHXONHCHXHNN 3 2 I CH^COOH Prolonged reflux O

CH3CONHCH2CHNNA. NH

Gentle reflux O + NaOH 0

O^N O CHO + NH^CH^CHNN A. NH

O O

0 ,N O CHNCH.CUNN A. NH

Scheme E. Preparation of N-(5-nitrofurfurylidenyl)aminoacetalaminohydantoin

EXPERIMENTAL METHODS 98 1. Sensitivities to nitrofurantoin of Escherichia coll DNA repair-deficient strains and their plasmid R46-containing derivatives

1.1. Introduction DNA repair-deficient bacterial mutants are more susceptible to DNA- damaging chemicals than repair-proficient wild-type strains (Jenkins & Bennett, 1976; Siccardi, 1969). Nitrofurans damage DNA and the effect of nitrofurantoin against wild-type and DNA repair-deficient Escherichia coll was therefore examined. Also, as some plasmids confer protection against DNA damage induced by radiation and alkylating agents (Molina ef a/., 1979; Mortelmans & Stocker, 1976; Pinney, 1980) and this could be significant in therapeutic resistance to nitrofurantoin, the effect of one such plasmid, R46, on the sensitivities to nitrofurantoin of these strains was also tested. The survival levels of the E. coll K12 strain AB1157, which is fully DNA repair-proficient, and a variety of DNA repair-deficient mutants, to lethal concentrations of nitrofurantoin were therefore investigated. The repair-deficient strains included TK702 umuC36 which lacks error-prone repair, AB2470 recB21 which lacks recombination repair, JG138 polA1 which is DNA polymerase I deficient, AB2494 lexA3 which is unable to express SOS inducible functions, AB1886 uvrA6 and JC3890 uvrB (DE301) which are deficient in excision repair, TK501 uvrB (DE301) umuCSG which lacks both excision and error-prone repair and AB2463 recA13 which is unable to carry out either recombination or error-prone repair functions. Plasmid R46 was then introduced into each of these strains as a representative of the many plasmids known to confer error-prone DNA repair capabilities on their host (Mortelmans & Stocker, 1976). UV-light was used as a model DNA-damaging agent to confirm the genotype of these strains and to confirm that plasmid-mediated DNA repair was expressed. It also enabled comparison of the relative sensitivities of these strains to UV light and nitrofurantoin.

1.2. Plasmid transfers Plasmid R46 (Materials & Apparatus Table 2.2.1) was transferred by conjugation (Experimental Methods 5) from E. coll 343/113(R46) thy erg lys into all

RESULTS TÔT nine of the E coli strains described above (Materials & Apparatus Table 2.1.1). The donor was counter-selected because of its requirement for lysine, which was not required by any of the recipients, and transconjugants were selected by the presence of tetracycline at 20 pg ml'\ to which R46 confers resistance. Ampicillin at 10 pg mM and sulphadiazine at 50 pg ml'^ were then used to check that the entire R46 plasmid had been transferred into the transconjugants. Significant, stable plasmid transfer occurred into all strains. Transfer frequencies per donor after 4 hour matings were 5.0 x 10'^ into AB1157 and varied between 1 .6 x 10'^ for po!A1 and 6.7 x lO'"* for lexAS for the repair-deficient strains.

1.3. Post-UV survival ofEscherichia coii and the effect of R46 E. coli AB1157, the DNA repair-deficient strains and the R46-containing transconjugants were exposed to UV light (Experimental Methods 4) to compare their relative sensitivities and to determine the effects, if any, of plasmid R46 on post-UV survival. As expected the various strains showed different sensitivities to UV light (Figure 4.1.1). The fully repair-proficient strain AB1157 was the most resistant, with the repair-deficient strains showing increased sensitivities according to which repair pathway was missing. The results are in agreement with the data of Upton & Pinney (1983) and confirm the genotypes of the strains. R46 did not protect AB2494 lexA (Figure 4.1.2) or AB2463 recA (Figure 4.1.3) which are uninducible for SOS functions and in which, therefore, the plasmid- mediated muc gene activity will not normally be induced (Waleh & Stocker, 1979). In repeated experiments R46 did not protect JG138 polA (Figure 4.1.4) either, contrary to the results reported by MacPhee (1974) and Upton & Pinney (1981; 1983). However, R46 did protect the other, SOS-inducible, DNA repair-deficient strains (Figures 4.1.5 to 4.1.10). The plasmid conferred the greatest level of UV resistance on the umuC-deficient strains (Figures 4.1.6 and 4.1.10) where survival of the R46-containing derivatives was about 100-fold that of the R~ strain. This is in agreement with Upton & Pinney (1983). However, unlike Upton & Pinney (1983) who only claimed an increase of approximately 1 0 -fold in the survival conferred by R46 on uvr strains, R46 conferred an increase of nearly 100-fold on the uvrA (Figure 4.1.8) and uvrB (Figure 4.1.9) strains in these experiments.

RESULTS ÏÔ2 From the results in Figures 4.1.2 to 4.1.10 it is concluded that plasmid- mediated DNA repair was expressed in all but one of the strains (polA) in which it might be expected to function.

1 1 0 3 CO

0 40 80 120 160 UV dose (J m“^)

Figure 4.1.1. Post-UV survival of Escherichia co//AB1157 and the DNA repair-deficient strains. (Mean results of 2 to 5 experiments.) # = AB1157; ■ = TK702 umuC\ ▼ = AB2470 recB\ ▲ = JG138 po/A; □ = AB2494 !exA\ V = JC3890 uvrB\ O = TK501 uvrB umuC\ O = AB1886 uvrA\ A = AB2463 recA

RESULTS 103 OJ 03 o O

CO CO

0 4080 120 160 0 20 40 60 80 UV dose (J m 4 UV dose (J m i

Figure 4.1.6. Post-UV survival of E. coli Figure 4.1.7. Post-UV survival of E. coli TK702 umuC and umuC{R46). AB2470 recB and æcfî(R46). ■ = umuC\ □ = umuC{R4Q) ▼ = recB\ V = recB{R46)

10'--

o u

ca ca

CO CO

0 4 8 12 16 0 4 8 12 16 UV dose (J m- 2\ UV dose (J m"^

Figure 4.1.8. Post-UV survival of E. coli Figure 4.1.9. Post-UV survival of E. coli AB1886 uvrA and uvrA{R46). JC3890 uvrB and uvrB{R46). # = uvrA] O = uvrA{R46) T = uvrB] V = uvrB{R46)

RESULTS 105 suspensions of 0/N cultures (Experimental Methods 1 & 6 ). MICs of nitrofurantoin for fully sensitive species normally fall betv^een 0.4 pg mM and 10.0 pg ml (Therapeutic Drugs, 1991 ). The MIC for AB1157 determined in this experiment was 10.0 pg ml'\ with all the DNA repair-deficient strains giving lower values (Table 4.1.1). Organisms with MIC below 32 pg ml'^ are considered susceptible (Martindale, 1996).

Table 4.1.1. Minimum inhibitory concentrations of nitrofurantoin for E. coli AB1157 and DNA repair-deficient strains

Minimum inhibitory concentration (pg mM) E. coli strain Genotype R- R46

AB1157 wild type 1 0 .0 1 0 .0

TK702 umuC 7.5 7.5

JG138 polA 5.0 5.0

AB2470 recB 3.0 3.0

AB2494 lexA 2 .0 2 .0

AB1886 uvrA 1 .0 1.0

TK501 uvrB umuC 0.75 1.5

JC3890 uvrB 0.5 1 0 .0

AB2463 recA 0.3 0.3

The most sensitive strain was AB2463 recA, a result which contradicts that of Hamilton-Miller et ai. (1977) who found that the sensitivities to nitrofurantoin of

E. CO//AB1157 and AB2463 recA did not differ. R46 made no difference to the MIC of nitrofurantoin for the AB1157, umuC, polA, recB, /exA, uvrA or recA strains, but doubled the MIC of the TK501 uvrB umuC strain and, surprizingly, increased the MIC of the JC3890 uvrB strain 20-fold (Table 4.1.1).

1.5. Bactericidal profile of nitrofurantoin Determination of MIC is a relatively coarse method for the comparison of drug sensitivity. Death-time curves give a more definite impression of drug sensitivity. The antibacterial effects of nitrofurantoin against AB1157, the DNA

RESULTS ÏÔ7 repair-deficient strains and the R46-containing derivatives were therefore compared. To find the optimum concentration of nitrofurantoin to use, 0/N cultures of E co//AB1157 were diluted 1 in 100 into nutrient broth containing nitrofurantoin concentrations of 0.0 (control), 10 (MIC), 20 (2xMIC), 50 (5xMIC), 100 (lOxMIC) or 200 (20xMIC) pg ml'\ Survival was determined hourly on nutrient agar

(Experimental Methods 3 & 8 ). Figure 4.1.11 shows the survival of strain AB1157 at all six nitrofurantoin concentrations tested. It was concluded that 200 pg mM was the most suitable concentration of nitrofurantoin to determine whether R46 conferred protection. However, for some of the repair-deficient strains, 200 pg ml'^ produced a rate of death that was too high for accurate measurement; no viable cells were recovered after 30 minutes (data not shown). Thus, as 200 pg mM was equivalent to 20xMIC of nitrofurantoin for AB1157, the 20xMIC concentration for each strain was tested to see if it would be suitable to determine whether the plasmid conferred resistance.

0 1 2 3 4 5 6 Time of exposure (hours)

Figure 4.1.11. Bactericidal activity of nitrofurantoin on E. co//AB1157. ■ = 0.0 pg ml'\ A = 10 pg ml'\ O = 20 pg ml \ O = 50 pg ml'\ T = 100 pg ml \ # = 200 pg ml'^

RESULTS 108 1.6. Sensitivities of DNA repair-deficient strains to 20xMIC of nitrofurantoin Volumes of nutrient broth containing 20xMIC of nitrofurantoin for each strain (Table 4.1.2) were inoculated 1 in 100 with 0/N cultures of all nine E. coli strains and survival was determined on nutrient agar (Experimental Methods 3 & 8 ).

Table 4.1.2.20 x minimum inhibitory concentration of nitrofurantoin for E. coli AB1157, DNA repair-deficient strains and R46-containing derivatives

E. coli strain 20xMIC

AB1157 AB1157(R46) 2 0 0 pg mM

umuC umuC{R46) 150 pg mM

polA polA(RA6) 1 0 0 pg mM

recB recB{RA6) 60 pg mr^

lexA lexA{RA6) 40 pg ml'^

uvrA uvrA(RA6) 2 0 pg mM

uvrB umuC uvrB umuC{RA6) 15 pg mM

uvrB uvrB{RA6) 10 pg mM

recA recA(RA6) 6 pg ml"''

Figure 4.1.12 illustrates the loss of viability, over a sixty minute period, of the R~ strains exposed to 20xMIC of nitrofurantoin. Even taking into account their varying sensitivities to nitrofurantoin, it is still very apparent how sensitive some of the derivatives are. For example, the survival of strain AB1886 uvrA after 60 minutes in 20 pg mM of nitrofurantoin has been reduced to 0.001% (Figure 4.1.12) compared with 0.1% survival of the repair-proficient AB1157 strain after 4 hours in 200 pg mM (Figure 4.1.11). Figures 4.1.13 to 4.1.16 show that plasmid R46 did not confer nitrofurantoin resistance on the DNA repair-deficient strains /exA, recA, recB or polA. However, it did confer some protection to the fully DNA repair-proficient strain AB1157 and the DNA repair-deficient strains umuC, uvrA, uvrB and uvrB

umuC (Figures 4.1.17 to 4.1.21).

RESULTS 109 c CD O

CD CL

Time of exposure (minutes)

Figure 4.1.12. Survival of Escherichia coli AB1157 and DNA repair-deficient strains in 20xMIC of nitrofurantoin. ■ = TK702 umuC, # = AB1157; □ = AB2494 /exA; ▼ = AB2470 recB\ A = AB2463 recA] A = JG138 po!A\ O = TK501 uvrB umuC\ V = JC3890 uvrB\ O = AB1886 uvrA

RESULTS 110 (D S o

CO CO

10'-

0 15 30 45 60 0 15 30 45 60 Time of exposure (minutes) Time of exposure (minutes)

Figure 4.1.13. Survival of E. co//AB2494 Figure 4.1.14. Survival of E. co//AB2463 lexA and lexA{R46) in 20xMIC of recA and recA(R46) in 20xMIC of nitrofurantoin. ■ = lexA\ □ = lexA{R46) nitrofurantoin. A = recA; A = æcA{R46)

03 0) O o

CO C/D

10'-

0 15 30 45 60 Time of exposure (minutes) Time of exposure (minutes)

Figure 4.1.15. Survival of E. co//AB2470 Figure 4.1.16. Survival of E co//JG138 recB and recB(R46) in 20xMIC of polA and po/A(R46) in 20xMIC of nitrofurantoin. ▼ = recB] V recB{R46) nitrofurantoin. A = po/A; A = po/A(R46)

RESULTS 111 1001

90-- 9 5 -

§ 90 - 0 Q. 13 1 8 5 - co

50-- 8 0 -

Time of exposure (minutes) Time of exposure (minutes)

Figure 4.1.17. Survival of E. co//AB1157 Figure 4.1.18. Survival of E. co//TK702 and AB1157(R46) in 20xMIC of umuC and umuC{R46) in 20xMIC of nitrofurantoin. nitrofurantoin. # = AB1157; 0 = AB1157(R46) ■ = umuC\ □ = umuC{R4E)

10’- ^ 10'- o0)

Time of exposure (minutes) Time of exposure (minutes)

Figure 4.1.19. Survival of E. co//AB1886 Figure 4.1.20. Survival of E co//JC3890 uvrA and uvrA{R46) in 20xMIC of uvrB and uvrB{R46) in 20xMIC of nitrofurantoin. # = uvrA] O = uvrA{R4Q) nitrofurantoin. A = uvrB] V = uvrB{R46)

RESULTS 112 10’-

o> o m

Time of exposure (minutes)

Figure 4.1.21. Survival of E. co//TK501 uvrB umuC and uvrB umuC{R46) in 20xMIC of nitrofurantoin. # = uvrB umuC\ O = uvrB u/T7uC(R46)

1.7. Summary Table 4.1.3 summarizes the order of sensitivities of AB1157 and the DNA repair-deficient derivatives to the bactericidal activity of ultra-violet light (Figure 4.1.1) and to the inhibitory (Table 4.1.1) and lethal (Figure 4.1.12) activities of nitrofurantoin. Allowing for errors introduced by the various techniques, the orders of sensitivity are remarkably similar. The protective effect of plasmid R46 was not observed in the AB2494 /exA, AB2463 recA or JG138 po/A strains for both UV and nitrofurantoin. R46 protected AB2470 recB against UV but not against nitrofurantoin. The plasmid protected all other strains against both UV and nitrofurantoin.

RESULTS 113 Table 4.1.3. Order of sensitivities of E co//AB1157(R") and DNA repair-deficient (Rr) strains to UV light and nitrofurantoin

Exposed to MICs of 20xMIC of Sensitivity UV light on nitrofurantoin in nitrofurantoin in solid medium solid medium liquid medium

Least sensitive AB1157 AB1157 umuC

umuC umuC AB1157

recB polA lexA

polA recB recB

lexA lexA recA

uvrB uvrA polA

uvrB umuC uvrB umuC uvrB umuC

uvrA uvrB uvrB

Most sensitive recA recA uvrA

RESULTS 114 2. Protective or sensitizing effects, to the lethai activity of nitrofurantoin, of a variety of plasmids from different incompatibility groups that either protect against, or sensitize to, ultraviolet light

2.1. introduction The data in the previous chapter have shown plasmid R46 not to affect the MICs of nitrofurantoin for AB1157 and the majority of DNA repair-deficient strains tested. However, R46 was shown to confer significant protection against the lethal effect of nitrofurantoin to several DNA repair-deficient strains. Approximately one in three plasmids tested from different incompatibility groups either protect against or sensitize to UV (Upton & Pinney, 1983). Since pathogenic bacteria will be predominantly wild-type with respect to DNA repair, such protection might confer a level of resistance that is significant in the therapeutic treatment of disease by nitrofurantoin. A collection of plasmids from a variety of incompatibility groups, known to affect survival after exposure to irradiation or UV-induced mutagenesis (Babudri & Monti-Bragadin, 1977; Pinney, 1980; Upton and Pinney, 1983) were transferred into strain AB1157 to test whether they affected survival of the organism after exposure to lethal concentrations of nitrofurantoin. The plasmids investigated were R16, R46, R124, R144, R391, R446b, R621a, R805a, pGW12, pGWIG, pKMIOI and pYDI (Materials & Apparatus Table 2.2.1). The strains were also exposed to UV to check the post-UV phenotypes conferred by the plasmids.

2.2. Plasmid transfers Most of the plasmids were transferred into AB1157 thr leu his arg pro by conjugation (Experimental Methods 5) with R"" strains of E. coll 343/113 thy arg lys (Materials & Apparatus Table 2.2.1). Mating mixtures were plated on Davis-Mingioli selecting medium, supplemented for strain AB1157 and containing an antibiotic to which the plasmid conferred resistance. Where possible an alternative antibiotic was used to check that the entire plasmid had transferred (Materials & Apparatus Table 2.2.1). For those plasmids only conferring resistance to one antibiotic, a high frequency of transfer as compared to the low spontaneous mutation frequency to drug resistance was accepted as indicating that the entire plasmid had transferred. Plasmid R805a was only available in E. coil strain TK501 thr leu his arg pro. Since

RESULTS ÏÏ5 this strain has the same nutritional requirements as strain AB1157, the plasmid was firstly transferred into E. coli J62-2 Rif^ (Materials & Apparatus Table 2.1.1) and from there into E. co//AB1157. E. co//AB1157 containing plasmids pGW12, pGW16 and pYDI were received perse from Dr. R. J. Pinney and plasmid presence was checked using the appropriate antibiotic(s) (Materials & Apparatus Table 2.2.1). Significant, stable plasmid transfer occurred into all strains. Transfer frequencies per donor after 4 hours mating were in the range 1.1 x 10'^ for R144 to 3.3x10'® for R621a.

2.3. Effects of plasmids on post-UV survival ofEscherichia co//AB1157 All twelve plasmid-containing E. co//AB1157 derivatives and the plasmid-free parent strain were exposed to UV light to determine the effects of the plasmids on post-UV survival (Experimental Methods 4). Plasmids RIG, R46, R124, R144, R446b, R621a, R805a and pKMIOI conferred protection against UV to E. coli AB1157 (Figures 4.2.1 to 4.2.4, 4.2.6 to 4.2.8 and 4.2.11), plasmid R391 conferred sensitization (Figure 4.2.5) and pGWIG conferred sensitization at lower doses and protection at higher doses (Figure 4.2.10). These results agree with the data of Babudri & Monti-Bragadin (1977), Little et ai. (1991) and Pinney (1980). Plasmid pGW12 conferred sensitization (Figure 4.2.9) and pYDI made little difference to the post-UV survival of AB1157 (Figure 4.2.12). Previously it had been claimed that plasmid pGW12 had a null effect (Little et a/., 1991) and pYDI conferred sensitization to UV (Ambler & Pinney, 1995).

2.4. The effects of plasmids on the minimum inhibitory concentration of nitrofurantoin for Escherichia co//AB1157 The minimum inhibitory concentrations of nitrofurantoin for E. co//AB1157 and its twelve plasmid-containing derivatives were determined on nutrient agar containing increasing concentrations of nitrofurantoin (Experimental Methods 6 ). Plasmid R46 had already been shown to have no effect on the MIC of nitrofurantoin against AB1157 (Table 4.1.1) and none of the other eleven plasmids had any effect on the minimum inhibitory concentration of nitrofurantoin. In this set of experiments, E. co//AB1157(R") and all of its plasmid-containing derivatives gave MICs of 7.5 pg ml'^ against nitrofurantoin (results not shown).

