THE IMPACT OF PHARMACOKINETICS ON THE EMERGENCE OF IN VITRO BACTERIAL RESISTANCE TO CEFOVECIN.

Dr Sarah A. Robson

BVSc(Hons) MANZCVS(pharmacology) MVS

Faculty of Veterinary and Agricultural Science

The University of Melbourne

Student Number: 206696

2017

1 ABSTRACT

Introduction Antibiotic resistance is a global issue in both human and veterinary medicine with substantial social and economic impact. Cefovecin, a third-generation cephalosporin antibiotic has a half-life of approximately 166 hours in the cat. This contrasts with the significantly shorter two-hour half-life of cephalexin, an oral first-generation cephalosporin. Sub-inhibitory antibiotic cultures were used to investigate the impact of cephalosporin antimicrobial concentration and time on the development of in vitro bacterial resistance. It was hypothesized that the long half-life of cefovecin would cause an increased emergence of bacterial resistance.

Materials & Method Five clinical isolates of Escherichia coli from the U-Vet Werribee Animal Hospital laboratory collection were subjected to sub-inhibitory conditions. Escherichia coli National Collection of Type Cultures England (NCTC) 10418 and NCTC 12441 (American Type Culture Collection (ATCC) 25922) reference strains were used as controls. Minimum inhibitory concentrations (MIC) of cefovecin and cephalexin were determined by broth microdilution techniques as described by the Clinical and Laboratory Standards Institute (CLSI) standards. Antibiotic concentrations of 0 - 0.95 x MIC in Mueller-Hinton cation adjusted broth (CAMHB) were used as sub- inhibitory culture conditions. The batch-cultures were incubated on an orbital shaking platform at 37 ◦C for a total period of 144 hours. The bacteria were diluted 1:1000 into fresh antibiotic media every 24 hours. Following this, the E. coli isolates from the 0.95 x MIC cefovecin sub-inhibitory experiments were cultured in antibiotic-free media for a further 144 hours to test the persistence of any increase in resistance.

2 Results E.coli isolates cultured in antibiotic-free CAMHB for 24 hours showed no significant change in MIC to either drug, however E. coli isolates cultured in sub- inhibitory antibiotic concentrations for periods of up to 144 hours showed up to 16 times increase in MIC for cefovecin and up to 8 times increase in MIC for cephalexin. Cultures with antibiotic concentrations closer to the MIC of the E. coli isolate increased their MICs both more rapidly and to greater multiples. The 0.95 x MIC cefovecin E. coli isolates that were cultured in antibiotic-free media for a further 144 hours showed either a 2-fold or 4-fold decrease in MIC, although none returned to the initial MIC.

This research provides preliminary evidence that cefovecin should be used with prudence to limit in vivo resistance emergence.

3 DECLARATION

I, Sarah Robson declare that this thesis is original work, submitted as 75% fulfilment of the requirements of Master of Veterinary Science (Research). The thesis contains no material previously submitted for any other academic degree. The thesis is less than 30,000 words excluding tables, figures and appendices.

4 TABLE OF CONTENTS

ABSTRACT ...... 2 DECLARATION ...... 4 TABLE OF CONTENTS ...... 5 LIST OF TABLES ...... 7 LIST OF FIGURES ...... 8 CHAPTER 1: INTRODUCTION ...... 10 CHAPTER 2: LITERATURE REVIEW ...... 12 Antimicrobial resistance ...... 12 Selective Window Theories and Mutant Prevention Concentration ...... 15 Sub-therapeutic Antibiotic Concentrations ...... 16 Persistence of resistance ...... 18 Resistance to Macrolides and Association to Pharmacokinetics ...... 19 Anti-malarial Drug Resistance and Pharmacokinetics ...... 20 Cephalosporins...... 21 Extended-spectrum β-lactamases ...... 22 The Impact of Cephalosporin Use...... 24 Cephalexin ...... 26 Cefovecin...... 27 Protein binding ...... 28 Conclusion ...... 30 CHAPTER 3: MATERIALS AND METHODS ...... 31 3.1 GLASSWARE AND WATER ...... 31 3.2 MEDIA ...... 31 3.3 ANTIBIOTIC STOCK SOLUTIONS ...... 32 3.3.1 Cephalexin ...... 32 3.3.2 Cefovecin ...... 33 3.4 BACTERIA SELECTION ...... 33 3.5 MICRODILUTION ...... 35 3.5.1 96 Well Plate Preparation ...... 35 3.5.2 96 Well Plate Inoculation ...... 36 3.5.3 Inoculum Testing...... 37 3.6 SUB-INHIBITORY CULTURES ...... 37 3.6.1 Cefovecin Sub-MIC Cultures ...... 37 3.6.2 Cephalexin Sub-MIC Cultures ...... 38 3.6.3 Cefovecin and cephalexin Sub-MIC Cultures with 5 E. coli isolates...... 39

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3.7 PERSISTENCE OF RESISTANCE CULTURES ...... 40 3.8 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY STABILITY STUDY ...... 41 3.8.1 Chromatographic system and conditions ...... 41 3.8.2 Chemicals used ...... 41 3.8.3 Sample preparation extraction procedure ...... 42 3.8.4 HPLC assay validation procedure ...... 42 3.8.5 Stability study procedure ...... 43 CHAPTER 4: RESULTS ...... 44 Broth Microdilution Initial MICs ...... 44 Sub-Inhibitory Cultures with One E. coli Isolate ...... 45 Sub-Inhibitory Cultures with Five E. coli Isolates ...... 47 Persistence of Resistance Cultures ...... 53 HPLC Results ...... 55 Validation of Assay: ...... 55 Stability Assay Results: ...... 63 CHAPTER 5: DISCUSSION ...... 65 Broth Microdilutions ...... 65 Sub-MIC Cultures ...... 67 Persistence of Resistance ...... 69 Repeatability ...... 72 Study Design ...... 72 HPLC Assay ...... 74 Significance of Results ...... 77 CHAPTER 6: CONCLUSION ...... 79 REFERENCES: ...... 83

6 LIST OF TABLES

Chapter 1

Chapter 2

Chapter 3

Table 3.1. E. coli isolate numbers, species of origin and type of specimen.

Chapter 4

Table 4.1. Cephalexin and cefovecin MICs for 5 E. coli isolates determined by broth microdilution technique (CLSI).

Table 4.2 Cefovecin Stability Assay over 24 hours in CAHMB incubated at 37°C.

Chapter 5

Chapter 6

7 LIST OF FIGURES

Chapter 1

Chapter 2

Figure 2.1 Cephalexin structure

Figure 2.2 Cefovecin structure

Chapter 3

Chapter 4

Figure 4.1a. Cephalexin MICs after culture in CAMHB with no cephalexin, 0.2 x MIC cephalexin, 0.4 x MIC cephalexin and 0.8 x MIC cephalexin.

Figure 4.1b. Cefovecin MICs after culture in CAMHB with no cefovecin, 0.05 x MIC, 0.1 x MIC, 0.2 x MIC, 0,4 x MIC and 0.8 x MIC.

Figure 4.2a. Cephalexin control- CAMHB and no cephalexin with 5 E. coli isolates

Figure 4.2b. Cefovecin control- CAMHB and no cefovecin with 5 E. coli isolates.

Figure 4.3a. Cephalexin 0.4 x MIC cultures with 5 E. coli isolates

Figure 4.3b. Cephalexin 0.8 x MIC cultures with 5 E. coli isolates.

Figure 4.4a. Cefovecin 0.8 x MIC cultures with 5 E. coli isolates

Figure 4.4b. Cefovecin 0.95 x MIC cultures with 5 E. coli isolates.

Figure 4.5. E.coli isolates post 144 h exposure to 0.95 x MIC cefovecin passaged through antibiotic free media.

Figure 4.6. Chromatogram Cefovecin 0.5 mg/L standard in mobile phase

Figure 4.7. Chromatogram Cefovecin 0.5 mg/L fully extracted CAMHB sample

8 Figure 4.8. Chromatogram Cefovecin 0.5 mg/L, after 24 hours incubation in CAMHB

Figure 4.9. Chromatogram Cefovecin 10 mg/L standard in mobile phase

Figure 4.10. Chromatogram Cefovecin 10 mg/L fully extracted CAMHB sample

Figure 4.11. Chromatogram Cefovecin 10 mg/L, after 24 hours incubation in CAMHB

Figure 4.12. Spiked CAMHB Cefovecin Standards Calibration Curve

Figure 4.13. Extracted CAMHB Cefovecin Standards Calibration Curve

Chapter 5

Figure 5.1. Reading 96 well cephalexin MIC plates on white background

Figure 5.2. Reading 96 well cephalexin MIC plates on black tiles with light

Chapter 6

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CHAPTER 1: INTRODUCTION

The primary objective of this research was to evaluate whether the pharmacokinetics of those cephalosporins which result in prolonged persistence at sub-inhibitory concentrations are more likely, than those with briefer persistence, to encourage development of bacterial resistance. Where resistance is defined as the acquisition of mechanisms allowing bacteria to grow in the presence of antibiotic concentrations higher than wild-type bacteria. Cephalexin and cefovecin were used as examples of first and third generation cephalosporins.

Aims for this research included:

Aim one: To determine the initial MIC for cefovecin and cephalexin for five E. coli isolates from clinical cases at the University of Melbourne veterinary teaching hospital.

Aim two: To perform cultures of five E. coli isolates in the presence of sub- inhibitory concentrations of cefovecin or cephalexin, for prolonged time periods and monitor any changes from baseline of the MICs.

Aim three: To culture a number of the isolates from the sub-inhibitory cultures in antibiotic-free media to assess the persistence of any increase in MICs.

Aim four: To develop and validate a high performance liquid chromatography (HPLC) assay to assess stability of cefovecin in CAMHB cultures.

The overall objective of this research was to identify factors that may help to improve veterinary antibiotic use, aiming to limit the emergence of antibiotic resistance and to minimise the impact of veterinary use of antibiotics on the development of multi-drug resistant bacteria in the human population.

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Hypotheses

H01: Prolonged exposure to sub-MIC concentrations of cefovecin does not alter the rate or frequency of the emergence of antimicrobial resistance in E. coli.

H02: Prolonged exposure to sub-MIC concentrations of cephalexin does not alter rate or frequency of the emergence of antimicrobial resistance in E. coli.

H03: Resistant E. coli lose their resistance after incubation in antibiotic-free media.

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CHAPTER 2: LITERATURE REVIEW

Antimicrobial resistance

Antibiotic resistance is a global issue in both human and veterinary medicine, with substantial health and economic impacts. It is amplified by the increasing use and misuse of antimicrobials in human, veterinary and agricultural fields. Antimicrobial resistance is one of the best examples of contemporary evolution1. It is demonstrated within the scientific literature that the use of antibiotics is associated with the selection of antibiotic-resistant bacteria2. Multi-drug resistant infections are becoming increasingly prevalent in cats and dogs with limited options for therapy3. Antimicrobial resistance is unique in that merely one bacterial cell is sufficient to transmit resistance genes to the recipient host; this is unlike many other infections or diseases3.

Development of resistance in zoonotic bacteria poses a human health threat. Possible reservoirs of zoonotic bacteria can include animal gastrointestinal tracts, sewage, water and soil. The role of antibiotic contamination of sewerage, water and soil in the transfer of antimicrobial resistance is largely unknown4. There have been reports of the transfer of zoonotic bacteria from food animals to humans, such as Salmonella isolates transfering from animals to humans through the food chain and tetracycline resistant E. coli strains5,6.

Emergence of bacterial resistance can be due to selection of pre-existing resistant subpopulations, selection of de novo random or induced mutations (which are more likely in large populations or at high cell density), acquisition of plasmids, phages or transposons, clonal spread or via induction7. Major resistance mechanisms include inactivation or increased efflux of the antibiotic, and blocking access to, or altering, the target site8. Resistance can be transferred by plasmids or other mobile genetic elements and is therefore not limited by bacterial species or genus. For example, mobile elements can transfer resistance from bacteria of

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animal origin to human pathogenic or endogenous flora. Plasmids can also carry resistance to a number of different antibiotic classes9. Plasmids can be integrated within the bacterial chromosome or separate and they have the ability to self- replicate10. They can transfer between bacteria through mobilisation by other plasmids, conjugation, natural transformation or transduction. Transposons are mobile DNA elements that can incorporate into plasmids or chromosomal DNA10.