RESULTS ÏÏ6 en CO

120 160 120 160 UV dose (J m"^ UV dose (J m"^)

Figure 4.2.1. R16 Figure 4.2.2. R46

CO CO

120 160 120 160 UV dose (J m‘ ^ UV dose (J m"^)

Figure 4.2.3. R124 Figure 4.2.4. R144

Figures 4.2.1,4.2.2,4.2.3 & 4.2.4. Post-UV survival of £ co//AB1157(R~) and AB1157(R^). • = AB1157(R-): 0 = AB1157(R")

RESULTS 117 CO

120 160 120 160 UV dose (J m"^ UV dose (J m"^)

Figure 4.2.5. R391 Figure 4.2.6. R446b

10° - -

CO co

120 160 120 160 UV dose (J m"^ UV dose (J m"^)

Figure 4.2.7. R621a Figure 4.2.8. R805a

Figures 4.2.5, 4.2.6, 4.2.7 & 4.2.8. Post-UV survival of E. co//AB1157(R“) and AB1157(R^). • = AB1157(R-); 0 = AB1157(R^

RESULTS 118 10°--

CO co

120 160 120 160 UV dose (J m"^ UV dose (J m"^)

Figure 4.2.9. pGW12 Figure 4.2.10. pGW16

10° - -

CO co

120 160 160120 UV dose (J m"^ UV dose (J m"2)

Figure 4.2.11. pKMIOI Figure 4.2.12. pYD1

Figures 4.2.9, 4.2.10, 4.2.11 & 4.2.12. Post-UV survival of E. coli AB1157(R“ ) and AB1157(R1. • = AB1157(R-): 0 = AB1157(R")

RESULTS 119 2.5. Survival of Escherichia coli AB1157, containing a range of different plasmids, in a bactericidal concentration of nitrofurantoin To determine the effect of plasmids on the survival of E. coli AB1157 in a bactericidal concentration of nitrofurantoin, volumes of nutrient broth containing 2 0 0 pg mM of nitrofurantoin, a concentration which had already been shown to be suitably bactericidal (Figure 4.1.11), were inoculated at dilutions of 1 in 100 with 0/N cultures of the test organisms and survival was determined on nutrient agar

(Experimental Methods 3 & 8 ). Some protection was conferred, against this bactericidal concentration of nitrofurantoin, upon E. co//AB1157 by plasmids R46, R446b, R621a, R805a and pKMIOI (Figures 4.2.14, 4.2.18, 4.2.19, 4.2.20 & 4.2.23), all of which also protected against UV light. Plasmids R144, R391, pGW12 and pGW16 (Figures 4.2.16, 4.2.17, 4.2.21, & 4.2.22) sensitized AB1157 to nitrofurantoin. Of these plasmids R391 (Figure 4.2.5) and pGW12 (Figure 4.2.9) sensitized to all doses of UV and plasmid pGWIS (Figure 4.2.10) sensitized to low doses but protected at higher doses of UV. However, R144 (Figure 4.2.4) protected against all UV doses tested. R16, R124 and pYDI (Figures 4.2.13, 4.2.15, & 4.2.24) neither protected AB1157 against nor sensitized it to nitrofurantoin, whereas R16 and R124 protected against UV light (Figures 4.2.1 & 4.2.3) and, in the current work, pYDI (Figure 4.2.12) was shown to have no effect on post-UV survival, whereas Ambler & Pinney (1995) claimed it to be UV-sensitizing.

RESULTS 120 10’ --

K 10°" -53 =

CO CO

300 time of exposure (minutes) time of exposure (minutes)

Figure 4.2.13. R16 Figure 4.2.14. R46

10’-

COCO

300 Time of exposure (mil Time of exposure (mil

F igure 4.2.16. R144Figure 4.2.15. R124 Figure 4.2.16. R144Figure

Figures 4.2.13, 4.2.14, 4.2.15 & 4.2.16. Effect of plasmids R16, R46, R124 and R144 on survival of E. co//AB1157 in 200 pg mM of nitrofurantoin in nutrient broth at 37°C. • = AB1157(R-): 0 = AB1157(R")

RESULTS 121 10'- 10’- (D

OO CO

300 Time of exposure (mil Time of exposure (minutes)

Figure 4.2.17. R391

10’- o;>

I 1 0 ° - 110 “ 13

CO CO

0 60 120 180 240 300 time of exposure (minutes) Time of exposure (minutes)

Figure 4.2.19. R621a Figure 4.2.20. R805a

Figures 4.2.17,4.2.18,4.2.19 & 4.2.20. Effect of plasmids R391, R446b, R621a and RBOSa on survival of E. co//AB1157 in 200 pg mM of nitrofurantoin in nutrient broth at 37°C. • = AB1157(R ); O = AB1157(R")

RESULTS 122 10'-

10“ - o> a>

time of exposure (minutes! time of exposure (minutes)

Figure 4.2.21. pGW12 Figure 4.2.22. pGW16

10'- 10'- o>

1 10“ - 15

GO OO

time of exposure (minutes) Time of exposure (mil

Figure 4.2.23. pKMIOI Figure 4.2.24. pYDI

Figures 4.2.21, 4.2.22, 4.2.23 & 4.2.24. Effect of plasmids pGW12, pGW16, pKMIOI and pYDI on survival of E. co//AB1157 in 200 pg ml ' of nitrofurantoin in nutrient broth at 37°C. e = AB1157(R-): O = AB1157(R1

RESULTS 123 2.6 Summary Table 4.2.1 summarizes whether the plasmids conferred protection against or sensitivity to UV light or nitrofurantoin on E. co//AB1157.

Table 4.2.1. Maximum protection against or sensitization to UV light or nitrofurantoin conferred by a variety of plasmids on E. co//AB1157 ^ = Survival of strain / Survival of R~ strain at stated UV dose (J m ^) or time of exposure (min)

Maximum^ protection against or sensitization to Plasmid UV light Nitrofurantoin

R16 Protection 8.1 (80) No significant difference

R46 Protection 10.4 (80) Protection 3.7 (180)

R124 Protection 3.5 (80) No significant difference

R144 Protection 9.5 (80) Sensitization 3.8 (135)

R391 Sensitization 5.2 (160) Sensitization 3.2 (135)

R446b Protection 11.0 (80) Protection 6.5 (270)

R621a Protection 4.3 (80) Protection 4.6 (270)

R805a Protection 4.5 (80) Protection 2.9 (270)

pGW12 Sensitization 53.0 (80) Sensitization 508 (225)

pG W ie Sensitization at low doses 3.2 (20) Sensitization 8.7 (135)

Protection at tiigh doses 3.8 (80)

pKMIOI Protection 12.3 (80) Protection 5.0 (270)

pYDI No significant difference No significant difference

The overall conclusion is that the levels of resistance or sensitivity conferred are not likely to be clinically significant, a conclusion also reached by Pinney ef a/. (1992) with regard to R46 and both nitrofurantoin and metronidazole.

RESULTS 124 3. Antibacterial action of nitrofurantoin against Escherichia coii when protein synthesis, RNA synthesis or cell division are inhibited

3.1. Introduction The emergence of quinolones as one of the leading groups of antlbacterlais of the late 2 0 th century has been made possible by chemical modification of the original nalidixic acid prototype discovered by Lescher and colleagues in 1962 (reviewed by Siporin, 1989). Nalidixic acid activity is limited to Gram-negative bacteria and its poor distribution within the body restricts its use to urinary tract and enteric infections, especially bacillary dysentery caused by Shigella species when other treatments have failed (Malengreau, 1984; McCormack, 1983; Panhotra & Desai, 1983; Parry, 1983). It took another decade to develop drugs chemically- related to nalidixic acid, such as acrosoxacin, cinoxacin, oxolinic acid, pipemidic acid and piromidic acid, but still they had poor pharmacokinetic profiles and limited antimicrobial activity. It was not until the 1980s that quinolones became fluorinated (fluoroquinolones). This structural change, together with other additional substituents, greatly increased the distribution, antibacterial spectrum, potency and oral bioavailability of these drugs. They now constitute a large, well-used group that includes amifloxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, pefloxacin, sparfloxacin and tosufloxacin (reviews by Siporin, 1989; Smith, 1984; von Rosenstiel & Adam, 1994). Nalidixic acid is only active against dividing bacteria undergoing RNA and protein syntheses (Deitz at ai, 1966; Gossat ai, 1964), a process described as Mechanism A by Smith (1984). However, the fluoroquinolones such as ciprofloxacin and ofloxacin, also possess Mechanism B, which is also active against non-dividing bacteria and does not require RNA or protein synthesis (Lewin at ai, 1991 ; Smith, 1984). Fluoroquinolones are also bactericidal to non-dividing cells by a mechanism that requires RNA and protein synthesis, a process described as Mechanism C (Ratcliffe & Smith, 1985). Like nalidixic acid, nitrofurantoin is only of therapeutic use in urinary tract infections (Martindale, 1996) and despite extensive chemical modification (Chamberlain, 1976) no effective systemic nitrofuran has been developed. Nitrofurans slumber on, to paraphrase Smith (1984). Quinolones target DNA as their site of action (Goss at ai, 1964) and induce DNA strand breakage (Fenwick & Curtis, 1973; Hill & Fangman, 1973). Similarly,

RESULTS nitrofurans bind to DNA and cause strand breakage (McCalla et a/., 1971, 1975; Touati at a/., 1989; Tu & McCalla, 1975; Wentzell & McCalla, 1980). However, unlike the quinolones, investigations of the mechanism of action of nitrofurans have not extended to a determination of the conditions necessary for their optimal activity. A series of experiments was therefore conducted to determine whether the inhibition of RNA or protein synthesis or cell division inhibited some or all of the activities of nitrofurantoin.

3.2. Antibacterial activity of nitrofurantoin in different liquid media and drug combinations Log phase cells (Experimental Methods 1 ) of the E. coli K12 strain AB1157, grown in nutrient broth, were diluted 1 in 1 0 0 into nutrient broth containing 2 0 0 pg mM of nitrofurantoin which was previously shown to be highly bactericidal (Results 1 Figure 4.1.11). Rifampicin at 160 pg ml''' and chloramphenicol at 20 pg ml'' (Smith, 1984) were used to inhibit RNA or protein synthesis, respectively. To obtain conditions in which cell division was inhibited but RNA and protein synthesis could continue, cultures were washed in phosphate buffered saline (PBS) and resuspended in PBS containing the required drug combinations. The log phase cells were diluted into nutrient broth or PBS containing different combinations of drugs (Table 4.3.1) at initial viable counts of 10® cells ml''. Survival was determined hourly by diluting in nutrient broth and plating on nutrient agar (Experimental Methods 3

& 8 ). Cells grew exponentially in nutrient broth (Figure 4.3.1 ) and neither grew nor died in PBS (Figure 4.3.2) over a six hour period. Controls showed that both chloramphenicol and rifampicin were bacteriostatic in nutrient broth (Figure 4.3.1), with chloramphenicol remaining bacteriostatic but rifampicin showing a slight lethal activity after 3 hours in PBS (Figure 4.3.2). The bactericidal activity of nitrofurantoin was similar in nutrient broth and PBS (compare Figures 4.3.1 and 4.3.2). The addition of chloramphenicol or rifampicin after two hours did not affect the bactericidal activity of nitrofurantoin in nutrient broth or PBS (Figures 4.3.3 and 4.3.4). Pre-exposure to rifampicin or chloramphenicol delayed the onset of nitrofurantoin-induced lethality by 1 or 2 hours respectively in nutrient broth (Figure 4.3.5). However, once death commenced, the rate was similar to that of the

RESULTS Ï26 nitrofurantoin control. Similarly in PBS, pre-exposure to chloramphenicol did not affect the bactericidal activity of nitrofurantoin whereas pre-exposure to rifampicin slightly increased the rate of kill of nitrofurantoin (Figure 4.3.6), possibly reflecting an additive effect with the slight bactericidal activity of this concentration of rifampicin in PBS (Figure 4.3.2). The presence of chloramphenicol or rifampicin together with nitrofurantoin from the start of the experiment made little difference to the lethal effect of nitrofurantoin in either nutrient broth or PBS (Figures 4.3.7 and 4.3.8).

10° - -

Time of exposure (hours) Time of exposure (hours)

Figure 4.3.1. NB Figure 4.3.2. PBS

Figures 4.3.1 & 4.3.2. Effect of chloramphenicol (□) or rifampicin (O) or nitrofurantoin (V) on cells incubated in nutrient broth (NB) or phosphate buffered saline (PBS). A = no drugs present

RESULTS 127 10’--

10° - - Î 10°- co co

Addition of Cm or Rf

1- 1 -- Addition of Cm or Rf

Time of exposure (hours) Time of exposure (hours)

Figure 4.3.3. NB Figure 4.3.4. PBS

Figures 4.3.3 & 4.3.4. Effect on the bactericidal activity of nitrofurantoin in nutrient broth (NB) or phosphate buffered saline (PBS) when chloramphenicol (□) or rifampicin (O) were added 2 hours after the start of the experiment. V = only nitrofurantoin present

Addition , of Nf Addition of Nf 10’-- 10’--

Time of exposure (hours) Time of exposure (hours)

Figure 4.3.5. NB Figure 4.3.6. PBS Figures 4.3.5 & 4.3.6. Effect of pre-exposure to chloramphenicol (□) or rifampicin (O), for 2 hours, on the bactericidal activity of nitrofurantoin in nutrient broth (NB) or phosphate buffered saline (PBS). V = only nitrofurantoin present

RESULTS 128 CO

1- 1 -

Time of exposure (hours) Time of exposure (hours)

Figure 4.3.7. NB Figure 4.3.8. PBS

Figures 4.3.7 & 4.3.8. Effect of chloramphenicol (□) or rifampicin (O) when present with nitrofurantoin in nutrient broth (NB) or phosphate buffered saline (PBS) from the start of incubation. V = only nitrofurantoin present

3.3. Summary These results contrast to those obtained with quinolones. Quinolone mechanism A is abolished in phosphate buffered saline whereas nitrofurantoin is as active in phosphate buffered saline as in nutrient broth. Quinolone mechanisms A and C are abolished by chloramphenicol or rifampicin whereas nitrofurantoin activity is unaffected by the copresence of chloramphenicol or rifampicin at the start of exposure to nitrofurantoin. Similarly, although delayed by pre-exposure to chloramphenicol or rifampicin in nutrient broth, nitrofurantoin lethality then proceeds at the same rate as in the control. Thus, unlike the quinolones, the bactericidal activity of nitrofurantoin is independent of RNA and protein syntheses and of cell division.

RESULTS 129 4. Plasmid stability to nitrofurantoin inEscherichia co/i AB1157

4.1. Introduction Plasmids can be eliminated from their bacterial hosts by growth in medium containing acridines (Hahn & Ciak, 1976; Salisbury et ai, 1972), sodium dodecyl sulphate (Tomoeda at si, 1968) or ethidium bromide (Bouanchaud at ai, 1968) and lost from laboratory strains not maintained on appropriate selective media (Dodd & Bennett, 1986; Godwin & Slater, 1979). Several therapeutically-used agents, which damage DNA or interfere with DNA synthesis, have also been found to cause elimination of plasmids (Bazzicalupo & Tocchini-Valentini, 1972; Hahn & Ciak, 1976; Hooper ef a/., 1984; McHugh & Swartz, 1977; Pinney & Smith, 1972b). E. coli strain AB1157 containing representative plasmids from a variety of incompatibility groups were examined to determine whether nitrofurantoin is a plasmid-eliminating drug. Methods were used which tested whether plasmids were eliminated from cells dying in broth containing lethal concentrations of nitrofurantoin or from cells growing in broth containing sub-inhibitory concentrations.

4.2. Elimination of plasmids from ceiis exposed to a lethai concentration of nitrofurantoin ‘Classic’ plasmid-curing techniques usually employ sub-inhibitory concentrations of the eliminating agent. However, it has also been shown that plasmids can be eliminated from cells exposed to bactericidal drug concentrations (McHugh & Swartz, 1977). 0/N cultures of E. co//AB1157 and twelve plasmid- containing derivatives, grown in nutrient broth, were diluted 1 in 1 0 0 into nutrient broth containing 0 .0 or 1 0 0 pg mM of nitrofurantoin, a concentration previously shown to be lethal (Results 1 Figure 4.1.11). Viable counts were made on MacConkey agar immediately and after 2 hours incubation. Approximately 1 to 2 % of all derivatives survived after this time (data not shown). Plasmid elimination was determined by replica-plating the colonies growing on the 2 hour plates, using a sterile, velvet pad, onto nutrient agar and onto nutrient agar containing an antibiotic to which the plasmid conferred resistance (Experimental Methods 10 & 11 ). Controls showed that replicated colonies of the and R~ strains of known resistance phenotype did, or did not grow, respectively, on the antibiotic-containing media. Plasmid elimination, determined as the number of colonies unable to grow on

RESULTS Ï3Ô antibiotic-containing agar, was expressed as a percentage of the total number of colonies growing on drug-free nutrient agar (Table 4.4.1).

Table 4.4.1. Plasmid elimination from E. co//AB1157 derivatives exposed for 2 hours to 0.0 or 100 pg ml^ of nitrofurantoin in nutrient broth ISO = isosensitest agar; subscript refers to concentration of antibiotic used (pg mM)

Elimination frequency Incompatibility (Number of colonies tested) Plasmid Antibiotic group 0 .0 pg ml’"' 1 0 0 pg mM

APao Kmzo R- Not applicable <0.99(101) <0.12(862) TC20 Tmgo ISO

R16 B AP20 0.63(158) <0.08(1186)

R124 FIV TC20 <0.42 (236) <0.07 (1380)

R144 la TC20 <0.49 (206) <0.06(1594)

R621a lY TC20 <0.40 (248) <0.10(1048)

R805a IC Kmzo 4.35 (253) 0.32(1241)

R391 J Kmzo <0.74(135) <0.07 (1350)

R446b M TC20 <0.81 (123) 0.17(1153)

R46 N TC20 <0.74(136) 49.44(1809)

pYDI Unknown Tmgo ISO <0.65(153) <0.09(1090)

Comparing the plasmid elimination frequencies obtained in 100 pg mM of nitrofurantoin with those obtained in drug-free broth, and accounting for the greater number of colonies tested from cells exposed to 1 0 0 pg mM of nitrofurantoin, it can be seen that a bactericidal concentration of nitrofurantoin eliminated approximately 50% of R46 from E. coli AB1157 but did not affect the stability of any of the other plasmids.

RESULTS 131 4.3. Elimination of plasmids from cells exposed to a sub-inhibitory concentration of nitrofurantoin 0/N cultures of E co//AB1157 and its plasmid-containing derivatives, grown in nutrient broth, were diluted 1 in 100 into nutrient broth containing 0.0 or 7.5 pg ml’’' of nitrofurantoin and incubated at 37°C. This concentration was previously shown to be sub-inhibitory (Results 1 Table 4.1.1). The approximate times at which the cultures became visibly turbid were 18 and 100 hours respectively. Plasmid elimination was determined by diluting the turbid culture and plating onto drug-free MacConkey agar to give approximately 100 viable cells per plate. These master plates were incubated overnight and the resulting colonies replica-plated (Experimental Methods 10 & 11) onto nutrient agar containing an antibiotic to which the plasmid conferred resistance. Once again controls showed that replicated colonies of the R* and R" strains of known resistance phenotype did or did not grow, respectively, on the antibiotic-containing media. The plasmid elimination frequencies are shown in Table 4.4.2. All plasmids were stable in cultures grown in drug-free nutrient broth (Table 4.4.2) and no significant evidence of elimination was obtained from cultures grown to turbidity in 7.5 pg mM of nitrofurantoin (Table 4.4.2).

4.4. Summary A lethal concentration of nitrofurantoin eliminated the N incompatibility group plasmid R46 from E. co//AB1157. However, the plasmid was stable to growth in a sub-inhibitory concentration of nitrofurantoin. Representative plasmids from seven other incompatibility groups were stable to both bactericidal or subinhibitory concentrations of nitrofurantoin.

RESULTS 132 Table 4.4.2. Plasmid elimination from E. co//AB1157 derivatives exposed to 0.0 or 7.5 pg mM of nitrofurantoin in nutrient broth for 18 or 100 hours respectively ISO = isosensitest agar used; subscript refers to concentration of antibiotic used (pg ml ^)

Elimination frequency Incompatibility (Number of colonies tested) Plasmid Antibiotic group 0 .0 pg mM 7.5 pg mM

A P io Ap20 R- Not applicable Km zo <0.22 (445) <0.64(156) TC20 Tm go ISO

R16 B AP 20 <0.24 (412) <0.88(113)

R124 FIV TC20 <0.31 (326) <0.55(182)

R144 let TC20 <0.71 (141) <1.01 (99)

R621a lY TC20 <0.24 (422) <0.48 (208)

R805a IC Km2o <0.24 (424) 0.26 (380)

R391 J K m jo <0.27 (442) 0.54(185)

R446b M TC20 <0.27 (377) 0.51 (198)

R46 N TC20 <0.28 (362) <0.36 (280)

pYD1 Unknown Tmgo ISO <0.27 (369) <0.57(174)

RESULTS 133 5. Mutation of Escherichia coli to nitrofurantoin resistance

5.1. Introduction Mutations occur spontaneously or can be induced. Spontaneous mutations originate as a result of normal cellular processes or random interactions with the environment (Lewin, 1994). Organisms that have spontaneously mutated and become resistant to a particular drug will grow on media containing concentrations of that drug above the MIC of the wild-type strain. Such mutants can therefore be identified and isolated. Plasmid R46 carries mutator mucAB genes (Molina etal., 1979; Perry & Walker, 1982) which increase the mutation frequency of cells harbouring R46 (Babudri & Monti-Bragadin, 1977; Mortelmans & Stocker, 1976) and could therefore increase the possibility of cells mutating to drug resistance. The frequency of spontaneous mutation of E. co//AB1157 to nitrofurantoin resistance was determined in the absence and presence of plasmid R46. Mutation is increased by exposure to mutagens, compounds that induce mutation. Nitrofurantoin is a mutagen (McCann et a/., 1975) and therefore the hypothesis, that exposure to nitrofurantoin would increase the frequency of nitrofurantoin resistance in exposed cultures, was tested. The resistant organisms could be identified because they would grow on media containing nitrofurantoin concentrations above the MIC of the wild-type (unmutated) strain. As the presence of R46 increases host cell mutagenesis following exposure to DNA-damaging agents (Drabble & Stocker, 1968; Mortelmans & Stocker, 1976; Pinney, 1980) cultures harbouring R46 were also tested.

5.2. Confirmation of the R46 mutator effect The mutator function of plasmid R46 was confirmed by assessing its effect on the frequency of spontaneous mutation to nalidixic acid resistance. A method similar to that of Ambler et al. (1993) was used. Viable counts of concentrated (ION), 0/N cultures (Experimental Methods 2) of E. coli AB1157 and AB1157(R46) were determined on nutrient agar and on nutrient agar containing 15 pg mM (5xMIC) of nalidixic acid (Experimental Methods 3 & 9). Mutation frequencies to nalidixic acid were expressed per viable cell plated (Table 4.5.1).

RESULTS 134 Table 4.5.1. Mutation frequencies to nalidixic acid resistance (5xMIC)

E. coli strain iviuiaiion Trequency oeiermineo oy AB1157 AB1157(R46)

This research 2.8 X 10-® 1.1 X 10-^

Ambler ef a/., 1993 2.0 X 10-® 9.9x10®

The frequencies of mutation to nalidixic acid resistance were very similar to those determined by Ambler etal. (1993). R46 increased the mutation frequency 4- fold (Table 4.5.1) compared to a reported 5-fold increase by Ambler etal. (1993). Since increases of 1.5-fold or greater are taken as being indicative of a positive mutator plasmid effect (Pinney, 1980), these data confirm that the mutator genes carried by R46 are present and functioning.

5.3. Spontaneous mutation of Escherichia coli to nitrofurantoin resistance Again, a method similar to that of Ambler et al. (1993) was used. Concentrated (ICON) suspensions (Experimental Methods 2) of E. coli AB1157 and AB1157(R46) were diluted and spread on nutrient agar containing nitrofurantoin at concentrations of 1 0 , 20, 50 and 100 pg ml'\ the MIC having been determined as 10 pg mM for both E. CO//AB1157 and AB 1157(R46) (Results 1 Table 4.1.1). Viable counts were also determined on drug-free nutrient agar (Experimental Methods 3). Mutation frequencies to nitrofurantoin resistance were calculated per viable cell plated (Table 4.5.2).

Table 4.5.2. Spontaneous mutation frequencies of E. co//AB1157 and AB1157(R46) to nitrofurantoin resistance determined on solid media

Mutation frequencies per viable cell E.coli strain plated at nitrofurantoin concentration AB1157 AB1157(R46)

10 pg mM Confluent growth Confluent growth

2 0 pg mM 1.3x10® 2 .0 x 1 0 ®

50 pg m|-^ 5.8x10-''° 1.3x10-°

1 0 0 pg m|-^ No growth No growth

RESULTS 135 Mutation frequencies could only be determined at nitrofurantoin concentrations of 20 and 50 pg ml'\ No resistant colonies were isolated at 100 pg ml'\ whereas confluent growth was obtained at 10 pg ml'\ The presence of R46 increased the mutation frequencies 1.5- and 2.2-fold at 20 and 50 pg mM of nitrofurantoin respectively (Table 4.5.2). Since, as previously mentioned, a 1.5-fold increase in mutation frequency is indicative of a positive mutator plasmid effect (Pinney, 1980), it may be concluded that the presence of R46 has a small effect on spontaneous mutation to nitrofurantoin resistance. Colonies isolated on nutrient agar containing 20 or 50 pg ml'^ of nitrofurantoin were then streaked onto plates containing a variety of concentrations of nitrofurantoin to confirm nitrofurantoin resistance phenotype and onto MacConkey agar to confirm they retained the AB1157 colonial phenotype (Table 4.5.3). All restreaked colonies were confirmed as AB1157.

Table 4.5.3. Number of E. coli AB1157 and AB1157(R46) colonies maintaining resistance to nitrofurantoin, nt = not tested

Colony isolated Number of colonies growing on E. coli Number on NA NA containing nitrofurantoin at AB1157 of containing strain colonies nitrofurantoin at 20 pg mM 50 pg mM 100 pg ml'^

R- 25 25 1 0 2 0 pg m r R46 24 24 5 0

R- 7 nt 4 0 50 pg ml''' R46 7 nt 4 1

All colonies of AB1157 and AB1157(R46) isolated on 20 pg ml'^ retained resistance to this concentration on restreaking, with 1 colony (4%) and 5 colonies (21%) respectively growing on 50 pg mM. When restreaked on 100 pg mM no colonies were found to retain resistance. Of 7 colonies isolated on 50 pg ml'\ 4 colonies (57%) of both AB1157 and AB1157(R46) retained resistance to this concentration on restreaking but only 1 colony (14%) of AB1157(R46) retained

resistance to 10 0 pg.

RESULTS 136 5.4. Mutation to nitrofurantoin resistance in nitrofurantoin-exposed cultures of Escherichia coii

0/N cultures of E. coli AB1157 and AB1157(R46) were diluted 1 in 100 into nutrient broth containing 0 .0 or 2 0 pg mM of nitrofurantoin to give a final volume of 150 ml. The mixtures were incubated in a static water bath at 37°C and hourly samples were diluted in nutrient broth and plated on nutrient agar to determine viable counts (Experimental Methods 3). The number of mutants was determined by concentrating samples by centrifugation and resuspending them in Davis- Mingioli minimal salts solution to one hundredth of the sample volume and plating onto nutrient agar containing 20 pg ml'^ of nitrofurantoin (Experimental Methods 9). The number of mutants was scored after three days incubation. Mutation frequencies of cultures in drug-free medium are an expression of spontaneous mutation. Mutation in drug-exposed cultures is expressed as ‘apparent mutation frequencies’ since it represents the proportion of resistant mutants per viable cell in a dying population (Ambler & Pinney, 1995). As expected, cells of both E. coli AB1157 and AB1157(R46) grew in the drug- free broth and the viable count decreased with time in broth containing 2 0 pg ml'"' of nitrofurantoin (Figure 4.5.1). Mutants were observed for both AB1157 and AB1157(R46) but only in the drug-free broth. The absence of mutants in broth containing 2 0 pg ml'^ of nitrofurantoin was probably due to susceptible cells dying more quickly than resistant cells were evolving. Mutation frequencies (Figure 4.5.2) for E. coli AB1157 rose from 1.6x10'^ after incubation for 1 hour to 1.5 x 10'® after 3 hours and then dropped to 3.7 x 10'^ after 5 hours. AB1157(R46) followed a similar pattern rising from 3.6 x 10"® after 1 hour to 6.0 x 10'® after 2 hours and decreasing to 4.6 x 10'® after 5 hours. The survival (compare Figures 4.1.17 and 4.5.1) and mutation frequencies (Figure 4.5.2) were unexpectedly higher for AB1157 than AB1157(R46).

RESULTS 137 10‘ H

1^0-7

CD 10'

CO 4= # IQ-

10- 9 1 2 3 4 5 Time of exposure (hoursi Time of exposure (hours)

Figure 4.5.1. Survival Figure 4.5.2. Mutation frequencies

Figures 4.5.1 & 4.5.2. Survival and mutation frequencies of E. coli AB1157 and AB1157{R46) in 0.0 or 20 pig mT^ of nitrofurantoin (I). = AB1157 at 0.0 pig ml '; □ = AB1157(R46) at 0.0 pig ml ' • = AB1157 at 20 m Q ml '; ■ = AB1157(R46) at 20 pig ml '

Since the higher the total cell count, the easier it is to detect mutants, the original method was modified in an attempt to increase both the growth rate and the final stationary phase concentration of cells. 0/N cultures were inoculated into nutrient broth which was drug-free or contained 20 pg ml''' of nitrofurantoin and treated as described above except that the mixtures were incubated in an orbital shaker. Samples were taken hourly for seven hours and then a sample was taken twenty-four hours after inoculation of the broths. Survival and mutation frequencies were calculated and are shown in Figures 4.5.3 & 4.5.4. Growth in drug-free nutrient broth was increased significantly by shaking (compare Figure 4.5.1 with 4.5.3) but the mutation frequencies were not (compare Figure 4.5.2 with 4.5.4). Cell death in nitrofurantoin was also increased by shaking (compare Figure 4.5.1 with 4.5.3) but nitrofurantoin resistant AB1157 mutants were not detected until 24 hours after inoculation of the broth. At this time the mutation

RESULTS 138 frequency for AB1157 was 9.13 x 10''' (Figure 4.5.4). As mutants were observed after twenty-four hours it is possible that any mutants present before this time were so low in number that they were below the limit of detection for the method used. No mutants of AB1157(R46) were detected at any time despite this strain having similar growth and death kinetics to the plasmidless AB1157 parent (Figure 4.5.3).

sio-

10’-

10-' 0 1 2 3 4 5 6 7 Time of exposure (hours) Time of exposure (hours)

Figure 4.5.3. Survival Figure 4.5.4. Mutation frequencies

Figures 4.5.3 & 4.5.4. Survival and mutation frequencies of E. coli AB1157 and AB1157(R46) in 0.0 or 20 pg ml ’ of nitrofurantoin (II). = AB1157 at 0.0 pg ml ’; □ = AB1157(R46) at 0.0 pg ml ’ # = AB1157 at 20 pg ml ’; ■ = AB1157(R46) at 20 pg ml ’

5.5. Summary The overall conclusion is that exposure to nitrofurantoin does not induce mutation to nitrofurantoin resistance in E. coli AB1157. Similarly, plasmid R46 does not significantly increase mutation to nitrofurantoin resistance in either control cells or in bacteria exposed to nitrofurantoin.

RESULTS 139 6. Preparation of novel nitrofuran compounds Nine novel nitrofuran compounds were prepared as described in Experimental Methods 14.1-14.9. All materials used are described in Materials and

Apparatus 6 & 8 .

6.1. Preparation of bis-5-nitrofurfurylidenylputrescine (1)

Figure 4.6.1. Bis-5-nitrofurfurylidenylputrescine

5-Nitrofurfuraldehyde diacetate (24.32 g, 0.10 mol) and putrescine (5.29 g, 0.060 mol), each dissolved in ethanol (30 ml), were reacted in the presence of 10% sulphuric acid (30 ml) as described in Methods 22.3. The solvent was evaporated and the resultant tar-like solid dried in a vacuum desiccator. This solid was dissolved in water, made basic (pH >7 and <9) and extracted into ethyl acetate (4 X 40 ml). The solvent was evaporated leaving a tarry, crystalline product which was triturated in methanol and filtered to yield a brown crystalline powder.

5 (80 MHz; CDCyDMSO-cfe) 8 .2 0 (s, CH=N), 7.59 (d, H;), 7.03 (d, H 3'), 3.91- 3.55 (b, CHz), 1.875-1.61 (b, CHg) (KBr disc) 1640 and 1480 (CH=N), 1350 (NO,) cm"' m/z(E.I.) 194(02NC4H20CHN(CH)2CHCH2), 181 ( 0 2 NC4H2 0 CHN(CH2)3),

153 (O2NC4H2OCHNCH2), 139(02NC4H20CHN), 84 (N(CH 2)4N), 70 ((CH 2)4N)

6.2. Preparation of bls-5-nltrofurfurylidenylspertnlne (n)

|_ j

Figure 4.6.2. Bis-5-nitrofurfurylidenylspermine

RESULTS Ï4Ô Heptaldehyde (1.13 g, 9.40 mmol) in isopropanol (1.5 ml) was added to spermine (1.00 g, 4.79 mmol) in water acidified with concentrated hydrochloric acid (4.5 ml) as described in Methods 22.4. The mixture was stirred for 5 hours at room temperature and then added to 5-nitrofuraldoxime (1.40 g, 8.89 mmol) together with concentrated sulphuric acid (3.5 ml) and stirred over a steam bath for 114 hours (Methods 22.4). The resultant bis-5-nitrofurfurylidenylspermine was filtered, washed with water and dried.

5 (250 MHz; DMSO-dg) 7.75 (s, CH=N), 7.74 (d, H/; J 4.085 Hz), 7.40 (d, H 3'; J 3.655 Hz), 3.40 (b, CH2) m/z (E.l.) 264 (0 2 NC4H2 0 CHN(CH2)3NH(CH2)3CN),

252 (02NC4H20CHN(CH2)3NH(CH2)4), 237 (02NC4H20CHN(CH2)3NHCH2CHCH2),

222 (02NC4H20CHN(CH2)3NCHCH2), 210 (02NC4H20CHN(CH2)3NHCH2),

180 (O2NC4H2OCHNCH2CHCH2), 153 (O2NC4H2OCHNCH2), 124 (O2NC4H2OC)

6.3. Preparation of bis-5-nitrofurfurylidenylspermldine (m )

. 0 O2 o N . O NO,

Figure 4.6.3. Bis-5-nitrofurfurylidenylspermidine

5-Nitrofurfuraldehyde (1.39 g, 9.85 mmol) and spermidine (1.00 g, 6 .8 8 mmol) were reacted as described in Methods 22.1. The solid was recovered after evaporation under reduced pressure at 45°C. As NMR of this product was not possible, due to its poor solubility in the commonly used solvents, and the mass spectrum gave few results, this product was accepted as being bis-5-nitrofurfurylidenylspermidine on the basis that its method of manufacture was the same as for compound I which gave bis-5- nitrofurfurylidenylputrescine.

m/z (E.l.) 182 ( 0 2 NC4H2 0 CHN(CH2)2CH3), 153 (O2NC4H2OCHNCH2)

RESULTS Î4T 6.4. Preparation of 4-(N-5>nitrofurfurylidene)amlnobenzoyl-2-acetylamlno- ethylamlde (IV)

^ ^—CH —N—^ ^—CO~NH—CH^CH^NH—CO —CH3

Figure 4.6.4. 4-(N-5-nitrofurfurylidenyl)aminobenzoyl-2-acetylaminoethylamide

5-Nitrofurfuraldehyde (4.01 g, 28.41 mmol) and 4-aminobenzoic acid (3.84 g, 28.00 mmol) were reacted in 96% ethanol as described in Methods 22.7 to give 5-nitrofurfurylidenyl-4-aminobenzoic acid. To 1 g (3.85 mmol) of the filtered precipitate suspended in 25 ml of toluene was added half of a molar equivalent (0.24 g, 1.92 mmol) of oxalyl chloride. This was heated gently at 50°C on a hotplate. 0.25 ml of pyridine (3.16 mmol) and N-acetylethylenediamine (0.40 g, 3.92 mmol) were added to the stirred mixture. The toluene layer was separated and the product extracted and dried as described in Methods 22.7.

5 (250 MHz; CDCy 9.85 (s, CH=N), 8.05 (d, H/), 7.75 (d, H 3'), 7.5-7 4 (m, 2HJ,

7.4-7.3 (m, 2HJ, 7.3-7 2 (m, 2NH), 3.65-3.45 (m, CHg), 2.02 (s, CH 3) m/z (E.l.) 259 (O2NC4H2OCHNC6H4CONH2), 177 (O 2NC4H2OCHNC3H2), 139 (O2NC4H2OCHN), 125 (O2NC4H2OCH), 112(02NC4H20),

84 (CH 3CONCHCH2)

6.5. Preparation of bis-5-nitrofurfurylidenylmethyllysine (V)

CO2CH 3 O.N.

Figure 4.6.5. Bis-5-nitrofurfurylidenylmethyllysine

5-Nitrofurfuraldehyde diacetate (9.73 g, 0.040 mol) and L-lysine methyl ester dihydrochloride (5.25 g, 0.023 mol), dissolved in ethanol (40 ml), were reacted in the presence of 10% sulphuric acid (20 ml) as described in Methods 22.2. The

RESULTS Ï42 solvent was evaporated and the resultant solid dissolved in water (40 ml), made basic (pH9) and extracted into ethyl acetate (3 x 40 ml). Evaporation of the solvent yielded a crystalline product.

5 (80 MHz; DgO) 7.57 (s, CH=N), 7.46 (s, H/), 6.75 (s, Hg'), 5.09 (s. CH), 4.48

(s, COOCH3), 3.09-2.79 (b, CHg), 2.125-1.14 (b, CHg) v^^ (KBr disc) 1 7 5 0 (0 0 0 ), 1530 (CH=N), 1480 and 1360 (NO 2) cm '

6.6. Preparation of 5-nitrofurfurylidenylglucosamine (VI)

I— OH p —\ HO HO OH = H

Figure 4.6.6. 5-Nitrofurfurylidenylglucosamine

Glucosamine hydrochloride (1.85 g, 8.42 mmol) in 96% ethanol (10.0 ml) and triethylamine (0.97 ml, 9.49 mmol) and 5-nitrofurfuraldehyde (1.54 g, 10.80 mmol) in 96% ethanol (10.0 ml) were reacted as described in Methods 22.1. The resultant solid was filtered and dried.