Consequences and trends of veterinary antimicrobial use do not differ from those in the human setting. The rate of resistance development depends on the amount and patterns of use of the antimicrobials3. One of the risk factors for increase in antimicrobial resistance is increased use of antibiotics; for example, between 1990 and 1998 the increased use of lincosamide in Sweden corresponded with an increase in resistance to lincosamides in Staphylococcus organisms isolated from canine pyoderma2. Also, the frequent use of antimicrobials in veterinary hospital intensive care units has compounded the emergence of multi-resistant nosocomial pathogens in dogs3 including Enterobacter11 and Salmonella species12 .

Long-term exposure to antibiotics and the treatment of large numbers of humans and animals provide greater selective pressure than short-course treatment of a single individual. In most countries reliable statistics of antimicrobials consumed by pet animals are not available. The selective pressure of companion animal antimicrobial use and its overall effect on development of resistance are therefore very hard to estimate.

Large antimicrobial concentration gradients are formed within the animal after antibiotic administration due to factors such as tissue diffusion, metabolism and protein binding. Low or high concentrations may be present at various times in different compartments13. Therefore, bacterial populations in the animal’s microflora may face a wide range of antibiotic concentrations after drug administration. Any antibiotic concentration that can inhibit growth of the susceptible population, but not of a variant harbouring resistance, has the ability to select for resistance14.

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The same resistance genes for antibiotic classes such as tetracyclines, penicillins, cephalosporins, macrolides and aminoglycosides have been identified in both human and veterinary medicine. Currently, there is a marked difference between the patterns of antimicrobial use in food animals versus companion animals. Virtually all classes of antimicrobials are used in companion animal medicine, including those banned in some countries for use in food producing animals such as third-generation cephalosporins and fluoroquinolones15. The extensive use of antimicrobial agents in companion animals and their close environmental cohabitation with humans could potentiate the spread of antimicrobial resistance8. This could include the direct spread of resistant zoonotic pathogenic bacteria from companion animals to humans. Transmission of resistant zoonotic bacteria to humans through the food chain has already been demonstrated5. There is also the potential for transmission of antimicrobial resistant genes through transfer of commensal bacteria between animals and humans.

The gastrointestinal tract is a large reservoir of bacteria and commensals can be reservoirs for antibiotic resistance plasmids, transposons and genes16. E. coli is a common gastrointestinal commensal bacteria in animals and humans. It can be transferred from animals to humans in food, water or by contact, and may colonize transiently. Antimicrobial therapy not only has effects on the target pathogens but can alter microorganisms in the gut and throat, disrupting the resistance to colonization. Antimicrobial therapy can eliminate sensitive bacteria allowing multi-drug resistant isolates to colonise and grow17.

Disruption of resistance to colonization reduces the minimal infective dose of a pathogen but also increases the amount and duration of bacterial shedding9. It has been suggested that resistant bacteria may have an increased biological fitness cost, yet it has been shown bacteria can also acquire fitness compensatory mutations18. Immunocompromised individuals have increased risks of potential transmission15.

As early as 1974 Burton and colleagues demonstrated that antibiotic resistance can be transferred between E. coli of animal origin and indigenous E. coli in the human intestinal tract after treatment with tetracycline19.

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Staphylococcus pseudintermedius, another commensal and pathogen of dogs and cats, has been isolated from owners and veterinary staff associated with dogs with atopic dermatitis. This bacterium is rare in humans, suggesting dog-human transmission. There is a risk that resistance genes could be passed from Staphylococcus pseudintermedius to human pathogenic Staphylococcus organisms20. There has also been a case of Salmonella Virchow transmitted from two pet dogs to an infant, confirmed by pulsed-field gel electrophoresis typing21.

Saeed and colleagues (1993) estimated that 6% of campylobacteriosis in humans has been transmitted from pets22. Stenkse et al (2009) looked at clonal relatedness and antimicrobial susceptibility of faecal E.coli between healthy dogs and their owners with pulsed-field gel electrophoresis. They found 9.8% prevalence of cross-species in household sharing, with no difference in antimicrobial susceptibility patterns. No behaviours were associated with increased sharing, yet lack of hand washing after patting dogs and prior to meals was associated with increased numbers of resistant E.coli isolates23.

It is often difficult to confirm where resistance genes first appeared and the exact direction transfer occurs3, yet these examples highlight that transfer of resistance genes between companion animals and owners and vice versa is highly plausible.

Selective Window Theories and Mutant Prevention Concentration

The probability that a resistant subpopulation exists at an infection site depends on the total population burden. When the bacterial load exceeds the inverse of the mutational frequency to resistance (10-6 to 10-8) by at least one order of magnitude there is a high chance that a resistant subpopulation will be present. This explains why antibiotic resistance is a common hurdle in pneumonia therapy as the bacterial load is often greater than 1010 bacteria24.

The selective window is traditionally defined as the range of antibiotic concentrations between the MIC of the most susceptible bacterial strain and the

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MIC of the most resistant strain. It was originally thought that concentrations below these could not select for resistance, as the susceptible bacteria growth would not be inhibited. Gullberg et al (2011) used antibiotic concentrations below the MICsusceptible to demonstrate that pre-existing and de novo mutants can be selected for with these sub-MIC concentrations of antibiotic. They defined sub-MIC selective window as the concentration between the minimal selective concentration (MSC), where fitness cost of the resistance is balanced with selection of the mutant, and the MICsusceptible. The MSC can be as low as 1/230 of the

MICsusceptible25. There is also no necessary correlation between the level of resistance selected and the selective antibiotic concentration26.

Mutant prevention concentration (MPC) is defined as the antibiotic concentration required to prevent single step mutations in a population of at least 1010 bacteria27. The majority of studies on MPC have been with fluoroquinolones, as chromosomal point mutations are their primary resistance mechanism. MPC studies with β- lactam antibiotics cannot predict the organism’s ability to acquire a β-lactamase enzyme and therefore may not adequately predict in vivo resistance mechanisms28. The mutant selection window is the antibiotic concentration between the MIC and MPC.

Sub-therapeutic Antibiotic Concentrations

One of the most important risk factors for emergence of resistance is prolonged exposure to suboptimal concentration of antibiotics29. Sub-lethal antimicrobial concentrations exert selective pressure on a pathogen population without eradicating it and these resistant mutants then replicate so as to dominate the population29. Dosing regimens aimed to reduce the duration of suboptimal antibiotic concentration exposure have been explored, as have pharmacokinetic and pharmacodynamic indices for the most effective dosing for different antibiotic classes30. The three commonly used indices include the ratio of the free drug concentration to the MIC, the ratio of the area under the free drug concentration-

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time curve to the MIC or percentage of a 24 hour time period that the unbound drug concentration exceeds the MIC31,32.

Reasons for fluctuating or suboptimal concentrations of antimicrobials in animals could include poor compliance, poor absorption, biofilms, intracellular bacteria, incorrect dosing schedules, selective compartments and long-acting antibiotic preparations which have prolonged terminal elimination pharmacokinetics 7, 33.

Suboptimal antibiotic concentrations can lead to an increase in resistance in several ways, including selecting for pre-existing resistant mutants and selecting for de novo mutants from a susceptible population25. Low antibiotic concentrations can also increase the rate of adaptive evolution, generate genetic and phenotypic variability and function as signalling molecules34.

Kohanski and colleagues (2010) investigated another possible mechanism for resistance development at sub-lethal antibiotic concentrations, namely, increased mutagenesis due to increased reactive oxygen species (ROS) production35. β- lactams and a number of other antibiotics can induce bacteria to produce ROS that can directly damage DNA, leading to mutations that can interfere with the organisms’ normal functions. E. coli cultures with sub-lethal concentrations of ampicillin showed an increase in mutation rate compared to a control. This increase in mutation rate was not evident when thiourea, a ROS scavenger, was added to the culture. The MIC of ampicillin to inhibit E. coli increased after cultures in sub-lethal concentrations of ampicillin for five days. Similar results were found for norfloxacin, tetracycline, chloramphenicol and kanamycin, also for E. coli35.

Tenney and colleagues in 1982 demonstrated increases in the norfloxacin MICs for Pseudomonas aeruginosa and E. coli isolates that were up to 512-fold higher than the original strains after serial passage on agar with sub-inhibitory concentrations of the drug36.

In vitro studies with sub-inhibitory β–lactam concentrations have shown that the sub-MIC concentrations have different effects on bacteria not previously exposed to antibiotic compared with those previously exposed to supra-MIC antibiotic

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concentrations. β-lactam antibiotics and bacterial cultures in the post-antibiotic phase exposed to sub-MIC concentrations showed a substantial prolongation of time before regrowth; in comparison cultures not previously exposed to antibiotics showed only a slight reduction in growth rates. It has been hypothesized that during the suprainhibitory concentration phase, the β-lactam antibiotics covalently bind to the penicillin binding proteins (PBPs). Synthesis of these PBPs continues during treatment and when the sub-MIC concentration is then applied, most of the PBPs are still inactivated so only a low concentration of antibiotic is needed to inhibit them37.

In clinical settings the rate of development of resistance has been greater than that predicted by in vitro experiments. Reasons for this could include high inocula infections such as pneumonia, fluctuating and suboptimal antibiotic concentrations in vivo, bacterial biofilms, immunosuppression, stress-induced efflux pumps and exposure of commensal organisms7.

Persistence of resistance

It has been suggested that most resistance mechanisms come at a fitness cost to the bacteria, which is normally observed as a slower growth rate, and that when the selection pressure is removed the susceptible bacteria will out-compete the resistant bacteria38. Most studies suggest that the reversibility of antimicrobial resistance, even when selection pressure is removed, is slow or absent at the community level38. Mathematical models have hypothesized that at sub-MIC concentrations the susceptible bacteria will not be inhibited, growth will only be slowed, thus resistant mutants with high fitness costs will not be selected, and only those with low or no cost will survive25. This differs from the resistant populations selected for in the traditional selective window where there is no competition from wild type strains and the mutant will be selected regardless of the fitness cost.

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Sub-MIC selection could therefore be a key factor in the persistence of long term resistance where very low antibiotic concentrations, present in the environment, can maintain the resistant populations25.

Resistant bacteria have been shown to appear rapidly following selection pressure, but then be slow to lose this resistance even in the absence of selection pressure, which highlights the minimal survival costs of some resistant strains39.

Persistence of resistance was confirmed in work by Kohanski and colleagues (2010) whereby E. coli was cultured for five days with sub-lethal concentrations of ampicillin. The E. coli showed an increase in MIC following these cultures; they were then passaged through drug-free cultures for a further two days. The increased MICs remained elevated and did not significantly change in the absence of ampicillin35.

Resistance to Macrolides and Association to Pharmacokinetics

Resistance patterns of Streptococcus pneumoniae, a common respiratory pathogen in humans, to macrolide antibiotics have been widely studied. Mechanisms include target site modification and active drug efflux40. Data from the Alexander project, a multicentre study established in 1992 investigating the prevalence of antimicrobial resistant pathogens in community-acquired respiratory tract infections, highlighted an increase in macrolide resistance to Streptococcus pneuomoniae. This increase coincided with increased use of macrolides, especially the newer longer acting macrolide agents14.

The longer acting macrolides include clarithromycin and azithromycin, with azithromycin having an elimination half-life of 2-3 days in humans41. Shorter acting macrolides such as erythromycin have a half-life of two hours42. The selective window model describes why longer acting antimicrobials may facilitate the selection of resistance, as resistant subpopulations of bacteria are selected for by a range of antimicrobial concentrations that suppress susceptible populations.

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Thus as selection of resistance occurs at concentrations that exert the maximum effect on the susceptible populations, longer acting antibiotics have a longer period in these sub-MIC concentrations and therefore a longer selective window, making resistance selection more likely 14.

Pankuch and colleagues (1998) incubated Streptococcus pneumoniae with azithromycin at three doubling dilutions below, and three above, the MIC. Five out of the six Streptococcus strains showed increased azithromycin MICs (0.5 to >256 mg/L) after 17-45 subcultures in sub-inhibitory antibiotic concentrations.43 While Kastner and colleagues (2001) investigated the influence of macrolide antibiotics on promotion of resistance in the oral flora of children. They found that the long elimination half-life of azithromycin allowed sub-inhibitory serum and epithelial lining fluid concentrations which promoted carriage of macrolide-resistant strains44.

The in vitro studies correlate with the clinical studies, showing that exposure to antimicrobial agents is a selection factor for resistance and incubation of bacteria at sub-MIC concentrations for long periods of time can increase the MIC of the bacteria significantly.