5 (250 MHz; DMSO-dg) 9.26 (s, CH=N), 6.39 (d, H J, 5.59 (d, H3'), 4.97 (m, HJ, 4.84 (m, Hb), 3.64-3.49 (b, 5H) m/z (E.l.) 231 (CeHiiOsNCHCOCH), 191 (GeHi^OgNCHs), 165 (CgHiiOg 2H+),

137 (O 2NC4OCHN)

6.7. Preparation of acetylated 5-nitrofurfurylidenylglucosamine (VU) A portion of the filtered solid (VI) was acetylated, as described in Methods 2.15, by dissolving in pyridine (10.0 ml) and adding 4 equivalents of acetic anhydride (2.63 ml, 25 mmol). The acetylated solid was filtered, washed with water and dried.

RESULTS 143 — OAc

OAc OAc O^N

Figure 4.6.7. Acetylated 5-nitrofurfurylidenylglucosannine

5 (250 MHz; DMSO-dg) 9.34 (s, CH=N), 7.71 (d, H J, 7.31 (d, Hg'), 5.55-5.40

(t, HJ. 5.05-4.90 (t, Hb), 4.30-4.15 (m, 5H), 3.45-3.10 (b. 4 COCH3)

6.8. Preparation of N-acetyl-N-(5-nitrofurfurylethenylidenyl)ethylenedlamlne (vm)

O 2N. .0^ ^CH,

Figure 4.6.8. N-Acetyl-N-(5-nitrofurfurylethenylidenyl)ethylenediamine

5-Nitrofuranacrolein (1.0 g, 6.98 mmol) suspended in ethanol was added to N-acetylethylenediamine (0.62 g, 6.03 mmol) also dissolved in ethanol and reacted as described in Methods 22.1. The solvent was evaporated leaving a solid which was dried in a vacuum desiccator before being dissolved in water (80 ml) and 1 M hydrochloric acid (5 ml). The aqueous phase was made basic and extracted with chloroform (2 x 25 ml). The solvent was evaporated and the product dried as described in Methods 22.1.

5 (250 MHz; C D C y 9.02 (d, CH=N), 7.1 (d, HV), 7.03 (d, -C H = ), 6.74 (d, H 3'),

6.10-6.05 (m, = C H -), 2.6 (d, CHg), 2.2 (d. CH^), 1.85 (s, COCH3)

RESULTS 144 6.9. Preparation of bls-5-nltrofurfurylldenylethylspermidine (IX)

Figure 4.6.9. Bis-5-nitrofurfurylidenylethylspermidine

5-Nitrofuranacrolein (1.58 g, 9.25 mmol) in 96% ethanol (15.0 ml) and spermidine (0.57 g, 3.61 mmol) in 96% ethanol (5.0 ml) were reacted as described in Methods 22.1. The resultant tarry mass was triturated in 80 ml of water and 5 ml of concentrated hydrochloric acid and filtered. The aqueous phase was made basic and extracted at pH7 and pH 10 with 2x25 ml portions of ethyl acetate as described in Methods 22.1. The organic solvent was evaporated under reduced pressure at 45°C and bis-5-nitrofurfurylidenylethylspermidine was dried.

Ô (250 MHz: DMSO-de) 8.10 (s, NH), 7.65 (m, CH=N), 7.30 (d, HV), 6.95 (d, HV), 6.85 (s, CH=CH), 4.20-4.10 (m, CHg)

6.10. Summary Nine compounds where therefore prepared for testing; four were polyamine conjugates (I, II, III and IX), one was an aminobenzoyl derivative (IV), one an amino acid derivative (V), two were glucosamine conjugates (VI and VII) and one was an acetylamine derivative (VIII).

RESULTS 145 7. Antimicrobial activities of novel nitrofuran compounds

7.1. introduction Nitrofurantoin is bactericidal to a variety of Gram-positive organisms such as enterococci and staphylococci and to most strains of the Gram-negative organism E. coli. However, Enterobacter and Klebsiella species are less susceptible and Pseudomonas aeruginosa is usually resistant (Martindale, 1996). Also, nitrofurantoin exhibits negligible antifungal activity (Paul & Paul, 1964). Thus, in order to test the antimicrobial effectiveness of the nine novel nitrofuran compounds (Figure 4.7.1), a collection of organisms was used to include those with known susceptibility or resistance to nitrofurantoin. The collection comprised of the Gram- positive bacteria Bacillus subtilis and Staphylococcus aureus, the Gram-negative bacteria E. coli, Klebsiella aerogenes. Pseudomonas aeruginosa and Salmonella typhimurium and the fungi Candida albicans and Saccharomyces cerevisiae (Materials & Apparatus Table 2.1.4). The antimicrobial activities were assessed by determination of MICs on solid nutrient medium (Experimental Methods 6 ) and/or by the agar diffusion technique (Experimental Methods 7). Quantities of some of the novel compounds were small and therefore it was not possible to screen all of them by both methods against each test organism.

7.2. Minimum inhibitory concentrations MICs of the novel nitrofuran compounds I (bis-5-nitrofurfurylidenylputrescine), II (bis-5-nitrofurfurylidenylspermine). III (bis-5-nitrofurfurylidenylspermidine), IV (4-(N-5-nitrofurfurylidene)aminobenzoyl-2-acetylaminoethylamide), V (bis-5-nitrofurfurylidenylmethyllysine), VI (5-nitrofurfurylidenylglucosamine), VII (acetylated 5-nitrofurfurylidenylglucosamine), V III (N-acetyl-N-(5-nitrofurfurylethenylidenyl)ethylenediamine), EX (bis-5-nitrofurfurylethenylidenylspermidine) and their parent compounds X (5-nitrofurfuraldehyde), XI (5-nitrofuranacrolein) and X II (5-nitrofuraldoxime) (Figure 4.7.1) were determined as described in Experimental

Methods 6 . The activity of nitrofurantoin (XIII), the most frequently used nitrofuran, and the polyamines putrescine, spermidine and spermine, XIV, XV and XVI respectively (Figure 4.7.1 ) were also assessed as controls. Solutions were prepared in DMSO (compounds I and IV - XVI) or in HOI neutralized with NaOH (compounds

RESULTS Ï46 II and III) and diluted in SDW before incorporation into the agar. The solvent systems were not inhibitory (data not shown). MICs are tabulated in Table 4.7.1.

7 W O NO,

7 ^ o 'NO2

,N 7 \\ 'NO2

IV o N O 2 ^ c h = n h C0-NH-CH2-CH2-NH-C0-CH3

CO2CH3 o . NO

OH OAc VI VII HO OAc OH OAc O.N ■=N 0 ,N —N

V III OgN^ N. .CH

IX o.N. . 0 . ^ O NO

OHO

O X II OgN^^O NOH NH

XIV HgN XV HgN

XVI HgN

Figure 4.7.1. Structures of novel nitrofuran compounds I - IX, parent compounds X - XII, nitrofurantoin (XIII) and the polyamines, putrescine, spermidine and spermine (XTV - XVI)

RESULTS 147 Table 4.7.1. Minimum inhibitory concentrations of the novel nitrofuran compounds, their parent compounds, nitrofurantoin and the polyamines, putrescine, spermidine and spermine, nt = not tested

Minimum inhibitory concentration (|jg mM) com pound B. S. E. coli K. aero P. aeru S. typhi C. S. cere subtilis aureus genes ginosa murium albicans visiae

I 2 2 2 2 2 2 15 4

II 2 4 4 4 4 4 2 0 10

III 2 2 2 4 2 2 2 0 10

IV 2 0 15 30 10 0 > 1 0 0 30 30 30

V nt nt nt nt nt nt nt nt

VI > 2 0 > 2 0 > 2 0 > 2 0 > 2 0 > 2 0 > 2 0 > 2 0

V II 30 30 > 1 0 0 > 1 0 0 > 1 0 0 1 0 0 > 1 0 0 > 1 0 0

VIII nt nt nt nt nt nt nt nt

IX nt nt nt nt nt nt nt nt

X 3 7.5 4 7.5 > 1 0 0 3 10 10

XI nt nt nt nt nt nt nt nt

XII 10 5 10 30 50 10 2 0 10

XIII 5 5 5 >75 >75 7.5 >75 >75

XIV > 1 0 0 > 1 0 0 > 1 0 0 >100 >100 >100 > 1 0 0 > 1 0 0

XV >100 >100 >100 >100 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0

XVI > 1 0 0 75 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0 > 1 0 0

Compounds I, n and m gave lower MICs against all of the organisms tested when compared with nitrofurantoin (compound X III), indicating that these compounds had improved antimicrobial activity. Unlike nitrofurantoin they were also broad spectrum’, giving good inhibition not only of Gram-positive bacteria but also Gram-negative bacteria and the fungi C. albicans and S. cerevisiae. The remaining novel nitrofuran compounds either gave higher MICs than those of nitrofurantoin, indicating that they were less active than nitrofurantoin, or results could not be obtained due to there being insufficient compound available for testing. These latter compounds were tested by the agar diffusion technique.

RESULTS Ï48 7.3. Agar diffusion Agar plates were seeded with the Gram-positive bacteria B. subtilis or S. aureus, the Gram-negative bacteria E. coli, K. aerogenes, P. aeruginosa or S. typhimurium and the fungi C. albicans or S. cerevisiae (Materials & Apparatus Table

2.1.4). Five wells, 6 mm in diameter, were cut in the agar of each plate and filled with different concentrations of solutions of the same compound. Stock solutions at a concentration of 1000 pg ml'^ were prepared in DMSO (compounds I and IV - XVI) or in HCI neutralized with NaOH (n and m). Dilutions were then made in SDW to give concentrations of 300, 100, 30 and 10 pg ml'\ The plates were incubated at 37°C (bacteria) or 25°C (fungi) for 1 or 2 days respectively (Experimental Methods 7). Plotting the diameter of the zone of inhibition against the logarithm of the concentration gives an impression of the qualitative difference in activities of the compounds (data not shown). In order to semi-quantify such data the critical inhibitory concentration (CIC) can be determined. This concept was proposed by Linton (1961 ) based upon the work of Abraham et al., 1941, Cooper (1955), Cooper & Gillespie (1952), Cooper & Linton (1952), Cooper & Woodman (1946) and Linton (1958). Linton (1961) stated that the border of ‘a zone (of inhibition) is formed when a critical concentration ... reaches for the first time a density of growing cells too large for it to inhibit'. Therefore, if the zone of inhibition (the square of the distance from the source of diffusion, i.e. the edge of the well, to the zone edge) produced by a specific concentration of compound is plotted against the logarithm of that concentration of compound (Figures 4.7.2 to 4.7.11 ), the CIC can be determined by extrapolating the calculated line of best fit and reading the concentration at which the zone of inhibition is equal to zero, i.e. if the well contained a solution of concentration equal to the CIC then no zone would be produced. The CIC results are shown in Table 4.7.2. All compounds were initially tested against E. coli, S. aureus and S. cerevisiae. Then, as sufficient of some of the compounds remained, their zones of inhibition were determined for the wider range of organisms.

RESULTS 149 4 0 -- Figure 4.7.2. Compound I

100 1000 Concentration of compound (pg ml

Figure 4.7.3. Compound II

100 1000 Concentration of compound (pg ml

100-h Figure 4.7.4. Compound IV

20 -

100 1000 Concentration of compound (pg ml

Figures 4.7.2, 4.7.3 & 4.7.4. Antimicrobial activities of novel nitrofuran compounds I, II and IV. • = S. subtilis] A = S. aureus] O - E. coli] A = K. aerogenes] V = P. aeruginosa] □ = S. typhimuhum] ▼ = C. albicans] ■ = S. cerevisiae

RESULTS 150 Figure 4.7.5. Compound VI

100 1000 Concentration of

32 + Figure 4.7.6. Compound VII

e 2 4 --

# 16-

100 1000 Concentration of compound (pg ml

Figure 4.7.7. Compound X

“ 6 0 -

1000 Concentration of compound (pg ml'

Figures 4.7.5, 4.7.6 & 4.7.7. Antimicrobial activities of novel nitrofuran compounds VI and VII and 5-nitrofurfuraldehyde (X). • = 8. subtilis] 0 = E. coli] A = K. aerogenes] V = P. aeruginosa] □ = S. typhimurium] T = C. albicans] ■ = S. cerevisiae

RESULTS 151 112+ Figure 4.7.8. Compound XI

100 1000 Concentration of

140+ Figure 4.7.9. Compound XII e 105-

100 1000 Concentration of compound (pg m f'

24+ Figure 4.7.10. Compound XIII E 1 8 -

# 1 2

I 6-

100 1000 Concentration of compound (pg ml' 1

Figures 4.7.8, 4.7.9 & 4.7.10. Antimicrobial activities of 5-nitrofuranacrolein (X I), 5- nitrofuraldoxime (X II) and nitrofurantoin (XIII). • = B. subtilis-, A = S. aureus; u = E. coli; A = K. aerogenes; V = P. aeruginosa; □ = S. typhimurium; T = C. albicans; ■ = S. cerevisiae

RESULTS 152 404-

e 3 0 -

' I ...... 1------H 10 100 1000 Concentration of compound (pg mF'

Figure 4.7.11. Antimicrobial activities of novel nitrofuran compound III, which was only active against S. cerevisiae and compounds V , V I II and IX which were only tested against E. coli, S. aureus and S. cerevisiae. O = III & S. cerevisiae A = V & S. aureus □ = VIII & E. coli V = V I II & S. aureus # = V I II & S. cerevisiae A = IX & S. cerevisiae

No zone of inhibition around a well indicated that the solution in the well was at a concentration equal to or below the critical inhibitory concentration. As the highest concentration used for the agar diffusion technique was 1000 pg m l'\ it can be assumed that the critical inhibitory concentrations for compounds not producing a zone of inhibition at this concentration is 1000 pg mM or greater. Due to small quantities, compounds V, VIII and IX were only tested against S. aureus, E. co// and S. cerevisiae. When compared with nitrofurantoin (XIII), V and VIII had improved antistaphylococcal activity but V had lost Gram-negative activity; IX had neither Gram-positive nor Gram-negative activity but showed surprisingly good antifungal activity against S. cerevisiae, VIII also showed good antifungal activity compared with nitrofurantoin. Of the remaining compounds, III showed no activity against any of the Gram- positive or Gram-negative organisms tested but was active against S. cerevisiae. Compounds I, IV, VI and VII retained activity against the two Gram-positive organisms as compared with nitrofurantoin. These four compounds also retained, and in the majority of cases improved, activity against the Gram-negative organisms, the only exception being compound II against S. typhimurium. They all showed better antipseudomonal activity than nitrofurantoin and gave good

RESULTS Ï53 antifungal activity.

Table 4.7.2. Critical inhibitory concentrations of the nine novel nitrofuran compounds, their parent compounds, nitrofurantoin and the polyamines, putrescine, spermidine and spermine, nt = not tested; nz = no zones produced

Critical inhibitory concentration (pg ml'^) com pound B. S. E. coli K. aero P. aeru S. typhi C. S. cere subtilis aureus genes ginosa murium albicans visiae

I 93 30 30 73 230 33 190 98

II 34 1 1 0 115 1 0 0 2 0 0 90 1 2 0 19.5

III nz nz nz nz nz nz nz 18.5

IV 7 11 12 27 260 9 175 94

V nt 2 0 nz nt nt nt nt nz

VI 37 8.5 40 30 1 0 0 30 nz 44

VII 29 15 40 51.5 79 33 nz 60

VIII nt 34 93 nt nt nt nt 40

IX nt nz nz nt nt nt nt 1.1

X 9.5 12 13 27 60 14.5 2 1 0 45

XI 15 16 12 80 nz 34 80 18

XII 27 90 34 34 85 34 95 38

XIII 1 0 0 93 80 230 nz 46 nz nz

XIV nz nz nz nz nz nz nz nz

XV nz nz nz nz nz nz nz nz

XVI nz nz nz nz nz nz nz nz

7.4. Summary Comparing Tables 4.7.1 and 4.7.2 it can be seen that the CIC values produced by the agar diffusion technique are higher, both for the controls and the test compounds, than the MIC values determined on solid medium. This is probably because the CIC test results are subject to the diffusion characteristics of the compound in the agar, whilst with MICs, provided the compound is in aqueous solution, the value obtained approximates to the true in vivo value. However, both

RESULTS Ï54 sets of results gave similar trends for the activities of the compounds against the test organisms.

RESULTS 155 8. Mutagenicity of novel nitrofuran compounds

8.1. Introduction Mutations induced by genotoxins and other DNA-damaging agents are involved in the initiation of carcinogenesis. Other agents, acting as promoters, then stimulate DNA synthesis and cell division in mutant pre-cancerous cells (Knudson, 1973; McCann & Ames. 1976; McCann et ai, 1975). Many nitrofuran derivatives are genotoxic carcinogens (reviewed by McCalla, 1983) and have therefore been removed from use. Of the nitrofurans that have been used clinically (Table 1.1.1) nitrofurantoin is mutagenic but does not appear to be carcinogenic (Cohen at si, 1973; lARC Working Group, 1990; McCann & Ames, 1976; McCann at si, 1975; McCalla, 1983), furazolidone is mutagenic but it's carcinogenicity has not been evaluated (McCalla, 1983; lARC Working Group, 1983), nitrofurazone is mutagenic and possibly carcinogenic (McCalla, 1983; lARC Working Group, 1990) and nifurimox is mutagenic but no conclusions have been reached on its carcinogenicity (McCalla, 1983). Mutagenicity of nitrofurans in E. coli or S. typhimurium appears unaffected by the presence of an exogenous metabolic system such as rodent liver (S9) extracts (Ebringer & Bencova, 1980; lARC Working Group, 1990; McCann & Ames, 1976; McCann at si, 1975). In seeking market authorisation, manufacturers of new nitrofurans, as with any new drug, food additive or chemical, would be required to provide basic mutagenicity information derived from the appropriate tests (DHSS, 1981; 1989; UKEMS, 1983; 1984; 1989). Those novel nitrofuran compounds, of which there was sufficient quantity (Figure 4.8.1), were therefore tested using the Ames Salmonalla mutagenicity assay and the SOS/umu genotoxicity test.

O NO

O.N. .0 0 NO

III O ^ N ^ O , N ^ // 0 NO

^ ^ ^—CH = N—^ ^—CO—NH—CH^CH^NH —CO—CH3

Figure 4.8.1. Structures of novel nitrofuran compounds I - IV RESULTS 156 8.2. Ames Salmonella plate-incorporation assay Bacteria are sensitive indicators of DNA damage and widely used in primary screening tests. The Ames Salmonella plate-incorporation assay is an internationally recognized test for the detection of potential mutagens. It was conducted as described by Maron and Ames (1983), with additional modifications as recommended by UKEMS (1990). 8.2.1. Preliminary toxicity tests were performed to determine MICs under Ames assay conditions. From these could be deduced the maximum sub-inhibitory concentrations of test compounds that could be used for the microbial mutagenicity test (Materials & Apparatus 7 and Experimental Methods 12). Stock solutions of the four novel nitrofuran compounds and nitrofurantoin were prepared. Compounds II and m were dissolved in HCI and then neutralized with NaOH and compounds I, IV and nitrofurantoin were dissolved in DMSO. Serial dilutions were then made in SDW (compounds II and III) or DMSO (compounds I, IV and nitrofurantoin) and 100 pi added to 2 ml of top agar (Experimental Methods 12) to give concentrations between 0.25 and 100 or 0.25 and 500 pg/plate respectively. 100 pi of an 0/N culture of the S. typhimurium tester strain TA98 (Materials & Apparatus Table 2.1.3 and Experimental Methods 12) and 0.5 ml of 0.2M buffer or S9-mix (Materials & Apparatus 7) were also added to the 2 ml of top agar, which was mixed and poured onto a solid Vogel-Bonner E minimal medium agar plate. Only one plate per concentration of test compound was made. After two days incubation the effects of the compounds on the lawn of bacterial growth were observed and the lowest concentrations giving no background lawn, or showing disruption of cells if the lawn was sparse, were recorded as the minimum inhibitory concentrations under these conditions (Table 4.8.1). S9-mix, the exogenous metabolic system derived from rodent liver, made no difference to the MIC of these compounds in S. typhimurium TA98. From these tests a concentration range, using the MIC as the maximum concentration, could be determined for use in the microbial mutagenicity tests. 8.2.2. Microbial mutagenicity tests were performed using the histidine- requiring S. typhimurium tester strains TA98, TA98NR and TA100 (Materials & Apparatus Table 2.1.3 and Experimental Methods 12). Reversion events occur in TA98 after frameshift mutation and in TA100 after base-pair substitution (Maron & Ames, 1984). Strain TA98NR, which has reduced nitroreductase activity, was used

RESULTS Ï57 to show whether the novel nitrofuran compounds need to be reduced to be active.

Table 4.8.1, Minimum inhibitory concentration of compounds I, II, III, IV and nitrofurantoin against S. typhimurium TA98 using the plate-incorporation method.

Minimum inhibitory concentration (pg/plate) Test Compound ------S9-mix + S9-mix

I 250 250

II 1 0 0 1 0 0

III 1 0 0 1 0 0

IV 250 250

Nitrofurantoin 10 10

Stock solutions of the four novel nitrofuran compounds and nitrofurantoin were prepared as described above and serial dilutions made to give concentrations between 0.5 and 100 pg/plate for compounds II and III and between 0.5 and 250 pg/plate for compounds I and IV. For nitrofurantoin, the concentrations used were between 0.5 and 10 pg/plate for strains TA98 and TA100 and between 0.5 and 20 pg/plate for TA98NR, as the absence of nitroreductase may increase the concentration of compound required to cause mutagenicity. Three plates at each concentration of test compound were prepared as described above. Six solvent plates and two plates of each positive control, i.e. 5000 pg/plate of cyclophosphamide, 10 pg/plate of 3-methylcholanthrene, 1 pg/plate of 2- nitrofluorene or 2 pg/plate of sodium azide (Experimental Methods Table 2.12.1), were also prepared. The presence of a light background lawn of growth, due to limited growth of non-revertant colonies before exhaustion of the trace amount of histidine, was confirmed for each plate by examination under a light microscope. Such a lawn was not present at inhibitory concentrations. The numbers of revertants per plate were then recorded. The critérium for a positive (mutagenic) response in this assay and for these strains is a two-fold increase in mean number of revertants over that observed for the solvent control (Experimental Methods 12). This was achieved for

RESULTS Ï58 all four positive controls (data not shown). The minimum concentrations at which the compounds doubled the number of revertants per plate, i. e. the concentration at which the compounds became mutagenic, were noted (Table 4.8.2) and the dose- mutagenicity relationships plotted (Figures 4.8.2 to 4.8.8).

Table 4.8.2. Minimum concentrations of compounds I, II, III, IV and nitrofurantoin which caused mutagenesis. Nf = nitrofurantoin; nt = not tested; B = became bactericidal before exhibiting mutagenic activity; > = the highest concentration tested did not cause mutagenicity

Minimum concentration (pg/plate) causing mutagenesis in Salmonella typhimurium tester strains Test comp' TA98 TA100 TA98NR

— S9~mix + S9-mix - S9-mix + S9-mix — S9~mix + S9-mix

Nf B B 0.5 0.25 2 0 > 2 0

I 37.5 37.5 15 15 5 37.5

II > 1 0 0 0.5 5 35 nt nt

III 75 0.5 > 1 0 0 > 1 0 0 nt nt

IV 5 37.5 >150 37.5 15 75

1500-

03 500-r

Concentration of compound (fjg per plate)

Figure 4.8.2. Dose-mutagenicity relationships for nitrofurantoin using S. typhimurium s train s TA98 and TA100 in the presence and absence of S9-mix. V= TA98 + S9; O = TA98 - S9; A = TA 100 + 89; □ = TA100 - S9

RESULTS 159 Figure 4.8.3. In presence of S9-mix g 300- C3l

(L>

200 - o> o

50 100 150 Concentration of compound (pg per plate]

Figure 4.8.4. In absence of S9-mix

0 3

100- -i

50 100 Concentration of compound (pg per plate)

Figures 4.8.3 & 4.8.4. Dose-mutagenicity relationships for novel nitrofuran compounds I, il, III and IV using S. typhimurium TA98 in the presence and absence of S9-mix. 0 = 1; V = II; A = III; □ = IV

RESULTS 160 Figure 4.8.5.

1500 + In presence of S9-mix

1000- O d> 5 0 0 -

100 150 plate)Concentration of compound plate)Concentration

Figure 4.8.6.

50 100 Concentration of compound Ipg per plate)

Figures 4.8.5 & 4.8.6. Dose-mutagenicity relationships for novel nitrofuran compounds II, III and IV using S. typhimurium TA100 in the presence and absence of S9-mix. 0=1; V = II; A = III; □ = IV

RESULTS 161 100 -

CO Û. 8 0 -

o 4 0 -

c 20 -

Concentration of compound (pg per platel

Figure 4.8.7. Dose-mutagenicity relationships for nitrofurantoin using S. typhimurium TA98NR in the presence and absence of S9-mix. O = + S9; □ = - S9

^ 4 0 0 - co CL

g 3 0 0 - (T3 I f 200- 05

100-

50 100 150 Concentration of compound (pg per plate)

Figure 4.8.8. Dose-mutagenicity relationships for compounds I and IV using S. typhimuhum TA98NR in the presence and absence of S9-mix. V = I + S 9 ; O = I - S 9 ; A = IV + S9; □ = IV - S9

RESULTS 162 It is usual in papers reporting mutagenic activities tc quote mutagenic potencies of the test compounds (for example: Jurado etal., 1994; McCann & Ames, 1976; McCann etal., 1975; Ni etal., 1987). Mutagenic potencies (Table 4.8.2) were therefore determined from the calculated slopes of linear regression lines fitted to the increasing portions of the dose-mutagenicity relationships. This is illustrated by replotting Figure 4.8.7 as an example (Figure 4.8.9).

100 - Lines of regression have the general (1) 8 0 - equation y = mx + b where m = slope and b = y-axis intercept

The equation for C is y = 1.3655X + 56.6463 4 0 - The equation for □ is y = 1.3869X + 24.0636 2 0 -

CD Units for the slope are thus revertants per pg

Concentration of compound (pg per platel

Figure 4.8.9. Slopes of the linear regressions fitted to the increasing portions of the dose- mutagenicity relationships for S. typhimurium TA98NR in the presence and absence of 89- mix. (Symbols as Figure 4.8.7)

In S. typhimurium TA98 nitrofurantoin became bactericidal before any evidence of mutagenicity was apparent whereas only a low concentration was required to initiate mutagenesis in S. typhimurium TA100 (Table 4.8.2). This suggests that base-pair substitution was the primary cause of mutagenesis rather than frameshift reversions. A background lawn of growth and revertant colonies of strain TA98NR were apparent at concentrations of nitrofurantoin up to the highest tested (Table 4.8.2 & Figure 4.8.7) indicating that nitroreductase activity, which is absent in TA98NR, is required for full antibacterial activity. 20 pg/plate of nitrofurantoin in the presence of S9-mix did not double the number of TA98NR revertants over the control, however, in the absence of S9-mix, 20 pg/plate of nitrofurantoin induced an average revertant plate count of 52 which is just over a doubling of the control plate count of 23 (Figure 4.8.7)

RESULTS 163 Table 4.8.3. Mutagenic potencies of the novel nitrofuran compounds and nitrofurantoin. Nf = nitrofurantoin; nm = non-mutagenic; nt = not tested

Revertants per pg of compound (Revertants per nanomole of compound) T ©st d Salmonella Salmonella Salmonella typhimuriumJA9Q typhimurium TA^OO typhimuriumJA9SHR

- S9-mix + S9-mix - S9-mix + S9-mix - S9-mix + S9-mix

417.49 470.08 1.39 Nf nm nm nm (1753.00) (1973.79) (5.87)

3.41 3.05 6.93 14.00 5.12 4.86 I (1 0 .2 0 ) (9.13) (20.72) (41.88) (15.32) (14.54)

0.31 1.71 3.09 II nm nt nt (0.69) (3.82) (6.90)

0.71 0.18 III nm nm nt nt (1.82) (0.46)

0 .6 8 1 .6 8 2 .8 8 9.37 2.49 1.58 IV (1.98) (4.87) (8.38) (27.21) (7.23) (4.59)

As nitrofurantoin was not mutagenic against S. typhimurium TA98, the mutagenic potencies of the four novel nitrofuran compounds in this strain could not be compared to nitrofurantoin. Compound II was also determined a non-mutagen in strain TA98 in the absence of S9-mix (Tables 4.8.2 & 4.8.3) and it can be seen from Figures 4.8.3 and 4.8.4 that although the other novel nitrofuran compounds gave reversion rates from which mutagenic potencies could be calculated (Table 4.8.3), compounds I and IV were highly mutagenic compared to compounds II and m. Compounds I and IV were also the most active mutagens against S typhimurium TA100 (Figures 4.8.5 & 4.8.6) although both were much less mutagenic than nitrofurantoin (Table 4.8.3). Compound III was non-mutagenic when tested against this strain (Table 4.8.3). The presence of S9-mix did not alter the mutagenicity of the novel nitrofuran compounds greatly. In strain TA98 the ratios of mutagenic potencies in the presence of S9-mix / absence of S9-mix were 0.89, 0.25 and 2.5 for compounds I, m and IV respectively whilst in strain TA100 they were 1.1., 2.0,

2.9 and 3.3 for nitrofurantoin and compounds I, II and IV respectively. The fact that the presence of S9-mix reduced the mutagenicity of

RESULTS Ï64 nitrofurantoin in strain TA98NR suggests that the exogenous enzymes present in the S9-mix metabolize the drug to non-active forms. Similar to their comparative activities in strain TA98, compounds I and IV were also more mutagenic than nitrofurantoin in TA98NR, suggesting that nitroreduction was not required for activity of the novel compounds.

8.3. SOSIumu test This bacterial test was developed by Oda et al. (1985) as another predictor of chemicals that might be mutagenic or carcinogenic in higher organisms. When bacterial DNA is damaged, the damage-inducible (din) genes umuD and umuC are expressed and error-prone SOS repair commences (Bagg at a!., 1981; Kato & Shinoura, 1977; Rajagopalan eta!., 1992). The test uses S. typhimurium JA^535 containing the multicopy plasmid pSKI 002 (Materials & Apparatus Table 2.1.3) which bears the umuDC operon fused to iacZ, the structural gene for p- galactosidase (Shinagawa at ai., 1983). Fusion of these genes brings iacZ under din control. Therefore, when the umuDC operon is induced so is iacZ and P- galactosidase is produced. Addition of a substrate for the enzyme enables P- galactosidase activity to be measured, which indicates the level of umuDC induction and therefore DNA damage. The microplate method (Experimental Methods 13) was developed by Reifferscheid at ai. (1991). Stock solutions of the four novel nitrofuran compounds, nitrofurantoin and the positive controls, ICR 191 and 2-aminoanthracene (Materials & Apparatus 7), were prepared as previously described. Two-fold serial dilutions were made of the novel nitrofuran compounds to give a total of ten test concentrations which started at 100 pg mM for compounds I and IV and 50 pg mM for compounds II and III. Two-fold dilutions were also made of nitrofurantoin

(highest test concentration of 2 0 pg mM), the solvents and the positive controls

(highest test concentrations of 10 pg ml"''), 2 -aminoanthracene in the presence of S9-mix or ICR 191 in the absence of S9-mix. All dilutions were tested in duplicate in microtitre plates. A total combined volume of 300 pi of drug solution, S. typhimurium TA1535(pSK1002) suspension and S9-mix or buffer was incubated for 2 hours (Experimental Methods 13). A sample of the drug-exposed bacteria was then transferred to fresh broth and allowed to recover for 2 hours. The absorbance

RESULTS Î65 of the recovered cells was measured at 600 nm (^^^ODgoo) before a sample was reacted with o-nitrophenyl-P-D-galactopyranoside (ONPG) in buffer for half an hour (Experimental Methods 13). After this time the reaction was stopped and the absorbance in each microtitre well measured at 600 nm (ODqoo) and 415 nm (OD 415). From these measurements the units of p-galactosidase activity were calculated according to the formula of Miller (1972), adapted for the microplate method.

Units of P-galactosidase activity = 1000 x OD^is - (1.75) (ODfinn) (t) (V) (^^^ODeoo) t = time (minutes) V = volume of culture used in assay (ml)

A positive result for umu induction was taken as a 2-fold increase in p- galactosidase activity over that produced by the solvent control (Oda et a/., 1985). The concentrations at which P-galactosidase activities were found to double are shown in Table 4.8.3.

Table 4.8.4. Lowest concentrations at which p-galactosidase activities were doubled by the novel nitrofuran compounds I, II, III, IV and nitrofurantoin in S. typhimurium TA1535(pSK1002)

Lowest concentration measured in pg mM causing doubling Test of p-galactosidase activity (nanomoles ml''') compound - S9-mix + S9-mix

Nitrofurantoin 0.25(1.0) 0.25(1.0)

I 6.25(18.7) 12.5(37.4)

II >50 (>111.5) >50 (>111.5)

III >50 (>127.8) >50 (>127.8)

I V______6.25(18.2) ______25 (72.6) ______

Dose-P-galactosidase activity relationships were then be plotted to show changes in p-galactosidase activity with concentration for each test compound. The activities of the novel compounds are compared with each other and the positive controls in Figures 4.8.10 and 4.8.11. None of the novel compounds are as genotoxic as the positive controls on a weight basis. Figures 4.8.12 and 4.8.13

RESULTS Ï66 compare the activities of nitrofurantoin with the respective positive controls in the presence or absence of S9-mix. Nitrofurantoin is a more active SOS inducer than the controls under both conditions.

Figure 4.8.10. In presence of S9-mix

§ 5000-

o 4000-

3000-

100 Concentration of compound Ipg per ml)

Figure 4.8.11. 5000- In absence of S9-mix

4000-

2000 - o

1 1000

100 Concentration of compound (pig per ml)

Figures 4.8.10 & 4.8.11. Dose-P-galactosidase activity relationships for novel nitrofuran compounds I, II, III, IV and positive control using S. typhimurium TA1535(pSK1002) in the presence and absence of S9-mix. (Data points are the average of two determinations.) 0 = 1; V = II; A = III; □ = IV; • = Positive control (2-aminoanthacene in presence of S9-mix or ICR 191 in absence of S9-mix)

RESULTS 167 Figure 4.8.12. 24000+ In presence of S9-mix

■ i 2 0 0 0 0 -

16000- o

12000-

Concentration of compound (pg per mil

Figure 4.8.13. 24000+ In absence of S9-mix

• i 2 0 0 0 0 -

:e 16000- 0 CO

% 12000- 1 JS.i 8 0 0 0 - CJ

Concentration of compound (pg per mil

Figures 4.8.12 & 4.8.13. Dose-P-galactosidase activity relationships for nitrofurantoin and positive controls (2-aminoanthracene in presence of S9-mix or ICR 191 in absence of S9-mix) using S. typhimurium TA1535(pSK1002). # = Positive control; ■ = Nitrofurantoin (Data points are the average of two determinations.)

RESULTS 168 In the absence of S9-mix, twenty-five times as much of compounds I and IV were required, compared to nitrofurantoin, before P-galactosidase activity doubled. Whereas, even 50 pg ml'\ the highest concentrations of compounds II and III tested, did not double p-galactosidase activity. The presence of S9-mix did not alter the concentration of nitrofurantoin required for positive umu induction, whereas, 2 - and 4-fold more of compounds I and IV respectively were required for positive P- galactosidase induction in the presence of S9-mix. These latter data indicate that S9 is not required for activity of the compounds.

8.4. Summary Nitrofurantoin was not mutagenic to S. typhimurium strain TA98 but was highly mutagenic to strain TA100. Novel nitrofuran compounds I and IV gave low levels of mutagenic activity against strain TA98 but were much less mutagenic against TA100 than nitrofurantoin. Compounds II was not mutagenic to strain TA98 in the absence of S9-mix whereas compound III exhibited slight mutagenicity but only at high concentrations. Compound III was non-mutagenic to strain TA100 either in the presence or absence of S9-mix wherreas compound II exhibited slight mutagenicity. These Ames assay results compare well with those of the SOSIumu test which indicated that compounds II and III did not induce the SOS response at any concentrations tested whilst compounds I and IV induced it but only at concentrations higher thanthose found positive for nitrofurantoin.

RESULTS 169 9. Activities of novel nitrofuran compounds against polyamine transport deficient mutants of Escherichia coii

9.1. Introduction One reason for synthesizing and testing conjugates of a nitrofuran with a polyamine was the proposal that this might increase the antibacterial activity of the nitrofuran by increasing its uptake into the cell via active transport systems for polyamines (Introduction 2.4). Of the compounds synthesized, compounds I, II, III and IV (Figure 4.9.1) were shown to be more active than nitrofurantoin (Figure 4.9.1 ) against E coli (Tables 4.7.1 and 4.7.2). If the above hypothesis is correct, polyamine conjugates, such as compounds I, II and III, should be relatively less active against polyamine uptake-deficient mutants of E. co//than wild-type strains. Whereas compound IV, which is a benzoyl conjugate, and nitrofurantoin itself might be predicted to have similar activities against polyamine-deficient and -proficient strains.

O NO

III O.N. , 0 o NO

^ Jj—OH—N—^ ^ —CO—NH—CH^CH^NH —CO—CH3

Nitrofurantoin r - i ° O2N. .0 , ,NH

Figure 4.9.1. Structures of novel nitrofuran compounds I - IV and nitrofurantoin. I = bis-5-nitrofurfurylidenylputrescine; II = bis-5-nitrofurfurylidenylspermine; III = bis-5-nitrofurfurylidenylspermidine; IV = 4-(N-5-nitrofurfurylidene)aminobenzoyl-2-acetylaminoethylamide

Kashiwagi et al. (1990) have isolated several polyamine transport deficient mutants of E. coli MA261 (Materials & Apparatus Table 2.1.1), a polyamine- requiring mutant. These are KK313 which is deficient in spermidine uptake, NH1596 which is deficient in spermidine and 90% deficient in putrescine uptake and KK313 potF/.Km which is deficient in spermidine and putrescine uptake.

RESULTS Î7Ô 9.2. Antibacterial activity of the novel nitrofuran compounds in liquid medium Volumes of nutrient broth containing 100 gg ml'"' of test compound or nitrofurantoin were inoculated with a 1 in 50 dilution of an overnight culture of the organisms. The concentration of 100 pg ml'^ was chosen because this concentration of nitrofurantoin is bactericidal to E. coli (Figure 4.1.11 ). Therefore, since the novel nitrofuran compounds have activities better than or similar to nitrofurantoin (Tables 4.7.1 and 4.7.2), the polyamine transport mutants should be more resistant to the respective novel nitrofuran compounds than the polyamine uptake-proficient parent strain. CompoundsI and I V were dissolved in DMSO, compounds I I and I I I were dissolved in HOI and neutralized with NaOH, and nitrofurantoin was suspended in water and dissolved by the addition of NaOH ( Materials & Apparatus 4). Drug-free nutrient broth and nutrient broth containing 2% DMSO were used as controls. All mixtures were held in a water bath at 37°C for five hours and viable counts determined hourly on nutrient agar (Experimental Methods 2.3). The effect of the novel nitrofuran compounds upon the survival of these strains is shown in Figures 4.9.2 to 4.9.8.