Anti-malarial Drug Resistance and Pharmacokinetics

Malaria is a blood infection with the parasite Plasmodium falciparum. There are three main groups of anti-malarial drugs; chloroquine, pyrimethamine- sulphadoxine and the artemisinin derivatives. Antimalarial drug resistance is usually due to changes in accumulation, drug efflux or reduced drug target affinity. Chloroquine has a terminal elimination half-life of one to two months 45, pyrimethamine-sulphadoxine 100 and 200 hours respectively46 and artemisinin derivatives approximately one hour47.

Hastings and colleagues (2002) built on previous mathematical models of anti- malarial drugs, adding elimination half-life as a new parameter in their model and

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three classes of susceptibility to the drug. ResO is the susceptible wildtype, Res1 is a group more tolerant of the drug but showing clinical resolution and Res2 is resistant to the drug. The model demonstrated that longer drug elimination half- lives lead to faster evolution of Res1 and Res2 resistance48.

The benefits of anti-malarial drugs with long half-lives include longer periods of protection (including against repeat disease exposure), fewer drug administrations and therefore potentially enhanced compliance. While drugs with longer half lives eliminate the parasite population and then ensure an infection-free period, the model suggests that this promotes resistance selection. Shorter half-life drugs may be more effective for treatment, and other strategies for preventing re-infection, such as mosquito nets, need to be explored49. A low level of drug persisting during slow elimination exerts selection pressure for drug resistance: it may be sufficient to eradicate new sensitive infections but incapable of clearing resistant infections50.

Spontaneously occurring parasite mutants have reduced susceptibility to the antimalarial drugs and become resistant populations. Shallow concentration-effect relationships and low drug clearance increase the likelihood of these resistant populations50.

Cephalosporins

Cephalosporins are semi-synthetic derivatives of cephalosporin C and are part of the β-lactam family. They elicit their bactericidal effect in a time-dependent manner by inhibiting the activity of transpeptidase and other penicillin binding proteins (PBP) which catalyse the glycopeptide cross linkages forming the bacterial cell wall. They are bactericidal on replicating cells, with slower kill rates than the aminoglycoside or fluoroquinolone classes51. Cephalosporins have a wide range of antimicrobial activity, excluding enterococci and methicillin-resistant staphylococci. Ceftazidime and fourth generation cephalosporins are effective against Pseudomonas aeruginosa51.

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There are a number of possible mechanisms of resistance to cephalosporins. These include β-lactamase production, enzymes which hydrolyse the β -lactam ring, PBP mutations, decreased penetration through the outer cell wall to access PBPs and increased efflux51.

β-lactamases can be chromosomally or plasmid mediated and can be moved by transposons. Classification systems have been based on molecular and functional characterization. The main groups are penicillinases, AmpC-type cephalosporinases, extended-spectrum β-lactamases (ESBLs) and carbapenemases. Resistance to third generation cephalosporins includes AmpC hyperproduction, ESBLs and metallo- β -lactamases.

Extended-spectrum β-lactamases

ESBLs are enzymes that have the ability to hydrolyse the β-lactam ring of third- generation cephalosporins and aztreonam52. ESBLs confer resistance to penicillins, most cephalosporins and aztreonam, but not to cephamycins or carbapenems. ESBLs are commonly derived from mutations in the amino acid configuration of TEM-, SHV- and OXA- plasmid-mediated β-lactamases, leading to their extended spectrum. ESBLs with cefotaxime hydrolysing capabilities (CTX-M) are suggested to have arisen from mobilisation of chromosomal bla genes from Kluyvera species then later diverged by point mutations like other ESBLs53. Only a few amino acid positions are critical to the hydrolysis of β-lactam rings, so the enzymes have a high capacity for remodelling54. ESBLs are produced by gram- negative bacteria such as E. coli, Klebsiella, Proteus, Salmonella and Shigella species.

ESBLs in humans are spread via direct or indirect contact with colonized or infected patients and contaminated environmental surfaces. ESBLs have been isolated from food-producing animals’ faeces and it is possible there could be contamination of meat55.

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In humans, TEM and SHV ESBLs have caused clonal epidemics, however the population structure of CTX-M ESBLs is more complex and associated with the spread of plasmids and mobile genetic elements54. It is important in nosocomial outbreaks to determine if the infection is being caused by the same clone and transferred horizontally between patients, or by different clones due to selection pressure such as antibiotic use.

It is often unknown where humans or animals first acquire ESBLs. Risk factors that increase the likelihood of human ESBL infections include previous antibiotic use, prolonged hospital stays, critically ill patients, invasive medical devices and recent surgery. It is expected that these factors would be similar in animals52.

As ESBLs are commonly plasmid encoded they can carry resistance genes to other drug classes such as fluoroquinolones, aminoglycosides, sulphonamides, tetracyclines and chloramphenicol55.

The blaCTX-M genes are often found in association with genetic structures such as sul1-type interferons, which are genetically linked to class 1 integrons known to integrate gene cassettes responsible for resistance to β-lactams, aminoglycosides, chloramphenicol, sulphonamides and rifampicin56.

ESBL-producing bacteria were first isolated from a dog in 1988 in Japan57. The dog was a laboratory animal kept for pharmacokinetic studies of β-lactam antibiotics and was infected with a CTX-M-1 type E. coli strain. In 1998, the first clinical ESBL producing strain was isolated from a dog in Spain with a recurrent urinary tract infection58.

A study in a human intensive care unit demonstrated that reduced use of third- generation cephalosporins was associated with a decrease in the rate of acquisition of ESBL-producing Klebsiella pneumonia in the respiratory tract of patients from 10.4% to 1.4%59.

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The Impact of Cephalosporin Use

Cephalosporin use is common in both the human and veterinary fields. Their use has been shown to impact significantly on cephalosporin resistance in faecal flora. Administration of ceftiofur, a third-generation cephalosporin, to feedlot cattle has been shown to transiently increase multi-drug resistant E. coli for a period of two weeks60 and cattle treated with ceftiofur also in a feedlot setting, have shown increased levels of faecal blaCMY-261. Subbiah and colleagues (2012) investigated ceftiofur metabolites in soil and found that under experimental conditions urine from ceftiofur-treated cattle drove selection for cephalosporin-resistant E. coli in soil62.

Cavaco and colleagues (2008) inoculated pigs with CTX-M-1-producing E. coli strains of pig origin and then treated with amoxicillin, ceftiofur or cefquinome. The half life of amoxicillin in pigs is 1.4 hours63, which is shorter than both desfuroyl- ceftiofur, the active metabolite of ceftiofur, (14.5 hours)64 and cefquinome (9 hours) 65. Significantly higher counts of CTX-resistant coliforms were observed in the treatment groups compared to controls, with higher counts in the ceftiofur and cefquinome groups that persisted for longer than the label withdrawal times. This increase could have been due to growth of indigenous CTX-M-producing strains or horizontal transfer, and highlights that cephalosporin use can influence CTX-M producing E. coli strains66.

Similar results have been found in studies with dogs, where cefotaxime resistant E. coli were isolated from 8 out of 13 dogs treated with cephalexin for pyoderma. All eight of these isolates carried the blaCMY-2 gene67. This gene confers resistance to all β-lactams registered for use in dogs and there have been reports of other opportunistic multi-drug resistant E. coli infections producing CMY β-lactamase in dogs68. Lawrence et al (2013) demonstrated the effect of cefovecin on the number and antimicrobial susceptibility of faecal enteric bacteria in healthy beagles. Results showed that the absolute number of cefovecin-resistant E. coli was higher in treated dogs compared to untreated dogs on days 7, 14 and 28. Resistance of

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Enterococcus species was not altered. Treated dogs were 3.25 times more likely to carry the blaCMY-2 resistance gene on day 28 than untreated dogs69. The blaCMY-2 is an ESBL gene that encodes cephalosporin resistance via AmpC β-lactamase and has been found in both commensal and pathogenic gastrointestinal bacteria. Thus it is evident from the literature that cephalosporins as a group exert profound influence on faecal and other flora in a variety of species.

In human medicine there are similar examples of the potential for, and risks of, developing resistance to third generation cephalosporins. Tenover (2006) reports the case of a child admitted to hospital with aplastic anaemia and bacteraemia. Blood cultures showed a single strain of E. coli resistant to ampicillin and narrow spectrum cephalosporins but susceptible to third generation cephalosporins. Over the following three weeks the child was treated with penicillins, aminoglycosides, cefotaxime, ceftazidime, clindamycin and vancomycin. In the fourth week, blood cultures showed E. coli isolates with increased resistance to third generation cephalosporins. In less than two months in the bloodstream, E. coli had acquired a new β-lactamase gene, mutated this gene to increase the level of resistance and down regulated its porins to increase resistance to cephalosporins and cephamycins70.

E. coli resistant to third generation cephalosporins, termed G3CREC, causing blood stream infections in hospitalised patients were investigated in 31 countries in 2007 and trends projected until 2015. In 2007 E. coli caused 163,476 blood stream infections, of which 15,183 were G3CREC. The G3CREC infections caused 2712 excess deaths compared to the non-resistant E. coli infections. The projections suggested the number G3CREC bacteraemias were likely to surpass the number of MRSA bacteraemias in the near future.71

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Cephalexin

Cephalexin is an oral first generation cephalosporin. It has a half-life of approximately two hours in domestic cats and is administered every 12 hours at 15-20mg/kg72. Cephalexin has an oral bioavailability of approximately 75% in dogs and cats73. Cephalexin is around 20% plasma protein bound74. Cephalexin is commonly used for the treatment of pyoderma75.

Fig 2.1) Cephalexin structure76

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Cefovecin

Cefovecin is a semi-synthetic, third generation, cephalosporin that has recently been developed for use in dogs and cats. Cefovecin is formulated as an aqueous solution, as a single stereoisomer and is administered as a subcutaneous injection. Cefovecin has been advocated for the treatment of urinary tract infections, pyoderma, wound infections and abscesses77-79.

Fig 2.2) Cefovecin structure80

The elimination half-life of cefovecin in dogs is 133 hours and 166 hours in cats81,82. Cefovecin does not undergo hepatic metabolism and is slowly cleared primarily by glomerular filtration unchanged and a smaller percentage by faecal excretion. The persistence of cefovecin in the plasma for long periods in these species has been attributed to its high plasma protein binding, slow clearance and possible reabsorption in the kidney tubules. Cefovecin is highly protein bound: 96- 98.7% in dogs and 99.5-99.8% in cats. A lower protein binding in tissue fluids ensures that this high plasma protein binding does not decrease antibacterial effectiveness in tissues81,82.

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Cefovecin’s high plasma protein binding in dogs and cats allows its slow release into tissue and ensures a long elimination half-life. Plasma protein binding as a fraction of total drug decreases with increasing cefovecin concentrations, suggesting saturation of binding sites81. Interestingly, in other species such as nonhuman primates, the half-life of cefovecin is substantially shorter, 2.6-8 hours, and protein binding is lower than in dogs83.

The MIC50 and MIC90 values for cefovecin against 483 E. coli pathogens isolated from dogs and cats have been reported to be 0.5 and 1 mg/L respectively with a range of 0.12- >32 mg/L. The MIC90 for cefovecin against Staphylococcus intermedius has been reported to be 0.25 mg/L84.

Stegemann and colleagues (2006) performed pharmacokinetic studies in cats and dogs and measured total and free cefovecin concentrations in plasma and transudate in a small number of cats and dogs using tissue cage models81,82. Total plasma concentrations (mean and standard deviations) of cefovecin 14 days post administration of subcutaneous 8mg/kg injection were 22.1 +/- 3.1 mg/L in cats and 10.1 +/- 2.1 mg/L in dogs, more than 20 and 10 times the E. coli MIC90 respectively. Free cefovecin concentrations, 14 days post dosing, in plasma measured by ultrafiltration were 0.035 +/- 0.009 mg/L in cats and 0.11 +/- 0.05 mg/L in dogs. Total transudate concentrations on day 14 were 16.4+/- 1.3 mg/L in cats and 7.5+/- 1.9 mg/L in dogs. Free transudate concentrations on day 12 were 0.28 +/- 0.26 mg/L in cats and 0.93 +/- 0.57 mg/L in dogs. It is important to bear in mind the small study numbers and large standard deviations in these results.