-9

— — I — ^ Time of exposure (hours) Time of exposure (hours)

Figure 4.9.3. 2% DMSO in nutrient broth Figure 4.9.2. Nutrient broth

Figures 4.9.2 & 4.9.3. Growth of E. coli strains MA261, KK313, NH1596 and KK313 pofF;: Km in drug-free nutrient broth or nutrient broth containing 2% DMSO. □ = MA261; 0 = KK313; V = N H 1 5 9 6 ; O = KK313 pofE:;Km

RESULTS 171 Cells grew exponentially and at similar rates in both drug-free nutrient broth (Figure 4.9.2) and in nutrient broth containing 2% DMSO (Figure 4.9.3) showing that 2% DMSO did not affect growth. The initial rate of growth of strain KK313 pofF::Km was higher than the other three strains.

Figures 4.9.4, 4.9.5 & 4.9.6. Survival of E. coli strains MA261, \ o KK313, NH1596 and KK313 potF:Km in 100 pg mT^ of nitrofurantoin or compounds I or II. □ =MA261; 0 = KK313; V = NH1596; O = KK313 pofF:;Km

c /5

Time of exposure (hours)

Figure 4.9.4. Nitrofurantoin

G Q------©------Q ------o

10’-

c /5 c/5

10"’-

Time of exposure (hours) Time of exposure (hours)

Figure 4.9.5. Compound Figure 4.9.6. Compound II

RESULTS 172 — Q------O------o ---O ------. O

10^-- 10’-

Time of exposure (hours) Time of exposure [hours]

Figure 4.9.7. Compound III Figure 4.9.8. Compound IV

Figures 4.9.7 & 4.9.8. Survival of E. coli strains MA261, KK313, NH1596 and KK313 p o tF /K m in 100 pg mM of compounds III or IV. □ =MA261; 0 = KK313; V=NH1596; O = KK313 pofF::Km

The kill kinetics of the polyamine transport proficient control strain MA261 in 100 pg ml'"' of nitrofurantoin were similar to those of E. coli AB1157, which had previously been used in bactericidal studies (compare Figures 4.9.4 and 4.1.11). Nitrofurantoin had similar activities against strains KK313 and KK313potF::Km but strain NH1596 was significantly more sensitive. The addition of 100 pg ml'"' of compound I, the putrescine conjugate (Figure 4.9.5) inhibited growth of strain KK313 potF::Km and was mildly bactericidal for strains MA261, KK313 and NH1596 (Figure 4.9.5). Since the compound is at least inhibitory to all four strains it can be assumed that it is able to enter cells of all of these strains regardless of their transport deficiency. Compounds II and III, the spermine and spermidine conjugates respectively, were also bactericidal against all of the strains (Figures 4.9.6 & 4.9.7). As it has been suggested that spermine is transported by the same system as spermidine (Kashiwagi eta!., 1986), neither of these conjugates should have been bactericidal to any of the polyamine transport-deficient mutants as they all lack the spermidine

RESULTS 173 transporter. However, due to the relatively bulky nitrofuran groups at both ends of the spermine or spermidine spacer, it is possible that these compounds were not transported via the spermidine transport system but by some other method. Compound IV, the benzoyl conjugate (Figure 4.9.8), gave results similar to compound I. It was bacteriostatic rather than significantly bactericidal against all strains over the five hour period. Since this compound is not a polyamine derivative it was expected that it might be as bactericidal as nitrofurantoin to all strains. However, it would appear that uptake of this benzoyl conjugate is otherwise affected.

9.3. Summary The four novel nitrofuran compounds did not behave as predicted against the three polyamine transport deficient strains of E. coli. It was expected that if the compounds were taken up only through active polyamine transport systems then the strains that were deficient in the particular transport mechanisms would be resistant to the respective compounds. However, since compounds I and IV are bacteriostatic and II and III are bactericidal to all strains, it must be assumed that alternate routes of uptake are available for these compounds.

RESULTS 174 PA R T FIVE DISCUSSION

DISCUSSION 175 1. Sensitivities to UV and nitrofurantoin of Escherichia coii DNA repair- deficient strains and the effect of R46 The fully DNA repair-proficient E. coli strain AB1157 and the DNA repair- deficient strains showed the expected order of sensitivities to UV light (Figure 4.1.1), dependent on their deficiencies; these were similar to those determined by Howard-Flanders (1968) and Upton & Pinney (1983). Strain AB2463 recA, which is deficient in both recombination and error-prone repair pathways and the strains deficient in the incision step of excision repair (AB1886 uvrA, JC3890 uvrB and TK501 uvrB umuC) were highly sensitive. Strains TK702 umuC (lacking error-prone repair), AB2470 recB (lacking recombination repair), JG138 polA (lacking the repolymerizing step of excision repair) and AB2494 lexA (lacking induction of error- prone repair) showed sensitivities increasing in the order given compared with AB1157. However, all were more resistant than AB2463 recA and the strains lacking the incision step of excision repair. Plasmid R46 carries mucAB genes, which are homologous to the chromosomal umuDC genes and are likewise under the control of lexA and recA (Introduction 4). R46 did not protect AB2494 lexA (Figure 4.1.2) or AB2463 recA (Figure 4.1.3) against UV damage. The LexA protein, determined by the /exA3 allele, does not autodigest in the presence of the RecA* coprotease (Walker, 1984). din gene expression is therefore not induced by DNA damage in this strain. Similarly, in the absence of a functioning RecA protein all SOS regulated genes remain uninduced. Without a functioning lexA or recA gene the plasmid muc genes are not expressed and cannot therefore increase the repair activities of their host (Mortelmans and Stocker, 1979; Waleh and Stocker, 1979; Walker, 1977). Unexpectedly, in repeated experiments, R46 did not produce any significant (by Student's t-test) difference in the survival of the polA strain at any of the UV doses tested (Figure 4.1.4). The polA mutant has reduced DNA polymerase I activity (Gross & Gross, 1969) and is more sensitive to UV irradiation (Monket ai., 1971) in comparison to the wild-type strain. Addition of plasmid R46 and therefore of muc genes has previously been shown to increase survival of polA mutants (MacPhee, 1974; Tweats at al., 1976; Upton & Pinney, 1981; 1983) and MacPhee (1974) even suggested that R46 encoded a DNA polymerase, though no DNA polymerase I activity was detected by Upton & Pinney (1981) in cell-free extracts of strain JG138 po/A(R46) after exposure to UV.

DISCUSSION 176 Varying levels of protection were conferred by R46 to the other, SOS- inducible, DNA repair-deficient strains (Figures 4.1.5 to 4.1.10), as the results of Oliver and Stacey (1977), Tweats et al. (1976) and Upton & Pinney (1983) have also shown. The greatest degree of protection was conferred upon strains TK702 umt/C (Figure 4.1.6) and TK501 uvrB umuC (Figure 4.1.10), giving survival levels approximately 100-fold greater than the R~ strain. Upton & Pinney (1983) also showed that maximum protection was conferred by R46 on the uvrB umuC mutant. The umuC derivative was restored to wild-type UV resistance by plasmid R46. Thus, the presence of R46 muc genes was replacing the absent umuC repair capacity. R46 also conferred an increase in survival of nearly 100-fold on the excision repair-deficient strains uvrA (Figure 4.1.8) and uvrB (Figure 4.1.9). The minimum inhibitory concentrations (MICs) of nitrofurantoin against all of the DNA repair-deficient strains were lower than that for the fully DNA repair- proficient AB1157 strain, indicating that the former were more sensitive to nitrofurantoin. Jenkins & Bennett (1976) found similar results with E. coli K12 recA13, recA56, recB21, recC22 and polA1 mutants. This also confirms that nitrofurantoin acts by damaging DNA, a view supported by lida and Koike (1977) and Lu etal. (1979). As all of the DNA repair-deficient strains tested were also more sensitive to UV irradiation it is likely that the decreased DNA repair efficiencies were also responsible for the increased sensitivities to nitrofurantoin. Since the excision and recombination repair-deficient strains were the most sensitive to nitrofurantoin (Table 4.1.1) it would appear that these are the repair pathways used for nitrofurantoin damage. These data, and those of Jenkins & Bennett (1976), contradict the results of Hamilton-Miller at al. (1977) who showed no difference in the MIC of nitrofurantoin against E. coli AB1157 and its recA13 mutant. It must be noted, however, that the MIC they reported for the wild-type strain fell at the lower end of the range for susceptible organisms; 0.5 pg ml'^ in a range of 0.4-10 pg ml'"' (Therapeutic Drugs, 1991), which is similar to the MIC determined for AB2463 recA in this thesis (Table 4.1.1). The incorporation of R46 made no difference to the MIC of nitrofurantoin for AB1157 or the majority of the DNA repair-deficient strains. However, it increased the MIC of the uvrB umuC strain 2-fold and of the uvrB strain 20-fold (Table 4.1.1), bringing the MIC of the latter strain up to that of the wild-type AB1157. This could

DISCUSSION Ï77 suggest that the absence of uvrB gene function increases expression of plasmid muc functions in the repair of nitrofurantoin-induced damage. Such heightening of effect is not seen with plasmid-mediated repair of UV-induced damage in the uvrB strain (Figure 4.1.9). Other authors have suggested that plasmids may carry genes determining nitrofurantoin resistance. Breeze & Obaseiki-Ebor (1983a; 1983c) and Obaseiki- Ebor (1984) isolated a plasmid, designated pBN105, which increases nitrofurantoin resistance in DNA-proficient and recA, uvrA and uvrA lexA deficient mutants of E. coli 35-3. The plasmid increases the MICs of the DNA-proficient strain 8 -fold and the DNA-deficient mutants 90-fold. Oliver (1981) has also shown that R46 itself confers protection against nitrofurantoin in recA and lexA mutants of E. coli, which he demonstrated was not due to altered cell permeability and, for reasons stated above, could not be due to induction of the R46-determined muc genes. Taking into account the different MICs of nitrofurantoin for each of the E. coli (R“) strains and therefore the bactericidal concentration of nitrofurantoin to which they were exposed in a liquid medium (20xMIC), the DNA repair-deficient strains showed considerably greater sensitivity than the wild-type AB1157 over a one hour exposure (Figure 4.1.12). R46 gave protection to strains AB1157, umuC, uvrA, uvrB and uvrB umuC (Figures 4.1.17 to 4.1.21) but not to any level that would be significant in clinical practice.

2. Effect of a variety of plasmids on the survival of Escherichia coli AB1157 In a lethal concentration of nitrofurantoin Plasmids R16, R46, R124, R144, R446b, R621a, R805a and pKMIOI conferred protection against UV to E. co//AB1157 (Figures 4.2.1 to 4.2.4, 4.2.6 to 4.2.8 and 4.2.11) as previously shown by Little et al. (1991) and Upton & Pinney (1983). Plasmid R391 conferred sensitization (Figure 4.2.5) as previously shown by Pembroke & stevens (1984) and Pinney (1980). Plasmid pGW16 conferred sensitization at lower doses and protection at higher doses (Figure 4.2.10) as shown by Little at al. (1991). However, plasmid pGW12 sensitized AB1157 to UV (Figure 4.2.9) contrary to the results of Little at al. (1991 ) who showed that it had a null effect. Walker (1978) stated that pGW12 had lost most of the ability of the parent plasmid pKMIOI to protect against UV not that it had lost all ability. Plasmid pYDI was shown to make little difference to the post-UV survival of AB1157 (Figure

DISCUSSION Î78 4.2.12), whereas previously it had been claimed to confer sensitization to E. coli and protection to a rifampicin resistant Shigella sonnei strain (Ambler & Pinney, 1995). None of the twelve plasmids tested had any effect on the minimum inhibitory concentration of nitrofurantoin. E. co//AB1157(R~) and all of the plasmid-containing derivatives gave MICs of 7.5 pg ml V However, the increments in concentration of nitrofurantoin used in this test were relatively wide, the next lowest concentration being 5 pg mM and the next highest 10 pg ml'\ It is therefore possible that any low levels of protection the plasmids may have conferred were not detected. It should be remembered that MIC is reported as the lowest concentration tested that inhibits growth. The real MIC is therefore somewhere between this concentration and the one below it that allows growth. As the interval between the concentrations increases, the error in MIC measurement also increases. Some protection was conferred, against a bactericidal concentration of nitrofurantoin, to E. coli AB1157 by plasmids R46, R446b, R621a, R805a and pKMI 01 (Figures 4.2.14, 4.2.18, 4.2.19, 4.2.20 & 4.2.23), all of which also protect against UV light. Plasmids R144, R391, pGW12 and pGW16 (Figures 4.2.16, 4.2.17, 4.2.21, & 4.2.22) sensitized AB1157 to nitrofurantoin. Of these, plasmids R391 (Figure 4.2.5) and pGW12 (Figure 4.2.9) sensitized to all doses of UV, plasmid pGWIG (Figure 4.2.10) sensitized to low doses but protected at higher doses of UV and R144 (Figure 4.2.4) protected against all UV doses tested. RIG, R124 and pYDI (Figures 4.2.13, 4.2.15, & 4.2.24) neither protected nor sensitized AB1157 against nitrofurantoin, whereas RIG and R124 protected against UV light (Figures 4.2.1 & 4.2.3) and in the current work pYDI (Figure 4.2.12) was shown to have no effect on post-UV survival. The level of protection conferred on strain AB1157 against nitrofurantoin by these plasmids was relatively small. As AB1157 is wild-type for DNA repair it may be that no further increase in repair capacity will help in the survival of the cells. Therefore, the conclusion reached is that the levels of resistance or sensitivity conferred by any of the plasmids is not likely to affect the clinical outcome of nitrofurantoin therapy.

3. Antibacterial action of nitrofurantoin against Escherichia co//when protein synthesis, RNA synthesis or cell division are inhibited Quinolones have three mechanisms of action. Mechanism A is active against dividing bacteria and requires RNA and protein synthesis. It is therefore abolished

DISCUSSION Ï79 in phosphate buffered saline or in nutrient broth containing either rifampicin or chloramphenicol. Mechanism B is active against dividing and non-dividing bacteria and does not require RNA or protein synthesis. Therefore, it is not abolished in phosphate buffered saline or nutrient broth containing chloramphenicol or rifampicin. Mechanism C is active against non-dividing cells and requires RNA and protein synthesis. It is still active on cells suspended in phosphate buffered saline, but is abolished in nutrient broth containing rifampicin or chloramphenicol. For a review of these mechanisms see Howard et al. (1993). Nitrofurantoin was equally bactericidal to E. co//AB1157 in nutrient broth and phosphate buffered saline (Figures 4.3.1 & 4.3.2). Its action was therefore not abolished in non-dividing cells. At the concentrations used, both rifampicin and chloramphenicol had a bacteriostatic action on organisms growing in nutrient broth (Figure 4.3.1). Suspension of cells in phosphate buffered saline abolished cell division but cells remained viable (Figure 4.3.2). The addition of rifampicin to phosphate buffered saline gave a slight bactericidal effect (Figure 4.3.2). Addition of rifampicin or chloramphenicol to the incubation medium, after 2 hours exposure to nitrofurantoin, did not affect the bactericidal activity of nitrofurantoin in nutrient broth or phosphate buffered saline (Figures 4.3.3 & 4.3.4). Thus, the absence of RNA or protein synthesis had no effect on the bactericidal activity of nitrofurantoin. Similarly, if cells in nutrient broth were pre-exposed to

rifampicin or chloramphenicol for 2 hours before the addition of nitrofurantoin, the

onset of nitrofurantoin kill was delayed for 1 or 2 hours but there was then no difference in the rate of death to that seen in the absence of rifampicin or chloramphenicol (Figures 4.3.5). In phosphate buffered saline, pre-exposure to chloramphenicol for 2 hours did not affect the activity of nitrofurantoin (Figure 4.3.6). Pre-exposure to rifampicin

in phosphate buffered saline for 2 hours, however, increased the bactericidal activity of nitrofurantoin (Figure 4.3.6). This parallelled the slight bactericidal activity seen with rifampicin alone in phosphate buffered saline (Figure 4.3.2) and indicates an additive effect of the activities of the two drugs. The concept of mechanisms A, B and C cannot, therefore, be applied to nitrofurantoin: its activity remains unimpaired under conditions that abolish the activities of some quinolones, such as nalidixic acid, and reduce the activities of

DISCUSSION Ï8Ô others. Nitrofurantoin, and therefore other nitrofurans, can thus be used without fear that a change in conditions may alter the activity of the drug.

4. Plasmid stability to nitrofurantoin inEscherichia co//AB1157 Plasmids are covalently closed autonomous circles of DNA whose replication depends on host-specified proteins, except for one essential plasmid-encoded replication protein, the repA gene product or its equivalent (Hardy, 1988; Langer et al., 1981). Plasmid replication is coupled with the synthesis of other cell components. Thus, the repA gene on the plasmid is transcribed for protein production of replication when the bacterial chromosome is replicating. Replication of the plasmid is then controlled in the same manner as for the bacterial chromosome. Plasmid replication is also linked to the cell cycle and division. The plasmid par gene product ensures that plasmids are partitioned efficiently at cell division (Hardy, 1988). This par mechanism is not well understood but it is believed that plasmid attachment to the cytoplasmic membrane of the cell ensures equipartition of replicated plasmids into daughter cells at division (Hardy, 1988). Thus plasmids are correctly replicated and partitioned. Anything that interferes with these procedures will reduce the likelihood of each cell in a culture carrying a plasmid. Some bacteria will be ‘cured’ of the plasmid. If the rate of curing becomes significant, then, for example in bacteria carrying R plasmids, the proportion of drug- sensitive bacteria will increase. Another way in which plasmids may be effectively eliminated from cultures is due to the fact that plasmids slightly decrease the growth rate of their host due to the additional metabolic burden they place on the host cell (Helling et a/., 1981 ). Plasmidless cells will therefore outgrow plasmid-carrying cells in a culture. Also, if a cell is undertaking repair of chromosomal DNA damage produced by, for example, the presence of a drug, then it has less resources available to provide for plasmid replication. If the plasmid is not replicated correctly, upon cell division one of the daughter cells will not contain a plasmid. The drug has therefore ‘cured’ the organism of the plasmid. Many chemicals, including some therapeutically useful drugs, have been shown to eliminate plasmids (Bazzicalupo & Tocchini-Valentini, 1972; Hahn & Ciak, 1976; McHugh & Swartz, 1977; Mitsuhashi et a!., 1961; Pinney & Smith, 1972b;

DISCUSSION isT 1973; Tomoeda etal., 1968; Watanabe & Fukasawa, 1960; 1961). Pinney & Smith (1971; 1972a) showed that R factor 1818, later renamed plasmid R46, was eliminated from thymine-requiring strains of E coli by thymine starvation. They went on to show (Pinney & Smith, 1972b) that the plasmid was also eliminated by trimethoprim treatment of thymine-proficient E. coll. The drug induces thymine deprivation in the wild-type strain. Bremer et al. (1973) showed that R-1818 was also eliminated by another DNA-damaging drug fluorodeoxyuridine. Novobiocin, an inhibitor of DNA gyrase, which is responsible for the supercoiling of DNA, also eliminates plasmids from a variety of E. coli strains in both sub-lethal and lethal concentrations (Hooper etal., 1984; McHugh & Swartz, 1977). It is therefore likely that anything affecting the synthesis or maintenance of DNA will effect plasmid elimination. Nitrofurans form adducts and intercalate into DNA (Mukherjee & Chatterjee,1992; Touati et al., 1989; Wentzell & McCalla, 1980), both of which inhibit replication (Lu et al., 1979; Wentzell & McCalla, 1980), nitrofurantoin was therefore tested for evidence of plasmid elimination. When plasmid-containing strains of E. coli were grown in broth containing a subinhibitory concentration of nitrofurantoin, plasmid elimination was not observed for any of the nine plasmids tested from at least eight incompatibility groups (Table 4.4.2). Therefore, any damage to the plasmid caused by the drug was repaired without interfering with plasmid replication or partitioning. Exposure of cells to a lethal concentration of nitrofurantoin, however, eliminated the N incompatibility group plasmid R46 from almost 50% of the surviving E co//AB1157 cells tested (Table 4.4.1). No other plasmid was eliminated at a significant frequency. Why nitrofurantoin only eliminates plasmid R46 from its host is speculative. However, when plasmids from a variety of incompatibility groups were tested for susceptibility to elimination by thymine starvation, only plasmids from the N incompatibility group, which includes R46, were found to be susceptible (Birks & Pinney, 1975).