Protein binding

In 1957 Robbins and Rall published the free hormone hypothesis, whereby biological thyroid hormone action is the function of free hormone in the blood, rather than the protein-bound plasma concentration. They concluded that as the

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free concentration was so minimal, the systems must be very sensitive or have their own concentrating mechanisms85.

There has been extensive debate surrounding the clinical relevance of plasma protein binding, and whether drug-drug interactions can occur in vivo with highly protein-bound drugs. As early as 1979, Sellers described that displaced highly plasma-bound drug could cause a relative increase in free fraction, which is then distributed throughout the body, making it more available for metabolism or filtration. Therefore, even if drug-drug interactions did occur they would be only transient in nature86. These protein-bound drug-drug interactions are based on the hypothesis that increasing unbound drug concentrations may cause adverse effects or toxicity.

One of the common examples of this phenomenon was elucidated in 1967 by Aggeler et al, who investigated the effects of administering warfarin with phenylbutazone, showing increased plasma concentrations of warfarin and increased prothrombin times. They also demonstrated that, in vitro, the higher affinity drug phenylbutazone displaced warfarin from albumin binding sites87. However, it was later determined that the observed increase in prothrombin time was due not to displacement of warfarin from its protein binding, but rather to inhibition by phenylbutazone of metabolism of the s-warfarin enantiomer, which is five times more potent as an anti-coagulant than the r-enantiomer88.

In vivo changes in the free drug fraction (free drug concentration/total drug concentration) can only occur through changes of total and or free drug clearances. For low extraction ratio drugs, which include the majority of drugs, changes in free fraction result in no change in free concentrations and changes in total concentrations because these drugs have a clearance of free drug that is independent of the free drug fraction and clearance of total drug that is proportional to the free fraction89.

The extent to which binding of antibiotics affects their penetration into interstitial fluid has been the subject of numerous conflicting reports in the literature. A study looking at six β-lactam antibiotics using a cantharides blister technique found a

29

linear relationship between protein binding and antibiotic penetration into the blister fluid, measured by area under the curve of the free antibiotic90. In contrast, Waterman et al (1976) looked at cephaloridine (10% protein binding) and cefazolin (80% protein binding) in dogs. They found despite the variation in protein binding the cefazolin level in the interstitial fluid during the first 15 minutes after administration was higher than the cephaloridine level and that the protein binding of cefazolin did not appear to affect interstitial fluid concentrations91. As previously discussed Stegemann and colleagues (2006) found free cefovecin concentrations to be considerably higher in transudate compared to plasma, despite being highly protein bound. They suggested this disequilibrium could be due to different binding sites in transudates leading to saturation of cefovecin binding or different binding properties in the tissues 81.

Conclusion

After examining previously reported literature surrounding antimicrobial resistance, particularly cephalosporins, and the role of sub-therapeutic antimicrobial concentrations and pharmacokinetics in the development and persistence of antimicrobial resistance, there are still a number of questions to be answered. This project was designed to start investigating the potential risks of long acting antimicrobial use in the veterinary industry, specifically long acting third generation cephalosporin antimicrobials by investigating the impact of cephalosporin antimicrobial concentrations and time on the development and persistence of in vitro bacterial resistance.

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CHAPTER 3: MATERIALS AND METHODS

The methods used for this research are described below; they involved broth microdilution to determine MICs and a number of sub-inhibitory antibiotic and antimicrobial-free cultures. An assay was developed using HPLC to assess the stability of cephalosporins in these cultures.

3.1 GLASSWARE AND WATER

Glass beakers, conical flasks and bottles were all washed in detergent and rinsed with purified Milli-Q Millipore water. The glassware was autoclaved at 121°C for 15 minutes, sealed and stored in glass cabinets before use. Calibrated Finnpippettes with sterile filter tips were used for pipetting. Microbiology grade deionised water was used for the preparation of antibiotic stock solutions.

3.2 MEDIA

1. Cation adjusted Mueller-Hinton broth (CAMHB) was prepared with 21gms Mueller Hinton powder (Oxoid) dissolved in 1 L of Milli-Q purified water in sterile glass bottles. 2. pH was checked using a calibrated pH meter. The pH was confirmed to be between 7.2-7.4 as specified by the manufacturer (Oxoid). 3. The preparation was then autoclaved at 121 °C for 15 minutes. 4. The media was cation adjusted by first preparing stock solutions of

a. 196 mg MgCl2.6H2O dissolved in 5 mL of microbiology grade deionised water

b. 184 mg of CaCl2.2H2O dissolved in 5 mL of microbiology grade deionised water

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5. The cation solutions were filtered with a 0.2 μm syringe filter 6. 180 μL of Ca2+ solution (to achieve 20-25 mg/L) and 62.2 μL of the Mg2+ solution (to achieve 10-12.5 mg/L) were added to the 1 L of broth. 7. This was then mixed well and stored at 4 °C in sterilized 250ml glass bottles.

University of Melbourne Media Preparation Unit prepared sheep blood agar (SBA) and MacConkey agar plates for cultures.

3.3 ANTIBIOTIC STOCK SOLUTIONS

3.3.1 Cephalexin

1. 0.1 mol/L phosphate buffer was prepared for use as diluent and solvent92. 2. 2.78 gm sodium dihydrogen phosphate (Acros Organics) was dissolved in 100 mL microbiology grade deionised water and 5.68 gm dibasic heptahydrate sodium phosphate (Acros Organics) was dissolved in 200 mL microbiology grade deionised water. 3. 43.7 mL of the monobasic solution and 6.15mL of the dibasic solution were combined with a further 50 mL of microbiology grade water. 4. pH of the phosphate buffer was measured at 6.2 with a calibrated pH meter. 5. 256mg of cephalexin powder (Sigma-Aldrich) was dissolved in 20 mL of the prepared phosphate buffer. 6. This was filtered with a 0.2 μm syringe filter. 7. The final concentration was 12800 mg/L. 8. 1mL aliquots of the cephalexin stock were stored in sterile plastic eppendorf tubes at -80 °C. 9. 2 of the aliquots were stored at -30 °C and 2 at 4 °C for use in stability studies.

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3.3.2 Cefovecin

1. Convenia (Zoetis) was used due to unavailability of analytical cefovecin powder. 2. Convenia product information states that adding 10 mL of sterile water to the lyophilised powder in the product makes 80000mg/L. 3. Presuming 800 mg active cefovecin sodium in the product bottle, 15.625 mL of microbiology grade deionised water was added to the product 4. The presumed concentration was 51200 mg/L 5. This was filtered with a 0.2 µm syringe filter. 6. 1mL aliquots of the cefovecin stock were stored in sterile polyethylene eppendorf tubes at -80 °C. 7. 2 of the aliquots were stored at -30 °C and 2 at 4 °C for use in stability studies.

3.4 BACTERIA SELECTION

Five isolates of suspected pathogenic Escherichia coli from the U-Vet Werribee Veterinary Hospital Laboratory archive were chosen as the test organisms (Table 3.1). These E. coli isolates were inherently sensitive to cephalosporins; this was tested by the Calibrated Dichotomous Sensitivity (CDS) disk diffusion technique. The E.coli were selected from both the digestive tract and urinary tract to allow for comparison and contrast of the different environments. The E. coli stocks were stored at -80 °C using a bead storage system (Protect). One bead was taken from each frozen stock and streaked onto a SBA plate which was incubated overnight at 37 °C. Two reference strain E. coli from commercial kits were used in the experiments, NCTC 10418 and NCTC 12441. These were inoculated onto SBA plates and incubated overnight at 37 °C.

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E. coli Isolate Number Species Specimen

NCTC 10418 - Provided by U-Vet Microbiology Laboratory

NCTC 12441 (ATCC 25922) - Provided by U-Vet Microbiology Laboratory

1 Feline Digestive system

2 Feline Bile

3 Feline Faeces

4 Feline Urine

5 Feline Urine

Table 3.1. E. coli isolate numbers, species of origin and type of specimen.

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3.5 MICRODILUTION

Broth microdilution techniques as described by the Clinical and Laboratory Standard Institute (CLSI)93 were performed to determine the minimum inhibitory concentration (MIC) of cefovecin and cephalexin to the five E. coli isolates and reference strains. Sterile, clear, flat-bottomed 96 well plates with lids (Nunc 167008) were used for the microdilution experiments. Plates were prepared on the first day of each 144 hour experiment period and then stored at -70°C with lids on, sealed with plastic (Parafilm M).

3.5.1 96 Well Plate Preparation

1. 1 mL aliquot of defrosted antibiotic stock solution was used to perform serial two-fold dilutions into sterile 50 mL plastic falcon tubes. 2. 50 μL of these dilutions were then pipetted into the appropriate well on the 96 well plate. 3. The last column comprised of the negative control (media only) and positive control (media and inoculum, no antibiotic). 4. The antibiotic concentrations in the wells were prepared to double the desired concentration, as 50 μL of inoculum would dilute these concentrations. 5. Cefovecin concentration range on the 96 well plate was 0.06 mg/L to 128 mg/L. 6. Cephalexin concentration range on the 96 well plate was 2 mg/L to 2048 mg/L.

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3.5.2 96 well plate inoculation

1. One E. coli colony was picked with a sterile loop from each of the SBA overnight cultures. The colony was placed into 10 mL of CAMHB and incubated on an orbital rocking platform at 37 °C overnight. 2. The prepared serial antibiotic dilution plates were defrosted. 3. The overnight E. coli cultures were diluted with CAMHB to a 0.5 McFarland turbidity standard (Bio-Merieux) by comparing the standard and inoculum with a bright light and white paper with black text by eye. The inoculum then approximated 1-2 x 108 CFU/mL. 4. This inoculum was then further diluted 1:200 in 2 mL of CAMHB to approximate 106 CFU/mL 5. 50 μL of this inoculum was then pipetted into each well of the 96 well plates, except the negative control wells. The final inoculum concentration was then approximated at 5 x 105 CFU/mL (accepted range 2-8 x 105 CFU/mL). 6. The 96 well plates were then covered with the plastic lid, wrapped in plastic (Parafilm M) and incubated in a sealed plastic container at 37 °C and stacked no more than 2 plates high for 16-20 hours as per CLSI standards92. 7. The wells were then checked for growth compared to the positive control. The lowest concentration of antibiotic that completely inhibited growth of the organism in the microtitre wells, detected by the unaided eye, was recorded as the MIC end point. 8. The MICs were performed in triplicate for each isolate and antibiotic; if there was discrepancy between the results, the highest concentration was recorded as the MIC.

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3.5.3 Inoculum testing

1. Inoculum testing was performed every 48 hours to ensure appropriate densities were present in the microdilution plates. 2. 10 μL was taken from one of the positive control wells directly after inoculation, this was diluted 1:1000 in CAMHB. 3. 100 μL of the dilution was spread using sterile glass beads onto a MacConkey agar plate and incubated at 37 °C for 16-20 hours. 4. The colonies were then counted. (Approximately 50 colonies represent 5 x 105 CFU/mL).

3.6 SUB-INHIBITORY CULTURES

3.6.1 Cefovecin Sub-MIC Cultures

Sub-MIC cefovecin cultures were then performed. E. coli isolate number 3 was selected from the previous MIC testing.

1. One colony of E. coli isolate number 3 was taken with a sterile loop from an overnight SBA culture. It was incubated in 10 mL CAMHB overnight on a rocking platform at 37 °C in a 50 mL sterile falcon tube. 2. 50mL sterile falcon tubes were prepared with sub-MIC cefovecin concentrations in 10mL broth. The concentrations prepared included 0.05 x MIC, 0.1 x MIC, 0.2 x MIC and one broth only control (no cefovecin). 3. 10μL of the E. coli overnight culture was inoculated into each of the cefovecin-containing and control tubes, which were then placed onto the rocking platform at 37 °C for 24 hours. 4. After 24 hours 10 μL of each overnight culture was also placed into a newly prepared 10 mL broth with the same antibiotic concentration to allow for dilution and continued bacterial growth. These tubes were returned to the rocking platform at 37 °C overnight.

37

5. The overnight cultures were diluted following the 96 well plate inoculation steps as described above for microdilution MIC testing. 6. The microdilution 96 well plates were read after 16-20 hours incubation as described above. 7. Steps 3-5 were repeated every 24 hours for a total of 144 hours.