5. Mutation of Escherichia coii to nitrofurantoin resistance

The traditional view of mutation is that it occurs without connection to any particular advantage it may provide. However, Cairns et al. (1988) showed that a strain of E coli, unable to metabolise lactose, undergoes an increased frequency of mutation to lactose utilisation when grown on media containing lactose as the

DISCUSSION Ï82 sole carbon source. This led to the proposal that mutations could occur in selected genes as a specific response to a non-lethal challenge; so called adaptive mutation. Nitrofurantoin causes numerous types of damage to DNA, such as strand breakage (McCalla et al., 1971; 1975; Tu & McCalla, 1975), adduct formation (Touati et al., 1989; Wentzell & McCalla, 1980), intercalation (Mukherjee & Chatterjee, 1992) and interstrand cross-links (Chatterjee etal., 1987). Some of the damage is removed by excision repair (Wentzell & McCalla, 1980) but other is persistent and causes daughter-strand gaps (Lu et al., 1979) which induce 80S error-prone repair (Bryant & McCalla, 1980; Chatterjee et al., 1983). This mechanism introduces mutations into the DNA, by incorporating incorrect bases opposite non-coding lesions, rather than leaving gaps in DNA, which would ultimately lead to cell death (Introduction 3.2.8). All alternate proposal by Echols (1981 ) suggests that mutagenic DNA repair increases the frequency of mutation, not to maintain the integrity of DNA itself, but to provide more opportunities for natural selection. This is supported by the small effect the absence of error-prone repair has on survival after DNA insult. Compare, for example, the survival of E. co//strain TK702 umuC, in which error-prone repair is non-functional, with the wild-type fully DNA repair proficient strain AB1157 (compare Figure 4.1.5 with 4.1.6 and see Figure 4.1.12). E. coli AB^^ 57 spontaneously mutated to nitrofurantoin resistance, as shown by growth of a small proportion of cells on solid media containing a lethal concentration of nitrofurantoin (Table 4.5.2). The mean spontaneous mutation frequency of E co//AB1157 to nitrofurantoin resistance, determined on solid media, was 6 .8 X l o t It has been suggested, in relation to quinolones, that plasmids may contribute to the development of chromosomal resistance by means of a mutator phenotype which increases mutation frequency in host cells (Crumplin, 1987). However, when R46, a mutator plasmid carrying genes analogous to those on the chromosome responsible for error-prone repair, was present in E. co//AB1157, its inclusion only increased mutation frequencies to nitrofurantoin resistance, on solid media, by an average 1.85-fold (Table 4.5.2). This is in contrast to the results of Ambler etal. (1993) using nalidixic acid, a drug which, like nitrofurantoin, induces

8 0 8 error-prone DNA repair, who showed that the presence of R46 increased the spontaneous mutation frequency to nalidixic acid resistance on solid media almost

DISCUSSION Ï83 5-fold. Hinton & Pinney (1997), also found that R46 increased the spontaneous frequency of mutation of E. coli AB1157 to nalidixic acid resistance 6 -fold over the plasmidless strain in liquid media, whilst Ambler & Pinney (1995) found a 12-fold increase. However, in the current work, the data indicated that R46 did not increase the spontaneous mutation frequencies to nitrofurantoin resistance in cultures growing in liquid media. Ambler & Pinney (1995) found that on exposure to a lethal concentration of nalidixic acid, twice the minimum inhibitory concentration (2xMIC), the presence of R46 increased mean apparent mutation frequencies 200-fold in E. coli AB1157. In the present thesis, no nitrofurantoin resistant mutants of AB1157(R46) were isolated from cultures exposed to lethal concentrations of nitrofurantoin, despite repeat experiments. Nitrofurantoin resistant mutants of the plasmidless AB1157 strain were detected but only after incubating for twenty-four hours. Comparing the relative survivals of AB1157 and its R46 derivative (Figures 4.5.1 & 4.5.3) it can be seen that R46 appears to sensitize AB1157 to 20 pg mM of nitrofurantoin whereas the plasmid protected its host against the higher concentration (200 pg mM) of nitrofurantoin previously tested (Figure 4.1.17). Khan & Hadi (1993), in using isolated plasmid DNA, claimed that different concentrations of nitrofurans cause different types of DNA damage with higher concentrations required for mutagenesis. It is therefore possible that low concentrations of drug are sufficient to kill cells but not to induce the chromosomal- or plasmid-encoded mutator opérons required for mutagenic activity. The data presented in Results 5 show that E. coli does mutate to nitrofurantoin resistance but only at very low frequencies. There is no evidence that the mutator plasmid, R46, significantly increases spontaneous or induced resistance in cultures of E. coli exposed to clinically-attainable concentrations of nitrofurantoin.

6 . Antimicrobial activities of novel nitrofuran compounds Measurement of minimum inhibitory concentration (MIC) is the most commonly used technique for quantifying the sensitivity of an organism to an antimicrobial. The agar diffusion technique is the classical method of assaying antibiotics (Abraham etal., 1941). The former has certain advantages over the agar

DISCUSSION Î84 diffusion technique. For example, the test compound is not required to diffuse to its site of action, but is present in solution throughout the medium. This enables compounds of large molecular size to be tested giving MICs which are a direct measure of inhibitory concentration. However, there are disadvantages in that the direct inoculation technique is more demanding of time and the concentration increments used may make the end point difficult to interpret. If concentration increments are too close together, growth diminishes over a range of concentrations without producing a clear end point and if test concentrations are too wide apart, the end point falls between two concentrations, producing a margin of error. Also, if the end point is taken as the absence of all growth as recommended by the BSAC Working Party (1991), then should the inoculum contain resistant mutants, the MIC determined would be that of the most resistant organisms. Lastly, dilute solutions of compounds incorporated into agar may lose their potency with time (Houang et al., 1983). One advantage of the agar diffusion technique over MIC measurement is that it tests the sensitivity of the organism to a concentration gradient of drug, thus avoiding the use of stepwise concentration increments. Plotting zone of inhibition (the square of the distance between the well edge and the zone edge) against log^o dose usually produces a linear relationship from which the critical inhibitory concentration (CIC) may be determined (Cooper & Woodman, 1946). The CIC is a measure of the sensitivity of the organism to the test antimicrobial. The sizes of zones of inhibition are dependent on many factors including the rate of diffusion of the drug molecules, incubation temperature, content of the test medium, inoculum size and its growth rate. The rate of diffusion of a compound varies with its molecular size and shape, its charge, and therefore its retention by the medium, and the concentration of compound at its source (Linton, 1961). CIC end points determined from zones of inhibition produced by slowly diffusing compounds cannot therefore be directly compared with those of faster diffusing compounds. The width of a zone of inhibition is a function of the diffusibility of the compound as well as its antimicrobial activity. Temperature affects both the rate of growth of the organism and the rate of diffusion and should therefore be kept constant. The test medium will affect diffusibility of the compound and thus strict comparisons cannot be made between results obtained on different media. The results obtained using nutrient agar are therefore not strictly comparable to those

DISCUSSION Ï85 produced with Sabouraud agar. The growth rates of different organisms vary and these can affect the zone size. The latter is dependent upon both the rate of diffusion, which determines the distance the CIC of a compound has diffused out into the agar, and growth rate, which determines the time taken to produce a visible density of cells at the zone edge. The majority of these factors, such as temperature, test medium and inoculum size were controlled, thus, the variables were the inherent growth rates of the various test organisms and the diffusibility of the compounds. MICs determined for nitrofurantoin (xm Table 4.7.1 ) were within the reported range of 0.4 -1 0 pg ml'^ for susceptible organisms (Therapeutic Drugs, 1991) and greater than 32 pg mM for the remaining organisms indicating that they were resistant (Martindale, 1996). The parent compounds, 5-nitrofurfuraldehyde (X ), 5- nitrofuranacrolein (X I) and nifuroxime (X II), which itself is used as a topical antibacterial, gave MIC (Table 4.7.1) or CIC (Table 4.7.2) values, against all organisms, that were similar to, or lower than, those of nitrofurantoin.

Of the polyamine conjugates, compounds I, II and I I I gave MICs lower than those of nitrofurantoin against all of the organisms tested (Table 4.7.1), showing that they were ‘broad spectrum’ antibacterials, with good antifungal activity.

Compared to their parents, X, X II and X respectively, these three compounds showed lower or identical MICs except compound I against C. albicans for which the

MIC was 1.5-fold that of its parent. The CICs of compoundsI and I I were similar to or lower than those for nitrofurantoin (Table 4.7.2). However, when compared to their parents (X and X II), the CICs of compounds I and I I were higher for the

bacteria tested. Compound I I I only gave a CIC against S. cerevisiae, which was

lower than that recorded for its parent (X), no zones of inhibition being recorded for

the other test organisms. MICs were readily determined for compound I I I (Table 4.7.1) and lack of demonstrable activity in the CIC test may reflect its poor diffusion characteristics.

It is interesting that compound IX , which is similar in structure to compound m, but has extra vinyl groups (Figure 5.7.1), gave an excellent CIC for S. cerevisiae (1.1 pg mM), the highest antifungal activity of any compound tested. Like compound

I I I it gave no zones of inhibition when tested against S. aureus and E. coll. The

good antifungal activities demonstrated by compounds I I I and IX might be worth

DISCUSSION Ï86 pursuing as nitrofurantoin(X III) gave no zones of inhibition in the CIC test against either of the fungi.

I ll O2N. .0 O NO

IX O2N. .0 O NO

Figure 5.7.1. Extra vinyl groups between the nitrofurans and the polyamine (spermidine)

Compound V III, which like compound IX has compound X I as a parent (Figure 4.7.1) and therefore an extra vinyl group, was tested against S. aureus, E. co//and S. cerevisiae. It gave antibacterial CICs similar to or less than nitrofurantoin and showed respectable antifungal activity. However, it was not as active as its parent.

Compound IV , the benzoyl derivative, gave MICs greater than those of its

parent (X ) for all of the organisms and greater than nitrofurantoin for all of the bacteria (Table 4.7.1). However, from the calculated CICs (Table 4.7.2), compound

IV appeared considerably more active against most of the organisms than its parent

or nitrofurantoin (XIII).

Compounds V I and V II, the two glucosamine derivatives (V II being acetylated) also gave higher MICs (Table 4.7.1) for all of the organisms when

compared to their parent (X ) or nitrofurantoin (X III). However, again these compounds appeared more active when comparisons were made using the CIC data (Table 4.7.2). Thus, conjugation of nitrofuran to glucosamine, a component of

cell membranes, may improve activity when compared to nitrofurantoin (XIII). Acétylation of the glucosamine group made little difference to the CICs measured.

Compound V, the methyl lysine derivative, gave a CIC value against S.

aureus which showed that it was more active than its parent (X ) and nitrofurantoin (x m ) against this organism. However, no CICs could be determined for compound

V against E. coli or S. cerevisiae (Table 4.7.2). Thus, overall, it can be seen that the compounds retain, and in the majority of cases also improve upon the activities of their parent nitrofurans against all of the

DISCUSSION 187 bacteria tested. As the antipseudomonal activity of nitrofurantoin is poor it is encouraging to see that the compounds tested show activity against P. aeruginosa. Some also exhibit good antifungal activity, which is not shown by nitrofurantoin.

7. Mutagenicity of novei nitrofuran compounds

DNA damage caused by genotoxic agents may lead to mutagenesis and ultimately carcinogenesis. Several tests for detecting genotoxic agents have been developed. Two such tests are the Ames Salmonella mutagenicity assay (Maron and Ames, 1983), from which the mutagenic potency of compounds can be calculated, and the SOSIumu test (Oda et al., 1985) which detects whether a compound induces expression of a P-galactosidase gene fused to another gene under SOS control, thereby giving indication of DNA damage. Several hundred nitrofurans have been synthesized and tested for use since World War Two. Many have been tried in clinical or veterinary medicine, or as food and drink preservatives. However, nitrofuran compounds are mutagenic, for example, furylfuramide (AF2) a food preservative had to be withdrawn from use in Japan (Tazima, 1979; Tazima etal., 1975). Some nitrofurans are also carcinogenic (Cohen etal., 1973; McCann & Ames, 1976; McCann etal., 1975). The mutagenicity of nitrofurans, as with their antimicrobial activity, is caused by transient intermediates of the nitroreduction process (Bryant & McCalla, 1980) and mutagenic potency has been found to relate to the extent of DNA-adduct formation (Wentzell & McCalla, 1980). Nitrofurans are activated by endogenous nitroreductases but their mutagenic potency is not affected by the presence of exogenous enzymes such as the rodent hepatic extract present in S9-mix, they are therefore classified as direct acting mutagens.

From the results of the Ames assay (Results 8 ) it can be seen that nitrofurantoin induced a high number of revertants in S. typhimurium strain TA100 but was non-mutagenic, in the sub-inhibitory concentrations used, against strain TA98 (Figure 4.8.6). This result confirms the data reported by Jurado et al. (1993) and indicates that base-pair substitution is the primary cause of mutagenicity by this compound. Koch et al. (1994) suggested that the predominant mutations induced by nitrofurantoin were GC to TA transversions. It should, however, be noted that although no concentration of nitrofurantoin tested was mutagenic against TA98, it

DISCUSSION Î88 was bactericidal to both TA98 and TA100, at concentrations greater than 5|jg/plate. The number of revertant colonies per plate of TA98NR, the nitroreductase deficient strain, growing in the presence of the highest concentration of nitrofurantoin tested in the absence of S9-mix (Figure 4.8.8) was just double that of the control, indicating slight positive mutagenicity. This may have resulted from adduct formation which does not require nitroreduction. The higher resistance of TA98NR to nitrofurantoin, compared to strain TA98, would allow detection of such low level mutagens. Comparison of the mutagenic potencies of the novel nitrofuran compounds with nitrofurantoin (Table 4.8.3) indicate that nitrofurantoin was more mutagenic against strain TA100 than the novel compounds but less mutagenic against TA98NR. It can also be seen that compound n is not mutagenic against strain TA98 and compound m is not mutagenic against strain TA100. CompoundsI and IV show mutagenicity against both strains TA98 and TA100. They also show higher mutagenic potencies against TA98NR than TA98 indicating that they do not require nitroreduction to be mutagenic. The decrease in mutagenicity of compounds I and IV observed in the presence of S9-mix may indicate that these compounds are metabolized to inactive species by exogenous enzymes present in the S9-mix. The SOS/umu test determines genotoxicity by measuring the activity of SOS- induced p-galactosidase. If DNA is damaged the SOS error-prone repair system is activated. As lacZ, the gene for P-galactosidase, is fused to the SOS-controlled umuDC operon on plasmid pSK1002, when UmuDC is produced so is LacZ. Provision of a substrate for the enzyme enables its activity to be measured as change in optical density of the reaction mixture. Nitrofurantoin was more genotoxic, as measured by induced p-galactosidase

activity, in the absence or presence of S9-mix, than ICR 191 and 2 - aminoanthracene, the two positive controls used in this test (Figures 4.8.12 & 4.8.13). In the absence of S9-mix, the minimum genotoxic concentration (MGC) of nitrofurantoin was 0.25 pg mM (Table 4.8.3), the lowest concentration tested. Pal et ai (1992) also showed nitrofurantoin to induce SOS/umu at 0.2 pg ml'\ The MGGs of novel nitrofuran compounds I and IV were both 6.25 pg mM indicating that

these compounds were at least 25-fold less genotoxic than nitrofurantoin. Novel nitrofuran compounds n and I I I did not induce genotoxicity at concentrations up to 50 pg ml'\ the highest concentration tested, which on these data indicate that they

DISCUSSION Ï89 are at least 200-fold less genotoxic than nitrofurantoin. In the presence of S9-mix, the genotoxic end points for nitrofurantoin and novel compounds II and III did not alter, whilst those for compounds I and IV increased 2- and 4-fold respectively. This is evidence that these compounds do not require any enzyme system present in 89- mix to be activated into genotoxins. In fact, the increased concentrations of compounds I and IV required to produce genotoxicity suggest that some component of 89 might be acting as a detoxicant. Thus, in general, the novel nitrofuran compounds have been shown to be significantly less genotoxic than nitrofurantoin and in the majority of cases are also significantly less mutagenic than nitrofurantoin.

8 . Activities of novel nitrofuran compounds against polyamine transport deficient mutants of Escherichia coli E. coli normally synthesizes putrescine and spermidine (Tabor & Tabor, 1984; Tait, 1985). The organism also imports putrescine, spermidine and spermine (Colbourn etal., 1961; Kashiwagi eta!., 1986) via transport systems (Furuchieta!., 1991; Kashiwagi et a!., 1991, 1992, 1993; Pistocchiet a!., 1993). To date, three transport systems have been described. There is a spermidine-preferential

spermidine/putrescine transporter ( 8 P transporter), a putrescine-only transporter (PO transporter) and a low activity putrescine transporter (putrescine/ornithine antiporter). The E. coli strains used were MA261 and its derivatives KK313, KK313 pofF::Km and NH1596. Mutations within the speB and speC genes of all strains make them polyamine-requiring. They cannot synthesize putrescine and are therefore dependent on an exogenous supply of putrescine from the medium in

which they are growing. 8 train MA261 is fully proficient for all polyamine transport systems, whereas the three derivatives are missing one or more genes encoding proteins required for polyamine transport (Kashiwagi et al., 1986; Pistocchi etal.,

1993). All derivatives have a defunct 8 P transporter and are therefore unable to import spermidine and spermine, as spermine is also likely to be transported by this

system (Kashiwagietal., 1986). 8 train KK313 poF;:Km and NH1596 have a defunct

PO transport system, in addition to their 8 P deficiency, and are therefore only able to transport putrescine through the low activity putrescine transporter. The results showed that, at the concentrations used, compound I, the

DISCUSSION Ï9Ô putrescine conjugate (Figure 4.9.5) and compound IV, the benzoyl derivative (Figure 4.9.8) were bacteriostatic and compound U, the spermine conjugate (Figure 4.9.6) and compound in , the spermidine conjugate (Figure 4.9.7) were bactericidal to all strains. If uptake of compounds II and III were dependent on the polyamine transport systems described above, they should have shown much reduced activity against all of the transport deficient strains compared to their activity against strain MA261. That their activity against the transport mutants was similar to that against MA261 suggests their uptake is not dependent on the SP transport system. If activity of compound I were dependent on active transport, it was expected to show low activity against KK313 potF::Km and NH1596 and higher activity against KK313 and MA261 as only the latter two strains have a functioning PO transporter. However, although not demonstrating much bactericidal activity, it showed the highest kill against strain NH1596. It was, however, only bacteriostatic against KK313 pofF:: Km. If uptake of the polyamine derivatives were solely dependent on the availability of active transport mechanisms, compounds II and III should not have shown activity against any of the transport deficient strains. They were, in fact, as bactericidal as nitrofurantoin (compare Figures 4.9.6 & 4.9.7 with Figure 4.9.4). It may be that other, as yet unknown, transport systems are functioning in the mutants, which could explain the activity of the polyamine conjugates. However, it is possible that, because of the bulky nitrofuran groups and/or the di-(nitrofuran) structure of compounds I, II and III, they are not transported into the cell via any specific polyamine transport system but gain access to their site of action via more general uptake mechanisms. The bacteriostatic activity of compound IV, the benzoyl derivative, indicates an alternate route of uptake is available and it may be that all of the novel nitrofuran compounds gain access to the cell via this system.