3.6.2 Cephalexin Sub-MIC Cultures

1. Sub-MIC cephalexin cultures were then performed. E. coli isolate number 3 was selected from the previous MIC testing. 2. One colony of E. coli isolate number 3 was taken with a sterile loop from an overnight SBA culture. It was incubated in 10mL CAMHB overnight on a rocking platform at 37 °C in a 50mL sterile falcon tube. 3. 50mL sterile falcon tubes were prepared with sub-MIC cephalexin concentrations in 10mL broth. The concentrations prepared included 0.2 x MIC, 0.4 x MIC and 0.8 x MIC and one broth only control (no cephalexin). 4. 10μL of the E. coli overnight culture was inoculated into each of the cephalexin containing and control tubes and these were then placed onto the rocking platform at 37 °C for 24 hours. 5. After 24 hours 10μL of each overnight culture was also placed into a newly prepared 10mL broth with the same antibiotic concentration to allow for dilution and continued bacterial growth. These tubes were placed back onto the rocking platform at 37 °C overnight. The overnight cultures were diluted following the 96 well plate inoculation steps as described above for microdilution MIC testing. 6. The microdilution 96 well plates were read after 16-20 hours incubation as described above. 7. Steps 4-6 were repeated every 24 hours for a total of 144 hours.

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3.6.3 Cefovecin and cephalexin Sub-MIC Cultures with 5 E. coli isolates

The five selected E. coli isolates were then cultured in sub-MIC antibiotic cultures including: cephalexin concentrations of 0.4 x MIC, 0.8 x MIC and control (no cephalexin) and cefovecin concentrations of 0.8 x MIC, 0.95 x MIC and control (no cefovecin) for each E. coli isolate.

1. One colony of each of E. coli isolate numbers 1-5 were taken with a sterile loop from overnight SBA cultures. They were incubated in five separate 10mL CAMHB overnight on a rocking platform at 37 °C in a 50 mL sterile falcon tube. 2. 50mL sterile falcon tubes were prepared with sub-MIC cephalexin and cefovecin concentrations in 10mL broth. The concentrations prepared included cephalexin concentrations 0.2 x MIC, 0.4 x MIC and 0.8 x MIC and one broth only control (no cephalexin). Cefovecin concentrations included 0.8 x MIC, 0.95 x MIC and control (no cefovecin) for each E. coli isolate. 3. 10 μL of the E. coli overnight culture was inoculated into each of the cephalexin and cefovecin containing tubes and control tubes and these were then placed onto the rocking platform at 37 °C for 24 hours. 4. After 24 hours 10 μL of each overnight culture was also placed into a newly prepared 10 mL broth with the same antibiotic concentration to allow for dilution and continued bacterial growth. These tubes were placed back onto the rocking platform at 37 °C overnight. The overnight cultures were diluted following the 96 well plate inoculation steps as described above for microdilution MIC testing. 5. The microdilution 96 well plates were read after 16-20 hours incubation as described above. 6. Steps 2-5 were repeated every 24 hours for a total of 144 hours.

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3.7 PERSISTENCE OF RESISTANCE CULTURES

The E. coli isolates from the 0.95 x MIC cefovecin culture from the experiment above were transferred to antibiotic-free CAMHB for a further 144 hours as per the procedure below.

1. 10 μL from each of the five E. coli isolate 0.95 x MIC cefovecin sub-inhibitory cultures were placed into five 50 mL falcon tubes with antibiotic-free CAMHB media, after 144 hours in the experimental conditions described above. 2. The cultures were then placed onto the rocking platform at 37 °C for 24 hours. 3. After 24 hours 10 μL of each culture was placed into a newly prepared 10mL antibiotic-free CAMHB. These tubes were placed back onto the rocking platform at 37 °C overnight. The overnight cultures remaining were then diluted following the 96 well plate inoculation steps as described above for microdilution MIC testing. 4. The microdilution 96 well plates were read after 16-20 hours incubation as described above. 5. Steps 3-4 were performed every 24 hours for 144 hours.

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3.8 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY STABILITY STUDY

High performance liquid chromatography (HPLC) was performed on broth with concentrations of cefovecin from 0.3 mg/L – 10 mg/L incubated in CAMHB at 37 °C in an orbital shaking incubator at 250 rpm over a period of 24 hours to determine stability of cefovecin in these conditions.

3.8.1 Chromatographic system and conditions

The HPLC system comprised of Poroshell 120 EC-C18 column 4.6 x 100 mm 2.7 micron (Agilent USA), waters 1525 binary pump and inline degasser, Waters 2707 autosampler and Waters 2998 PDA detector with absorbance at 270 nm. The column was maintained at room temperature. The mobile phase was aqueous ammonium acetate buffer as eluent A and 100% methanol as eluent B. The rates were 0.32 mL/min eluent A and 0.08 mL/min eluent B for a flow rate of 0.4 mL/min. Total run time of the assay was 27 minutes and the injection volume was 20 μL.

3.8.2 Chemicals used

Cefovecin (Zoetis) product was used, as there was no available analytical grade cefovecin. HPLC grade methanol was from Chem-supply, Australia, and water was purified by Milli-Q reverse osmosis (Merck Millipore, Bayswater, Vic., Australia).

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3.8.3 Sample preparation extraction procedure

1. 500µL of sample was pipetted into Amicon Ultra 2mL 3000NMWL Ultracel filter tubes (Merck Millipore Ltd Tullagren Ireland) 2. 1500µL of 100% propanol was then pipetted into each filter tube. 3. The cap was placed on the filter tubes and they were placed into a swinging bucket centrifuge (Allegra CR 600) at 2500G for approximately three hours at 20 degrees. 4. The filtrate in the collection tubes was transferred into 10mL falcon tubes and refrigerated. 5. Another 1500µL of 100% propanol was added to each filter tube. 6. The samples were then centrifuged for a further 3 hours. 7. The filtrate samples from steps 4 and 6 were combined into the collection tubes. 8. The collection tubes were then placed into a speedvac (Savant AES2000) to dry on low setting for approximately three hours or until dry. 9. Samples were reconstituted with 500µL of 20% methanol (mobile phase)

3.8.4 HPLC assay validation procedure

1. Cefovecin standards were prepared in 20% methanol (mobile phase) in the following concentrations: 0.3 mg/L, 0.5 mg/L, 1 mg/L, 5 mg/L and 10 mg/L. 2. HPLC analysis as per the above method was performed on these samples on each day the assay was run. Multiple injections of each of the standards were performed. 3. 500μL cefovecin samples in CAMHB were then prepared at the same concentrations as in step 1: 0.3 mg/L (x 1 sample), 0.5 mg/L (x 5 samples), 1 mg/L (x 1 sample), 5 mg/L (x 1 sample) and 10 mg/L (x 5 samples). Six blank 500μL CAMHB samples were also prepared. 4. The extraction procedure described in 3.8.3 was then performed on the samples from step 3 above.

42

5. The blank CAMHB samples following extraction were spiked with the following concentrations of cefovecin 0.3 mg/L, 0.5 mg/L, 1 mg/L, 5 mg/L and 10 mg/L. 6. Cefovecin HPLC analysis was performed on these samples as per the protocol described above. A set of the cefovecin standards (3.8.4.1) was run before and after this analysis and one cefovecin standard injection was also performed after every 5 injections of samples.

3.8.5 Stability study procedure

1. 12 x 50mL falcon tubes were prepared with the following cefovecin concentrations in CAMHB: 0 mg/L (x 2 samples), 0.3 mg/L (x 2 samples), 0.5 mg/L (x 2 samples), 1 mg/L (x 2 samples), 5 mg/L (x 2 samples), and 10 mg/L (x 2 samples). 2. The samples were placed in the orbital shaking incubator at 37 °C and 250 rpm for a period of 24 hours. 3. A 500 μL sample was taken from all the tubes at 0 hours, 1 hour, 3 hours, 6 hours, 12 hours and 24 hours. 4. The samples were frozen at -80 °C. 5. After the 24 hour samples were collected all the samples were defrosted. 6. The extraction procedure described in 3.8.3 was performed on the samples. 7. The cefovecin HPLC analysis procedure as described above was then performed on the samples. Cefovecin standards in mobile phase were run before and after the samples. A cefovecin standard in mobile phase was also run between every 5 injections of samples.

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CHAPTER 4: RESULTS

Broth Microdilution Initial MICs The initial MICs determined for cephalexin and cefovecin for the five E. coli isolates are listed in table 1.

Cephalexin MIC (mg/L) Cefovecin MIC (mg/L)

E. coli Isolate 1 32 0.25

E. coli Isolate 2 16 0.5

E. coli Isolate 3 16 0.5

E. coli Isolate 4 8 0.5 E. coli Isolate 5 16 0.25

Table 4.1. Cephalexin and cefovecin MICs for 5 E. coli isolates determined by broth microdilution technique (CLSI).

The cephalexin MIC determined for the reference E. coli NCTC 10418 was 16 mg/L. The reference strain E. coli NCTC 12441 (ATCC 25922) MIC for cephalexin was 8 mg/L and cefovecin was 0.5 mg/L.

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Sub-Inhibitory Cultures with One E. coli Isolate Figure 4.1a shows the MICs determined for control (no antibiotic), 0.2, 0.4 and 0.8 x MIC cephalexin concentration cultures at 24-hour intervals. The control culture showed no change in MIC over the 124 hour period, whereas the 0.2, 0.4 and 0.8 x MIC concentration cultures showed increased MICs with increasing concentrations of antibiotic. The E. coli cultured in 0.2 x MIC concentration of cephalexin showed a two times increased in MIC after 24 hours and this was stable until the end of the experiment. The 0.4 x MIC cephalexin culture also showed a two times increase in MIC after 96 hours. The 0.8 x MIC cephalexin culture E. coli had an increase in MIC by 4 times the initial value after 96 hours.

Figure 4.1b shows the cefovecin subcultures with one E. coli isolate in a culture with no antibiotic (control), 0.05, 0.1, 0.2, 0.4 and 0.8 x MIC concentrations of cefovecin. Similarly to the cephalexin culture, the control with no antibiotic showed no change in MIC. The E. coli MICs increased more with increasing concentrations of cefovecin in the CAMHB, with the 0.4 and 0.8 x MIC both increasing by 8 times, this occurred for the 0.4 x MIC culture after 120 hours and after 96 hours for the 0.8 x MIC culture. The 0.05 and 0.1 x MIC concentration cultures showed an increase in E. coli MIC by 2 times and the 0.2 x MIC cefovecin concentration culture by 4 times.

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Cephalexin MICs After Sub-MIC Static Culture

140

120 Control 100

MIC (mg/L) 80 0.2MIC

60 0.4MIC

40 0.8MIC

20

0 0 24 48 72 96 120 Time (hours)

Fig 4.1a) Cephalexin MICs after culture in CAMHB with 0, 0.2, 0.4 & 0.8 x MIC of cephalexin.

Cefovecin MICs After Sub-MIC Static Culture

2.5

2

Control

MIC 1.5 0.05 MIC (mg/L)

0.1 MIC 1 0.2 MIC

0.4 MIC 0.5 0.8 MIC

0 24 48 72 96 120 144

Time (hours)

Fig 4.1b) Cefovecin MICs after culture in CAMHB with 0, 0.05, 0.1, 0.2 0,4 & 0.8 x MIC of Cefovecin

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Sub-Inhibitory Cultures with Five E. coli Isolates Figures 4.2a and 4.2b show the controls for the cefovecin and cephalexin culture experiments with the five selected E. coli isolated from the collection. Controls contained no antibiotic in the CAMHB. A term called resistance factor was used to measure change in MIC; this was defined as the current MIC at that time point divided by the initial MIC of the E. coli isolate at the start of the experiment. The cephalexin control experiment showed no change in the MIC over 144 for E. coli isolates 1, 2 and 4. E. coli isolates 3 and 5 showed an increase in MIC by a factor of 2 (a one well increase on the serial dilution plate) after 96 and 72 hours respectively. E. coli isolates 1 and 5 were cultured for 120 hours only, thus are not featured at the 144-hour time point in figure 4.2a. The cefovecin controls showed no change in the initial MIC over the time course of 144 hours for E. coli isolates 1,2,3 and 4. E. coli isolate 5 showed an increase in MIC by a factor of 2 after 120 hours but this reverted to the original MIC at 144 hours.

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Control Cephalexin Cultures

8 E.coli isolate 1 7

Resistance 6 E.coli isolate 2 Factor 5 E.coli isolate 3 4

3 E.coli isolate 4

2 E.coli isolate 5 1

Initial MIC0 0 24 48 72 96 120 144

Time (hrs)

Fig 4.2a) Cephalexin control- CAMHB and no cephalexin with 5 E. coli isolates

Resistance Factor = Current MIC at time point / Initial MIC at zero hours

Control Cefovecin Cultures

8

7 E.coli isolate 1

6 Resistance E.coli isolate 2 Factor 5

4 E.coli isolate 3

3 E.coli isolate 4 2

E.coli isolate 5 1 Initial MIC 0 0 24 48 72 96 120 144

Time (hrs)

Fig 4.2b) Cefovecin control- CAMHB and no cefovecin with 5 E. coli isolates.

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Figures 4.3a and 4.3b depict the results from the 0.4 x MIC and 0.8 x MIC cephalexin cultures with 5 E. coli isolates. In the 0.4 x MIC culture isolate 1 MIC increased by a factor of 2, isolates 2,3 and 5 increased by 4 and isolate 4 increased MIC by a factor of 8 after 144 hours. The 0.8 x MIC cultures showed an increased in MIC by a factor of 4 in all 5 E. coli isolates. Isolate 1 was cultured for a period of 120 hours in these experiments and the other 4 isolates were cultured for 144 hours.

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0.4 x MIC Cephalexin Cultures

8

7

6 E.coli isolate 1

Resistance 5 Factor E.coli isolate 2 4 E.coli isolate 3 3

E.coli isolate 4 2

1 E.coli isolate 5

Initial MIC 0 0 24 48 72 96 120 144 Time (hrs)

Fig 4.3a) Cephalexin 0.4 x MIC cultures with 5 E. coli isolates

Resistance Factor = Current MIC at time point / Initial MIC at zero hours

0.8 x MIC Cephalexin Cultures

8

7

E.coli isolate 1 6

5 E.coli isolate 2 Resistance Factor 4 E.coli isolate 3

3 E.coli isolate 4 2

E.coli isolate 5 1

Initial MIC 0 0 24 48 72 96 120 144

Time (hrs)

Fig 4.3b) Cephalexin 0.8 x MIC cultures with 5 E. coli isolates.

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Figures 4.4a and 4.4b show the 0.8 x MIC and 0.95 x MIC cefovecin concentration culture results. The 0.8 x MIC cefovecin culture experiment shows a maximal increase of 16 times the original MIC for E. coli isolate 3 at 96 hours. After 144 hours isolates 1 and 3 showed an 8 times increase in MIC from time point zero and isolates 2, 4 and 5 showed a 4 times increase in MIC. Isolates 3,4 and 5 showed the 4 times increase at 24 hours followed by isolates 1 and 2 at 72 hours.

When the concentration was increased to 0.95 x MIC all five E. coli isolates showed an 8 times increase in MIC after 144 hours. Isolates 3,4 and 5 MICs increased to 4 times the initial after 24 hours, replicating the results from the 0.8 x MIC culture. Isolates 1 and 2 showed a 4 times increase at 72 hours also replicating the 0.8 x MIC results. Isolates 1 and 3 both showed 8 times increase in MIC after 0.8 x MIC and 0.95 x MIC experiments, this increase occurred at 96 and 120 hours at 0.8 x

MIC and 48 and 72 hours at 0.95 x MIC.

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0.8 x MIC Cefovecin Cultures 1610

9

8 E.coli isolate 1

7 E.coli isolate 2 Resistance 6 Factor 5 E.coli isolate 3

4 E.coli isolate 4 3

2 E.coli isolate 5

1

Initial MIC 0 0 24 48 72 96 120 144

Time (hrs)

Fig 4.4a) Cefovecin 0.8 x MIC cultures with 5 E. coli isolates

Resistance Factor = Current MIC at time point / Initial MIC at zero hours

0.95 x MIC Cefovecin Cultures

9

8

7 E.coli isolate 1

6 Resistance E.coli isolate 2 Factor 5

4 E.coli isolate 3

3 E.coli isolate 4 2

E.coli isolate 5 1

Initial MIC 0 0 24 48 72 96 120 144 Time (hrs)

Fig 4.4b) Cefovecin 0.95 x MIC cultures with 5 E. coli isolates

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Persistence of Resistance Cultures

Figure 4.5 shows the results of the persistence of resistance experiment. The starting MICs at -144 hours represent the initial E. coli isolates MICs. The MICs at time point zero represent the MIC after exposure to 144 hours in 0.95 x MIC cefovecin concentration CAMHB (from time point 144 hours in figure 4.4b). These MICs were then measured every 24 hours after passage through antibiotic-free CAMHB for 144 hours.

As shown previously in figure 4.4b, the isolates all increased in MIC by 8 times after culture in 0.95 x MIC cefovecin for 144 hours. All five isolates then decreased one well, or two times the concentration, after 24 hours in the antibiotic-free broth. Isolates 1, 2 and 5 then stayed at this MIC for the further 120 hours of the antibiotic-free culture. Isolate 3 showed a one well decrease in MIC at 24 hours like the other isolates, decreased another well in MIC at 120 hours, before increasing 2 times again at 144 hours. Isolate 4 showed a one well decrease at 24 hours, another well decrease at 72 hours and continued at this MIC for the remainder of the experiment.

E. coli isolate 4 was the last isolate to increase MIC to 8-fold in the previous experiment (fig 4.4b) and the only isolate to decrease MIC by 2 wells in the persistence of resistance experiment.

E. coli isolate 3 in the 0.8 x MIC experiment (fig4.4a) showed the most inconsistent results with an increase from the initial MIC to 4 times increase at 24 hours then 16 times increase at 96 hours and back to an 8 times increase at 120 hours. Isolate 3 was also the only isolate to both decrease and increase MIC in the persistence of resistance experiment seen in fig4.5.

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E.coli E.coli E.coli

E.coli E.coli

Fig 4.5 ) E. coli isolates post 144 h exposure to 0.95 x MIC cefovecin passaged through antibiotic free media. -144hr = initial cefovecin MIC 0hr = cefovecin MIC after 144 hour exposure to 0.95 x MIC 0-144hr = cefovecin MICs after culture in plain CAMHB

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HPLC Results

Validation of Assay:

The chromatograms for 0.5 mg/L and 10 mg/L cefovecin standards in mobile phase are shown in figures 4.6 and 4.9. The peak shapes are largely symmetrical with no peak fronting and minimal peak tailing. Figures 4.7 and 4.10 show the chromatograms from the fully extracted 0.5 mg/L and 10 mg/L cefovecin samples in CAMHB. The chromatograms in figures 4.8 and 4.11 show the cefovecin peaks after incubation of 0.5 mg/L and 10 mg/L cefovecin samples in CAMHB for 24 hours and then full extraction.

Calibration curves were prepared using extracted standards and extracted plain CAMHB spiked with cefovecin post extraction in the following concentrations: 0.3 mg/L, 0.5 mg/L, 1 mg/L, 5 mg/L and 10 mg/L. Figures 4.12 and 4.13 show the spiked and extracted validation curves.

The lowest limit of quantification was set to 0.3 mg/L, and upper limit of quantification was 10 mg/L. The coefficient of determination (r2) was >0.99. Specificity was determined by assessing the spectral data from the PDA detector to identify peaks. Intraday precision was assessed by analysing multiple samples at the lower limit of quantification 0.3 mg/L (LLOQ) and the upper limit of quantification 10 mg/L (ULOQ) (n=5) and determining the coefficient of variation, which was 0.6% at the LLOQ and 0.21% at the ULOQ. Inter-assay precision for the LLOQ was 5.46% and 1.43% for the ULOQ. The recovery was determined by comparing peak area of the spiked extracted CAMHB standards with the fully extracted CAMHB standards, with the mean recovery being 81.04%.

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0.004

0.003

0.002 20.975 AU

0.001

0.000

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 Minutes

Peak Name Retention Time Area Height

Cefovecin 20.975 50078 2210

Figure 4.6) Chromatogram Cefovecin 0.5 mg/L standard in mobile phase

56

0.030

0.025

0.020

AU 0.015

0.010

0.005 21.153

0.000

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 Minutes

Peak Name Retention Time Area Height

Cefovecin 21.153 39440 1688

Figure 4.7) Chromatogram Cefovecin 0.5 mg/L fully extracted CAMHB sample

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0.012

0.010

0.008

0.006 AU

0.004

0.002 21.347

0.000

-0.002 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 Minutes

Peak Name Retention Time Area Height

Cefovecin 21.347 40576 1690

Figure 4.8) Chromatogram Cefovecin 0.5 mg/L, after 24 hours incubation in CAMHB

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0.040 21.349

0.035

0.030

0.025

AU 0.020

0.015

0.010

0.005

2.356 2.746

3.055

26.467

4.225 4.709 14.314

3.338 3.731 0.000

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 Minutes

Peak Name Retention Time Area Height

Cefovecin 21.349 1093290 42718

Figure 4.9) Chromatogram Cefovecin 10 mg/L standard in mobile phase

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0.15

0.10 AU

0.05 21.406

0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 Minutes

Peak Name Retention Time Area Height

Cefovecin 21.406 843162 33376

Figure 4.10) Chromatogram Cefovecin 10 mg/L fully extracted CAMHB sample

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0.15

0.10 AU

0.05 21.397

0.00

2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 Minutes

Peak Name Retention Time Area Height

Cefovecin 21.397 601620 23994

Figure 4.11) Chromatogram Cefovecin 10 mg/L, after 24 hours incubation in CAMHB

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Spiked CAMHB Cefovecin Standards

1100000

1000000

900000 Cefovecin 800000 Standards

700000

600000 Area Under Linear Peak (μV*sec) 500000 (Cefovecin Standards) 400000 300000 y = 100910x - 541.69 R² = 0.9998 200000 100000

0 0 2 4 6 8 10 12 Cefovecin Concentration μg/ml

Fig 4.12) Spiked CAMHB Cefovecin Standards Calibration Curve

Extracted CAMHB Cefovecin Standards

1000000 900000 800000

700000 Cefovecin Standards 600000 Area Under 500000 Peak (μV*sec) 400000 Linear (Cefovecin 300000 Standards)

200000 y = 84525x + 3114.6 100000 R² = 0.9972 0 0 2 4 6 8 10 12 Cefovecin Concentration μg/ml

Figure 4.13) Extracted CAMHB Cefovecin Standards Calibration Curve

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Stability Assay Results:

Results of the stability study in table 4.2 show the cefovecin peak area at time 0, 1, 3, 6, 12 and 24 hours for concentrations 0.3, 0.5,1,5 and 10 mg/L in duplicate. These concentrations were chosen on the basis that the cefovecin MICs for the five E. coli isolates used in the sub-inhibitory culture and persistence of resistance experiments varied between 0.25-4 mg/L. Peak areas at 24 hours were compared to peak areas at 0 hours to assess stability, these percentages showed some variability and ranged from 79.929% to 125.234% with an average of 97.786%. Due to some unanticipated problems with the Amicon ultra filter tubes in the sample preparation, two of the samples did not pass through the filters after the centrifuge steps. These samples could therefore not be extracted or run through the HPLC assay.

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Stability Assay Results

Cefovecin Cefovecin Percentage: Starting concentration Peak Area at 24 hours/ Concentrations 0 Hrs 1 Hr 3Hrs 6 Hrs 12 Hrs 24 Hrs mg/L at 24 hours Peak Area 0 hours

0.3 mg/L 18520 15291 16359 25561 13701 16567 0.145 89.454

0.3 mg/L 20672 11557 20547 36249 11908 16523 0.145 79.929

0.5 mg/L 22731 28348 33259 24912 27464 28467 0.286 125.234

0.5 mg/L 25026 26866 31948 --- 25987 27569 0.275 110.161

1 mg/L 64649 59138 53826 62585 64380 65712 0.727 101.644

1 mg/L 67094 67121 67956 59496 53017 64523 0.713 96.168

5 mg/L 326893 325267 314142 341613 305613 334367 3.911 102.286

5 mg/L 315973 330159 307543 347746 299692 320700 3.749 101.496

10 mg/L 729136 650785 694193 656720 605160 601620 7.077 82.511

10 mg/L 734892 666711 723742 691099 --- 653905 7.697 88.980

Table 4.2. Cefovecin Stability Assay over 24 hours in CAHMB incubated at 37 °C. --- = filter error, no results were obtained for these samples.