DISCUSSION 191 PART SIX UMMARY CONCLUSION,

SUMMARY AND CONCLUSIONS 192 The mechanism of action of nitrofurans, such as nitrofurantoin, results, as with quinolone antibacterials from the binding of the drug or its metabolites to DNA. This stalls DNA replication and induces the SOS response, which can lead to mutagenic error-prone DNA repair. The summation of these processes is cell death. However, unlike quinolones, nitrofurantoin is fully active when RNA and protein syntheses or cell division are inhibited. It has also been shown that nitrofurantoin is much less susceptible than the quinolones to the development of resistance due either to spontaneous or induced mutations. The carriage of mutator plasmids also has little effect on the development of nitrofurantoin resistance. Polyamines are specifically and actively transported into bacterial cells. The functions of polyamines suggest they are closely associated with DNA in the intracellular environment. Nitrofurans are reduced by endogenous nitroreductases to active metabolites which, as stated above, bind to DNA. Thus, conjugation of a nitrofuran with a polyamine might enable specific uptake of the nitrofuran and its targeting to microbial DNA, thus increasing the selective toxicity of this underdeveloped group of antibacterials. Nine novel nitrofuran compounds (I - IX) were synthesized and tested for antimicrobial activity. Compounds I - IV were also tested for both mutagenic and genotoxic potential and for activity against polyamine-requiring polyamine transport- deficient strains of E. coli. The four novel nitrofuran compounds (I - IV) did not behave as predicted against the polyamine transport deficient mutants. It was expected the compounds would only exhibit significant antibacterial activity against the transport-proficient parent strain. However, as compounds I and IV were bacteriostatic and II and III were bactericidal to all of the transport-deficient mutants, it must be assumed that alternate routes of uptake were available for the novel nitrofuran compounds. Antimicrobial activities were determined as minimum inhibitory concentrations (MICs) on solid medium and as critical inhibitory concentrations (CICs) calculated from agar diffusion experiments. Compounds I, II, III, IV, VI and V II showed broad spectrum antimicrobial activities against all of the organisms tested. Antimicrobial activities against nitrofurantoin-sensitive Gram-positive and Gram-negative bacteria were improved, with compounds I, II, III, IV and V I also showing antipseudomonal and antifungal activity, neither of which was exhibited by nitrofurantoin. When

SUMMARY AND CONCLUSIONS 193 compared to nitrofurantoin, compound V showed improved antistaphylococcal activity, compound V III showed improved antistaphylococcal and antifungal activities and compound IX showed improved antifungal activity. Compounds I - IV were also tested for mutagenic and genotoxic potential using the Ames Salmonella mutagenicity assay and the SOSIumu induction test respectively. From the Ames assay it was observed that nitrofurantoin was not mutagenic in S. typhimuhum strain TA98 or TA98NR but was highly mutagenic in TA100, indicating it induced base-pair substitution mutations rather than frameshifts. Compounds I, II and IV gave mutagenic potencies for S. typhimuhum strain TA100 much lower than that of nitrofurantoin. Compound III was not mutagenic in TA100. In TA98, compounds I, III and IV were mutagenic whereas nitrofurantoin and compound II were not mutagenic. From the SOS/umu test results, compounds II and III were not observed to cause induction of the SOS response even at the maximum concentration tested. This concentration was at least 200 times that of the minimum concentration of nitrofurantoin shown to cause induction of the SOS response. The concentrations of compounds I and IV required to induce the SOS response were 25-fold above that shown by nitrofurantoin. Thus all four novel nitrofuran compounds tested were considerably less genotoxic than nitrofurantoin. Of the compounds synthesized numbers I, II and III, the polyamine conjugates, appear most promising. They have broad spectrum antimicrobial activity, including inhibition of P. aeruginosa, K. aerogenes and the fungi, C. albicans and S. cerevisiae, against which nitrofurantoin has no activity. These compounds also show reduced genotoxic and mutagenic potency as compared to nitrofurantoin. The next step towards development of this new group of antimicrobialsmight be to conduct preliminary pharmacokinetic experiments in animals to show if their distribution would permit use as systemic anti-infectives.

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REFERENCES 231 ApBeinlix A

Each novel nitrofuran compound was characterized using one or more of the following spectroscopy techniques; nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR) and mass spectroscopy. These techniques also give an indication of the purity of the compounds. The data listed in Results 6.1 to 6.9 is explained below using novel nitrofuran compound I (bis-5- nitrofurfurylidenylputrescine) as an example.

NMR data In a strong magnetic field and with the application of energy the spins of nuclei of atoms assume different energy levels (resonance). The nuclei of protons (^H) are usually studied and in organic molecules they resonate at slightly different energies depending on the atoms to which they are attached and the presence of nearby protons and electrons. The resultant spectrum shows peaks of absorption at different chemical shifts (Ô) and is unique for the compound.

TMS tnhWiOM rt*H C ÎC U rervioie

J\K Hb

Figure A1. NMR spectrum for compound I (bis-5-nitrofurfurylidenylputrescine)

APPENDIX A 232 The NM R spectrum for compound I (Figure A1), dissolved in CDCI3 and

DMSO-cfg, shows various single (s), double (d) and broad (b) peaks and can be abbreviated to: 5 (80 MHZ; CDCI3/DMSO-cfe) 8.20 (s, CH=N), 7.59 (d, H;),

7.03 (d, H3), 3.91-3.55 (b, CHg), 1.875-1.61 (b, CHg) This means that the single peak at 8.20 ppm is consistent with hydrogen atoms in CH=N groups, the double peaks at 7.59 and 7.03 ppm are consistent with hydrogen atoms H 3 and H4 on the nitrofuran and the broad peaks at 3.91-3.55 ppm and 1.875- 1.61 ppm are consistent with aliphatic hydrogens Mg and attached to the polyamine. The integrals of the peaks are proportional to the number of protons causing absorbance. Figure A1 shows integrals in the ratio 2:2:2:4:4 corresponding to the two CH=N, two H4, two H3, four Mg and four groups (Figure A2).

N NO.

Figure A2. Compound I (bis-5-nitrofurfurylidenylputrescine) showing positions of hydrogen atoms

IR data The basic function of an infrared spectrophotometer is to measure and record the extent to which a sample absorbs infrared energy as a function of frequency (v^g^). The result is a spectrum as shown in Figure A3 and, as with peaks on NMR spectra, troughs produced on IR spectra are characteristic of the sample. The IR spectrum for compound I showed three major troughs which can be abbreviated to: v^ax (KBr disc) 1640 and 1480 (CH=N), 1350 (NOg) cm ^ The troughs at 1640 and 1480 cm'^ suggest the presence of CH=N groups and the trough at 1350 cm^ suggests the presence of a NOg group.

APPENDIX A 233 i

1 7 0 0 1 4 0 0 1 100 3 0 0

Figure A3. IR spectrum for compound I (bis-S-nitrofurfuryiidenylputrescine)

Mass spectroscopy The production of fragments, by electron impact (E. I.), of a neutral substance is another technique for the identification of compounds. The results (Figure A4) appear as a histogram showing the percentage of each fragment of specific atomic mass (m/z). The data can be abbreviated as follows: m/z(E. I.) 194(02NC4H20CHN(CH2)2CHCH2), 181 ( 0 2 NC4H2 0 CHN(CH2)3),

153 (O2NC4H2OCHNCH2), 139 (O2NC4H2OCHN), 84 (N(CH 2)4N), 70 ((CH 2)4N) and shows the structures of the fragments expected at the atomic masses stated. E.g. a fragment of atomic mass 194 would have a structure of (02NC4H20CHN(CH2)2CHCH2).

APPENDIX A 234 C2H079B: Scan 123-128 (14.35 14.93 utn) B aia: 79.00 In t: 1 .15399e->006 S a n fle : El Spectre#, B TOeV, 100*

95%

90%

85%

80%

75%

70%

65% Sample 6 E IW

60%

55%

50%

45%

40%

35%

50% SO I 39

25%

20%

15%

10%

5% 0* 75

Figure A4. Mass spectrum for compound I (bis-5-nitrofurfurylidenylputrescine)

APPENDIX A 235 PAPER, RESISTANCE OF R PLASMID-CONTAINING STRAINS OF ESCHERICHIA COLI TO NITROFURANTOIN

C A Sharpe and R J. Pinney, Microbiology Section, Department o f Pharmaceutics, The School o f Pharmacy, University o f London, Brunswick Square, London WC IN 1 AX

DNA repair-deficient strains o f bacteria are more sensitive than repair- Nitrofurantoin MICs varied by greater than 30-fold, depending on the proficient strains to some drugs that damage DNA. However, Pinney et al repair deficiency o f the strain (Table I). Plasmid R46 increased the MICs (1992) found little difference in sensitivities to nitrofurantoin (NF). o f strains TK50I and JC3890, but had no effect on the MICs o f the other Repeating these experiments on a wider range o f Escherichia coli strains, strains. The order o f resistance o f the strains was similar to their order o f using smaller concentration differences, we report newly determined resistance to DNA damage induced by ultraviolet light (UV). When minimum inhibitory concentrations (MICs), and use these to test survival strains were exposed to 20 times their respective MICs, surv ival after 1 of strains into which a DNA damage-protecting plasmid has been hour still varied by more than lOLfold (Fig. 1). The fully DNA repair- introduced. proficient strain A B l 157 (1) was still the most resistant, but resistances then diminished in the order AB2494 (2) >AB2470 (3) >AB2463 (4) MICs in nutrient agar, were determined for undiluted and for 10'^ and 10 ^ >JG138 (5) >TK501 (6) >JC3890 (7) (Fig. 1). Recombination repair- dilutions o f overnight nutrient broth cultures. Sensitivities to lethal deficient strains appeared, therefore, more resistant to bactericidal concentrations in nutrient broth were obtained by plating dilutes samples concentrations o f NF than to UV or to growth inhibition by NF. on nutrient agar. Once again, plasmid R46 only 100 protected strains TK 50I and Table 1. Sensitivities to nitrofurantoin JC3890. These extended data Strain Genetic Repair MIC (mg L ’ confirm the conclusion o f Pinney defect deficiency nitrofurantoin) et al (1992), that plasmid- R- +R46 mediated resistance to nitrofurantoin is unlikely to be of A B I157 - none 10 10 TK702 umuC EPR 7.5 7.5 clinical significance. It remains to JGI38 polA ER 5 5 be seen whether the mcrease in GO AB2470 recB RR 3 3 mutation frequency mediated by AB2494 lexA EPR 2 2 DNA-protecting plasmids has TK501 uvrB umuC EPR&ER 0.75 1.5 0.01 significant effect on mutation to 0 15 30 45 60 JC3890 uvrB ER 0.5 10 nitrofurantoin resistance. Time (min) AB2463 recA EPR,EP&RR 0.3 0.3 Sensitivities o f Pinney, R. J. et al (1992) J. Pharm. RR = recombination repair, EPR = error-prone ^ repair strains to NF. Pharmacol. 44: 1057 SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF NITROFURAN-POLYAMINE CONJUGATES

R. A. Watt, Y. M. B. O'Reilly, B J. Williams, *C A. Sharpe and *R J. Pinney, Department o f Pharmaceutical Chemistry, and *MicrobioIog>' Section, Department o f Pharmaceutics, The School o f Pharmacy, University o f London, London W C IN I AX, UK

Polyamines, such as putrescine and spermidine are widely distributed in Concentrated solutions o f all four derivatives and o f nitrofurantoin (5) as nature In bacteria they are synthesized via amino acid decarboxylation, or control were prepared in dimethylsulphoxide. These were diluted further in are taken into the cell by active transport. Their function is uncertain, but water and tested for antimicrobial activity. One hundred m illilitres o f molten they associate, as polycations, with polyanionic DNA Nitrofuran nutrient agar for bacteria, or Sabouraud dextrose agar for yeasts, were cooled antimicrobials (McOsker & Fitzpatrick 1994), which have limited use to 55 °C and seeded with 0.1 mL of overnight cultures of Staphylococcus because o f toxicitv and poor solubility, also interact with DNA. We report aureus NCTC 6571, Escherichia coli KL16, or a laboratory strain o f the synthesis and activit\' o f some nitrofuran-polyamine conjugates, prepared Saccharomyces cerevisiae. Twenty m illilitre quantities were poured into 9 to test for increased uptake, leading to improved action and reduced toxicitv, cm diameter Petri dishes and allowed to set. Wells, cut into the agar with a compared with currently-used nitrofurans. sterile cork borer, were filled with antimicrobial solution. After incubation, the diameters o f any zones o f inhibition were measured; data points in Fig. 5-Nitrofurfuraldehyde, or its stable diacetyl equivalent, was reacted in 1. represent the means o f three determinations. ethanol solution at temperatures between 0 and 80 °C with putrescine, 1 25 glucosamine, methyl lysine or N-acety lethylenediamine to yield the A ll compounds were active against S. conjugates 1-4 respectively. The products were isolated as solidds by aureus (Fig. 1). Nitrofurantoin extraction and chromatography, and characterised by IR, NMR and mass showed greatest effect on E. coli, spectroscop). with compound 3 being inactive. Neither compound 3 nor nitrofurantoin were active against S. cerevisiae, whereas compounds 1, 2 0;N XIL O CH=N(CH2),N=CH r iiO NO^ (1) and 4 were inhibitor) . These data confirm that nitrofuran-polyamine n r i (3) conjugates retain good antibacterial 0;N O CH=N(CH2)^CHN=CH O NO; activity. Some are active against N=CH O NO CO;CH, Concentration (pg mL"’) fungi. Fig. 1. Susceptibilities o f S. aureus to nitrofurantoin (5) McOsker, C C , Fitzpatrick, P. M. (1994) J. Antimicrob. Chemother. 0;N j n O CH=N(CH;);NHCOCH; (4) and to the nitrofuran-polyamine conjugates 1-4. 33(SupplA): 23-30 ANTIBACTERIAL ACTION OF NITROFURANTOIN AGAINST ESCHERICHIA COLI WHEN PROTEIN SYNTHESIS, RNA SYNTHESIS OR CELL DIVISION ARE INHIBITED

C A. Sharpe and R J. Pinney, Microbiology Section, Department o f Pharmaceutics, The School o f Pharmacy, University o f London, Brunswick Square, London WCIN I AX, UK.

Both quinolone and nitrofuran antibacterials bind to DNA causing strand breaks. or die in NB containing Cm (3) or R f (4), nor in PBS (9). The addition o f Cm (5) or Their precise bactericidal actions are unknown, but modem fluoroquinolones exhibit R f (6) after 2 h. did not affect the bactericidal activity o f N f (2). Pre-exposure to R f three mechanisms o f activity referred to as A, B and C. Mechanism A is abolished if (8) or Cm (7) delayed the onset o f Nf-induced lethality by 1 or 2 h. respectively. cells cannot divide, or synthesise RNA or protein. Mechanism B occurs in the absence However, once death commenced, the rate was similar to that o f the N f control (2). o f RNA or protein synthesis or cell division. Mechanism C also occurs in cells that Incubation in PBS (10) increased the lethal effect o f N f over that seen in NB (2). cannot divide but requires RNA and protein synthesis (Smith & Lewin. 1988). We report experiments to determine whether the bactericidal activity of a nitrofuran is These results contrast to those similarlv affected. obtained with the quinolones. Mechanism A is abolished in Log phase cells o f Escherichia coli A B I 157 grown in nutrient broth (NB) were PBS whereas the activity o f N f exposed to nitrofurantoin (Nf) at 200 jig mL ’ in NB. Rifampicin (Rf) at 160 does not require cell division. |ig mL'* and chloramphenicol (Cm) at 20 Mechanisms A and C are - 3 .4.9 Table I. Incubation mixtures. jig mL'* were used to inhibit R N A or 10 abolished by Cm or R f due to the Antibiotic added at protein synthesis respectively. NB-grown requirement for inducible protein synthesis (Howard et al., 1993), IM 0 hours 2 hours log phase cells were also washed twice in -- whereas N f activity is delayed by 1 NB phosphate buffered (normal) saline (PBS) 2 NB N f - pre-exposure to Cm (7) or R f (8) and resuspended in PBS. This inhibits cell 3 NB Cm - but then proceeds at the same rate division but allows R N A and protein 4 NB R f - as control (2). Thus, unlike the 5 NB N f Cm synthesis to continue. Bacteria were 10 2 ,5.6 quinolones, the bactericidal suspended in incubation mixtures (IM , 0 2 4 6 6 NB N f R f Time of exposure (hours) activity of nitrofurantom is 7 NB Cm N f Table 1) at initial viable counts of 10^ independent o f RNA and protein 8 NB R f N f organisms mL . ^rvival was determined Fig. I. Survival o f E. coli A B I 157 syntheses and o f cell division

9 PBS -- by diluting in NB and plating on nutrient 10 PBS N f - agar. Howard, B.M.A. et al. (1993) Arzneim.-Forsch./Drug Res. 43 (II): 1125-1129 Smith, J. T., Lewin, C. S. (1988) In: Andriole, V. T. (Editor) The Quinolones. London: Academic Press Ltd.: 23-82 From Fig. 1 the following conclusions can be drawn. Cells did not grow Antimicrobial Activities of Novel Nitrofuran-Polyamine Conjugates C.A. SHARPE, R.A. WATT and R.J. PINNEY, The School of Pharmacy, University of London, Brunswick Square, London WCIN I AX, UK

Polyamines are actively transported into bacteria and bind to DNA. We report the synthesis and antimicrobial activities of some nitrofuran-polyamine conjugates, designed to take advantage of the cellular properties of polyamines to target the antimicrobial nitrofuran to its site of action

Condensation of 5-nitrofuraldehyde with putrescine, spermidine or spermine, followed by extraction into IM hydrochloric acid and then ethyl acetate yielded 6As-5-nitrofuryl-putrescine

(I), /?/'ç-5-nitrofuryl-spermidine (II) and 6zj-5-nitrofuryl-spermine (III) respectively. Structures were confirmed by nuclear magnetic resonance and mass spectroscopy. Concentrations that inhibited the growth of isolated inocula (MICs) were determined on nutrient agar for bacteria and on dextrose Sabouraud agar for fungi, and compared with those of nitrofurantoin (IV), putrescine (V), spermidine (V I) and spermine (V II).

V, VI and V II were not inhibitory (MICs > 75 mg L'^ for all organisms tested). MICs for IV against Escherichia coli. Pseudomonas aeruginosa. Staphylococcus aureus and Candida albicans were 5, >75, 5 and >75 mg L’‘ respectively Similar data for I for the same range of organisms were 1,1,1 and 7.5 mg L'* respectively; for II were 1,1,1 and 10 mg L ' respectively; and for III were 1,1,1 and 10 mg L ' respectively

The conjugates are thus more active than nitrofurantoin against all organisms tested, showing particularly good antipseudomonal and antifungal activities.