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CHAPTER 5: DISCUSSION

Broth Microdilutions

The broth microdilution CLSI technique used for MIC determination in these experiments showed repeatable results, with the reference E. coli strain MICs consistently falling within published acceptable ranges. For E. coli reference strain NCTC 10418 the cephalexin MIC determined in this experiment was 16 mg/L, with a published acceptable reference range of ≤16 mg/L. For strain NCTC 12411, our MIC was 8 mg/L for cephalexin (published acceptable range of 4-16 mg/L) and 0.5 mg/L for cefovecin (published acceptable range in the Zoetis Canada Convenia label of 0.5-2 mg/L) 92,94.

The initial MIC for the five pathogenic E. coli isolates used in this experiment fell in the range of 0.25-0.5 mg/L for cefovecin. This is in agreement with Stegemann and colleagues’ 2006 study, of antimicrobial activity of cefovecin against 483 E. coli isolates from the United States and Europe, which found a mode MIC of 0.5 mg/L, range 0.12->32 mg/L, with 90% of MICs below 1 mg/L. The initial MIC range for cephalexin against the five pathogenic E. coli isolates was 8-16 mg/L, which also corresponded with the Stegemann study, in which the MIC for cephalexin was below 16 mg/L for 90% of E. coli isolates (range of 3->32 mg/L)84.

Interpretation of the cephalexin plates was initially difficult, as determining growth with the unaided eye proved less obvious than for the cefovecin plates. This was consistent across all strains and all experiments. No contaminants were found in the plates, and the positive and negative controls performed as expected. This challenge was overcome by reading the plates under a lamp against a black tile backdrop, which greatly improved the readability of the plates as evident in figures 5.1 and 5.2.

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Figure 5.1. Reading 96 well cephalexin MIC plates on white background.

Figure 5.2. Reading 96 well cephalexin MIC plates on black tiles with overhead lamp.

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Sub-MIC Cultures

The results highlight that both first generation and third generation cephalosporin cultures at sub-MIC concentrations over periods of 144 hours can substantially increase the MIC of pathogenic E. coli isolates, where a substantial increase was defined as more than a one well or 2-fold increase in MIC.

Incubation in sub-MIC cephalexin increased E. coli MICs by up to 8 times (Fig 4.3a) and with cefovecin increased E. coli MICs by up to 16 times (Fig 4.4a). It took 72 hours for some of the cephalexin sub-MIC cultures to increase the MIC 4-fold, whereas with the cefovecin sub-MIC cultures MICs increased by 4-fold as early as 24 hours into the culture. It was hypothesised that the resistance mechanisms selected for were likely induction of efflux pumps, due to the magnitude of MIC increase, although inducible β-lactamase production is also a possibility. These results suggest that sub-MIC concentrations of third-generation cephalosporins can induce resistance faster and to a greater degree than first-generation cephalosporins. This pattern of results is in agreement with Carsenti-Etesse and colleagues (1995) who studied the in vitro development of resistance to β-lactam antibiotics in Streptococcus pneumoniae, showing that selection pressure of sub- inhibitory concentrations of the different antibiotics varied. Five Streptococcus strains were passaged though sub-inhibitory concentrations of different antibiotics. Cefadroxil, a first generation cephalosporin, sub-inhibitory cultures showed three strains increase MIC by 4-fold, one by 8-fold and one by 16-fold. In comparison cefixime, a third generation cephalosporin, sub-inhibitory cultures showed three strains increase MIC by 32-fold and two by 16-fold95.

This data is consistent with observations in the human field where broad spectrum cephalosporin use has been identified as a risk factor for resistance development96. Chow and colleagues (1991) investigated the effect of previously administered antibiotics on the susceptibility of Enterobacter species isolated from patients with bacteraemia. They demonstrated a 19% emergence of resistance following third- generation cephalosporin therapy compared with 0.01% for aminoglycoside

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therapy and 0% for other β-lactam therapy97. Third generation cephalosporin resistance is a serious public health concern and organisms that are resistant to these broad spectrum cephalosporins are often multi-drug resistant96. Studies have shown a median increase in hospital stay of 9 days, increase in hospital charge of $29,379 and increased mortality rates for human patients with Enterobacter infections resistant to third generation cephalosporins98.

The results for the sub-MIC cultures for both cephalexin and cefovecin showed an increase in MIC across the 144-hour experiment period, regardless of the sub-MIC concentration. As the sub-MIC concentrations of cephalexin and cefovecin increased there was a trend towards a faster development of higher MICs. The 0.8x MIC cefovecin culture showed two of the E. coli isolates MIC increased by 8-fold by the end of the 144 hours and the other three isolates by 4-fold after 144 hours. In contrast, the 0.95x MIC cefovecin culture showed all five E. coli isolates MICs increase by 8-fold. This data is consistent with the selective window theory, which suggests that concentrations closer to the MIC inhibit more susceptible bacteria, but are lower than the MIC of the resistant bacteria allowing resistant populations to grow99. The results are also in agreement with macrolide resistance trends, where Streptococcus pneumoniae resistance was noticed to spike with the increased use of longer acting macrolide antibiotics14.

The control E. coli cultures with no antibiotic mostly stayed at the same MIC throughout the experiments. In two isolates in the cephalexin experiment and one isolate in the cefovecin experiment, the MIC increased by two-fold or one well, and in one of these cases, reverted to the original MIC at the next time point. It would be expected that there would be minimal selection pressure on the isolates in CAMHB with no antibiotic present. These slight variations in MIC in the antibiotic- free cultures may be due to variability in pipetting, laboratory conditions, inconsistency when reading the plates, or natural variability in the MIC.

MIC is not an absolute value; the true MIC is between the lowest antimicrobial concentration that inhibits growth and the next lowest concentration in two-fold dilutions. Under even strictly controlled laboratory conditions dilution tests may

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not yield the same MIC value every time: the acceptable reproducibility in a laboratory dilution MIC test would be within one two-fold dilution either side of the endpoint93,100,101. Experimental protocol factors such as duration of the measurement, density of the starting culture, the use of optical density or cell counts to determine growth can cause differences of MIC by a factor of 8, this should not be a problem in reporting trends using the same laboratory protocol but may be more of an issue when comparing absolute values between laboratories102. The recommended media is CAMHB as it has demonstrated minimal batch variance in dilution MIC testing. The use of a spectrophotometer and an automatic plate reader may have made this study more accurate by reducing variability in reading plates and errors in transcription of results103.

We hypothesised that longer exposure inside the sub-MIC selective window would lead to increased amplification of resistant bacteria. This was evident in the 0.95 x MIC cefovecin culture where two isolates had shown an 8- fold increase in MIC by 48 hours and after 144 hours all five of the isolates had an 8-fold increase in MIC. Infrequent dosing with long acting antibiotic formulations results in antibiotic concentrations falling within these sub-MIC selection windows for much greater periods of time than compounds with short half-lives and multiple day dosing. Therefore, following these principles and our in vitro results, long-acting compounds are likely to lead to more rapid and greater resistance development in vivo.

Persistence of Resistance

Our persistence of resistance experiments showed only one of the five E. coli isolates had a significant decrease in MIC after serial passage through antibiotic- free media and none returned to their initial MICs. These results are in agreement with previous work published on β-lactam antibiotics; Kohanski and colleagues (2010), cultured E. coli in sub-lethal ampicillin concentrations for 5 days, MICs increased initially and then stayed stable when passaged through antibiotic-free

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media35. Wise and colleagues (1978) found similar results when testing a new parenteral cephalosporin, HR756. They performed seven passages of strains of E. coli, Klebsiella and Staphylococcus aureus through media containing HR756, which led to emergence of strains with resistance to HR756. This resistance was retained for at least 7 transfers through plain media104. Carsenti-Etesse and colleagues (1995) showed increased cefadroxil MICs after sub-inhibitory culture with Streptococcus pneumoniae were stable after 10-passaged on drug free agar, yet increased amoxicillin MICs after sub-inhibitory culture decreased after 10 passages on drug-free agar95.

In 2011 Van der Horst and colleagues investigated de novo acquisition of resistance to amoxicillin, enrofloxacin and tetracycline by E. coli. Sub-MIC static or stepwise increasing concentration cultures were performed. Amoxicillin MICs increased from 4-32 mg/L in the static culture and to 512 mg/L when the exposure was increased in a stepwise fashion. The static culture increase in MIC was reversed after culture in antibiotic-free media yet the stepwise increased MIC was maintained. They hypothesized this was due to a genetic change in the stepwise isolate compared to a phenotypic adaption only in the static isolate. The MIC changes induced by enrofloxacin were also permanent but the MIC increase by tetracycline was rapidly reversible. It was suggested this was due to the bactericidal versus bacteriostatic nature of the antibiotics and the permanent change induced by the bactericidal antibiotics was due to SOS-induced mutagenesis. This study showed cross-resistance developed for the bactericidal antimicrobials but not the bacteriostatic105. A further step in this research would be to investigate any cross-resistance induced by cefovecin or cephalexin after the sub-inhibitory cultures. The stepwise manner in which E. coli isolate 4 in our persistence of resistance experiment decreased MIC in antibiotic-free media, may signify individual adaptations being reduced or lost over time in the absence of selective pressure or SOS-induced mutagenesis.

Bacteria that develop antibiotic resistance are usually subject to a fitness cost, often manifested as a reduction in bacterial growth rate38. This is due to the fact that resistance mechanisms not only effect the interaction between the

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microorganism and the antibiotic but also the host and the environment106. The extent of this fitness cost drives the stability of the resistance in the absence of antibiotic selection pressure and thus the persistence of resistance. Antibiotic resistance is directly related to the amount of antibiotic use and inversely related to the fitness cost the resistance places on the bacteria106. Thus, reducing the amount of antibiotic use would suggest that susceptible bacteria could outcompete the resistant mutants. Compensatory evolution and genetic co-selection complicate this process, however. Co-selection is common with horizontal gene transfer and confers resistance to more than one antibiotic. The time required to reduce resistance is thought to be inversely related to the cost of resistance38. Resistant bacteria are more likely to undergo compensatory mutations to reduce the fitness cost than revert to drug sensitivity107. Environmental conditions can also have an effect on fitness and some mutations that show little cost in vitro have been shown to have high costs in laboratory mice108.

Compensatory mutant selection during serial passage depends on mutation rate, fitness of the mutations and bottlenecks38. A population bottleneck describes a large reduction in the bacterial population, which, in vivo, may be due to extreme environmental changes or competition. In our in vitro experiment population bottlenecks occurred at frequent known time intervals and fixed dilution ratios of 1:1000, selecting a random population sample to continue in the passage109. Mathematical models have shown that beneficial mutations may not survive bottlenecks, as mutations often occur just before bottleneck when the population is at its largest, and may offer only a slight selective advantage110. Lower fitness mutants can therefore survive a bottleneck if they form at a higher rate than higher fitness mutants106. There is also a possibility that after the initial growth phase following dilution there is a stationary phase where some of the E. coli may die, possibility skewing the end results.

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Repeatability

The repeatability of the results was assessed in these experiments. In this study E. coli isolate three was cultured in 0.2 x MIC of cefovecin, 0.8 x MIC cefovecin and 0.8 x MIC cephalexin in two separate experiments under the same laboratory conditions. The 0.2xMIC cefovecin culture showed E. coli isolate three to have a 4- fold increase in MIC at 120 hours in both experiments. The 0.8xMIC cefovecin culture showed an 8-fold increase in MIC both times, the first time this first occurred at 72 hours and the second time at 96 hours. The 0.8xMIC cephalexin culture showed a 2-fold increase in MIC initially and a 4–fold increase in MIC the second time it was performed at 96 and 120 hours. These results show that there was minimal variability between the sub-inhibitory cultures when repeated with the same E. coli isolate, conditions and antibiotic concentrations.

Study Design

This study is limited by its in vitro design. In vitro studies do not take into account complex interplays between the microorganism, the in vivo environment and the immune system. However mutation rates in vivo have been reported to be greater than in in vitro studies due to fluctuations in pH, temperature, oxygen concentration, antibiotic concentration, biofilm formation, immunosuppression, exposure of commensal flora and high inoculum levels in certain infections such as pneumonia7. It has also been shown that in vitro studies and in vivo studies may cause differing fitness-compensating mutations in antibiotic resistant populations111.

It is important to remember this in vitro study investigates the effects of cefovecin and cephalexin at sub-MIC concentrations on E. coli that has not been exposed to supra-MIC antibiotic concentrations. This is not the same scenario as culturing

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bacteria in sub-MIC concentrations after supra-inhibitory β-lactam concentrations and the associated post-antibiotic effect37.

While serial passage experiments can assess the probability of emergence of mutation-driven resistance, they do not assess the risks of resistance via horizontal gene transfer, the stability of the increased resistance or the fitness cost. Fitness costs are a combination of phenotype and environment, so must be assessed in the relevant environment and clinical setting in vivo 112.

In vitro studies do not look at phenotypic, or non-inherited resistance, which can occur in vivo in different states of infection. Slow growing bacteria might be resistant to antibiotics that kill actively growing bacteria and can form resistant biofilms, which are common in surface-associated infections. This is known as ‘drug indifference’ where the antibiotic is only active in specific bacterial physiological conditions; it was first identified for β-lactam antibiotics and their inactivity against non-dividing cells. Bacteria have also been documented to change their permeability to antibiotics based on environmental factors such as temperature, reactive oxygen species and presence of specific inducers113. These factors are not assessed in in vitro studies. Natural compounds have also been documented to induce resistance such as bile salts in the gastrointestinal tract inducing MDR pumps in Salmonella typhimurium 112, 114.

Inoculum size, an important factor in the development of resistance, is another variable that is not replicated in in vitro experiments. The bacterial load at the site of infection may be higher than in the laboratory tests and thus the actual MIC of the population may be higher. The inoculum effect was first described for β-lactam antibiotics, due to β-lactamase enzyme production degrading the antibiotic at a faster rate when there where more cells present115. It has since been described for other classes of antibiotics and possible mechanisms for this include a reduction in the antibiotic concentration at high cell densities due to binding of the antibiotic to cell debris or due to less molecules of antibiotic being present per cell at high inoculum infections113.

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HPLC Assay

The HPLC assay developed for this experiment assessed stability of cefovecin in CAMHB incubated at 37 °C for extended periods of time. The media CAMHB is composed of primarily dehydrated beef proteins in addition to casein and starch and previous work using tissue chambers in cats showed cefovecin to remain above 0.06 mg/L in transudate for at least 14 days post subcutaneous dosing82. Thus, it was hypothesised that cefovecin in a broth composed of largely water and protein would behave similarly to cefovecin in the cat’s extracellular fluid which is of similar temperature and composition116.

There was difficulty encountered in sample extraction from the CAMHB. The majority of techniques described in the literature are for biological fluid analysis such as plasma or urine and use mass spectrometry; there are no published studies to the author’s knowledge for cephalosporin extraction from microbiological growth mediums. Cox and colleagues in 2014 published the first validated method for cefovecin HPLC analysis with internal standard, without using mass spectrometry. The assay involved plasma sample extraction using hydrophilic lipophilic balanced (HLB) cartridges, ammonium acetate and acetonitrile as the mobile phase, a C18 column and flow rate of 0.85mL/min117. Cefovecin concentrations have also been determined using HPLC and mass spectrometry in a number of studies in monkeys, macaques, baboons, dogs and cats, and without mass spectrometry in green iguanas, hens and alpacas118,81,82,119,120.

In 2003 Lin and colleagues described an HPLC assay to determine the concentration of colistin in CAMHB, and Wright and colleagues in 1998 published an HPLC assay for fluoroquinolone detection in CAMHB121,122. Both the colistin and fluoroquinolone methods used C18 columns and mobile phases consistent of acetonitrile and a buffer. The colistin sample extraction from CAMHB used C18 solid phase extraction (SPE) cartridges and the fluoroquinolone method used acetonitrile to achieve deproteinization and filtration with a 0.45 μm syringe filter

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for sample extraction121,122. The extent of protein binding of colistin and fluoroquinolones are however significantly lower than cefovecin123,124.

Based on this literature a number of SPE cartridges were trialled without success for this assay. Cefovecin was poorly retained on C18 SPE cartridges and binding on HLB cartridges only occurred under acidic conditions when the analyte was protonized. Heavy washing with acid-methanol solutions was necessary to remove matrix interference after the HLB cartridge extraction and this also removed a large proportion of the cefovecin analyte. A dispersive silica-based primary and secondary amine (PSA) sorbent that relies on weak anion exchange was also trialled; the PSA has sorbent functional groups that are ionized at pH of less than 8. Major matrix interference peaks did not bind to the PSA yet binding of cefovecin, pKa 8, was limited due to minimal ionization at a pH of less than 8. The final assay used centrifugal filter tubes with a 3000 dalton molecular weight limit, which successfully reduced proteins from the broth matrix while allowing unbound cefovecin with a molecular weight of 453.488 g/mol125 to pass through and be collected as the supernatant. This produced narrow cefovecin peaks with good symmetry, little peak fronting and very minimal peak tailing. It is important that HPLC assay peak shapes resemble Gaussian peaks as closely as possible to improve resolution and ensure accurate quantification.

Results of the cefovecin stability assay showed that the mean percentage of cefovecin detected after 24 hours incubation in CAMHB compared to the starting concentration was 97.786%. The loss could signify degradation or insufficient deproteination of samples prior to analysis. Degradation of drugs is defined as an irreversible change in the structure of the organic molecule. Hydrolysis and oxidation are the most common forms of degradation and the stability of the drug is based on the rate these reactions occur. As a general rule for pharmaceuticals 90% of the specified label potency is the minimum acceptable level, therefore no more than 10% degradation can occur126.

Cefovecin is documented to have poor stability in the aqueous form after extended periods of time and cephalosporins in general are known to be less soluble and

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unstable in aqueous solutions therefore are often prepared as sodium salts which have increased stability at lower temperatures127. Cefovecin is therefore not available commercially as a ready-to-use injection but rather comes as a lyophilised, dehydrated powder and diluent containing benzyl alcohol and water for injection128.

The only published literature concerning cefovecin stability pertains to the commercially available cefovecin product, Convenia. The stability studies for registration were performed for the lyophilised powder and the reconstituted form. Degradation was observed to occur in aqueous solution and elevated temperatures. Stability data from both active ingredient and the lyophilised cefovecin powder in accelerated conditions at 25°C and 60% relative humidity for 6 months showed no significant changes, where significant changes are defined as more than 5% change in assay from initial value, any degradation exceeding acceptance criterion, failure to meet acceptant criteria for appearance, physical attribute and functionality129. The lyophilised powder stability studies, although not significant, demonstrated the content of cefovecin decreased slightly with increasing temperature and the degradation products increased slightly with increasing temperature128.

Accelerated conditions are often used for pharmaceutical product testing; this involves stressing products at different conditions such as higher temperatures, pH and moisture. This can then allow degradation at the recommended storage conditions to be predicted using relationships between degradation rate and acceleration factor, based on the Arrhenius equation which describes reaction rate in relation to temperature dependence130. Thus, even though the cephalosporins are not a particularly stable chemical group, the stability of cefovecin in aqueous solution and elevated temperature in this work, aiming to replicate the in vivo environment, appears adequate for these in vitro experiments and is in agreement with the described scientific literature.

Cephalexin stability in CAMHB was not assessed by HPLC in this project due to resource limitations. Cephalexin, a deacetoxycephalosporin, has been shown to

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degrade via hydrolysis at the β-lactam bond. Semilogarithmic plots of cephalexin degradation are linear, suggesting the degradation follows first-order kinetics131. In 1976 Yamana and colleagues reported that after cephalexin was incubated at 35°C and pH 8.02 for 30 hours, there was at least 80% of the initial cephalexin concentration remaining determined by HPLC analysis132. Lallemand and colleagues (2016) investigated in vitro degradation of antimicrobials uding borth microdilution methods. They observed a degradation of 56% of cephalexin after 24 hours at 37°C with no bacteria present. They then tested E.coli MICs with either the standard microdilution protocol or with ad hoc addition of cephalexin to account for degradation and found no significant difference in MICs between the two133. Thus suggesting that some level of antimicrobial degradation may not have significant impact on MIC values, regardless of the absence or presence of bacteria.

Significance of Results

The registration application advice summary form for cefovecin in Australia states that the National Health and Medical Research Council (NHMRC) expert advisory group on antimicrobial resistance assessed the antibiotic resistance aspects of cefovecin sodium and expressed concern at the lack of antimicrobial resistance data. It was decided that the product presented low and acceptable risks as it was an injectable product for companion animals only and they advised the label must state: “Do not use in food-producing species of animals and for use only in dogs and cats where indicated by antibiotic sensitivity testing according to principles of prudent use”. The European dossier summary stated, “the particular impact of cefovecin on the human and animal gut flora in respect of the development of antimicrobial resistance has not been investigated as the product is not indicated for treatment of food-producing animals”.

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While antimicrobial use in food-producing animals may pose more of a risk to antimicrobial resistance and the spread of this resistance to humans, this statement is concerning given the amount of literature now describing transmission of pathogenic bacteria from companion animals to humans including Campylobacter species, Salmonella species, Staphylococcus pseudointermedius and E. coli 20-23.

The β-lactam antibiotics are defined as concentration independent, or time dependent antibiotics, where the key parameter associated with clinical cure is the amount of time the drug level at the site of infection exceeds the MIC. This is different for each pathogen and maximal effect may be when the concentration is above the MIC from anywhere from 40-100% of the dosing interval29. Alou and colleagues (2005) used an in vitro model to compare intermittent and continuous infusions of ceftazidime against Pseudomonas aeruginosa. Against susceptible and intermediate strains there was no difference between the dosing regimens, only the continuous infusion achieved bactericidal activity for the resistant strain, stressing the importance of time above MIC134. Therefore, it is logical that antibiotics that minimize development of resistance when given more frequently are not appropriate pharmacodynamically for long-acting antibiotic preparations33.

While the limitations of in vitro resistance testing and the methods used in this research have been discussed, the results of these sub-inhibitory cultures and analysis of the current literature suggest that the pharmacokinetics of cefovecin in companion animals presents an increased risk for development of antimicrobial resistance compared with shorter acting cephalosporin products.

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CHAPTER 6: CONCLUSION

There were four main aims for this project, all of which were completed. A successful MIC method was performed to determine MICs for cefovecin and cephalexin for E. coli isolates and fulfil aim one. Aims two and three included performing sub-inhibitory cefovecin, cephalexin and antibiotic-free cultures and the results of aim four indicated that cefovecin was stable enough to use in in-vitro cultures.

It was demonstrated that prolonged exposure to sub-MIC concentrations of both cephalexin and cefovecin resulted in the emergence of antimicrobial resistance in E. coli. Sub-inhibitory cephalexin cultures at 0.8 x MIC increased E. coli MICs by 4 times (in 2/5 isolates), after 72 hours and by 4 times in all 5 isolates by 96 hours. In contrast, sub-inhibitory cefovecin cultures at 0.8 x MIC increased E. coli MICs by up to 4 (in 3/5 isolates), 16 (in 1/5 isolates) and 8 (in 2/5 isolates) times after only 24, 96 and 120 hours respectively. The action of cefovecin on these 5 isolates of E. coli was both more rapid and to a larger extent than the similar action of cephalexin. This study shows that exposure to sub-MIC concentrations of cephalexin or cefovecin pose a great risk for the development of resistance, and that the risk was higher for cefovecin than for cephalexin for these 5 isolates of E. coli.

Although the initial increases in MIC may be by only a few orders of magnitude, if this increase in MIC occurs in commensal flora and regular therapeutic doses of antimicrobials are used, the organisms may become increasingly resistant.

According to published cefovecin pharmacokinetic studies, when cats are dosed with cefovecin according to the label dosage of 8 mg/kg by subcutaneous injection, the total plasma concentration would still be 50 x MIC 21 days post dosing for E. coli isolates 1 and 5 from our project and 25 x MIC for our isolates 3, 4 and 5. Free plasma concentrations would fall below the MICs after 24 hours, yet free cefovecin concentration in transudate would remain above our E. coli isolate 1 and 5 MICs

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for more than 16 days. Cephalexin pharmacokinetic studies in cats show peak plasma concentrations between 11.1 and 28.6 mg/L, two hours post 25mg/kg oral dosing72. Thus the feline plasma concentration would be above the MICs of our projects E. coli isolates 2-5 for less than 3 hours. Thus as it took 72 hours in our study to show an increase of MIC by more than 2-fold for cephalexin in 0.8 x MIC cultures it is unlikely this will occur in vivo when following conventional dosing. Cefovecin however showed an increase of MIC by more than 2-fold after as little as 24 hours in the 0.8 x MIC cefovecin culture in our experiment, thus in vivo with free transudate concentrations being above 0.8 x MIC for more than 16 days similar results are feasible.

This research highlights the need for in vivo studies involving long-acting cephalosporin antibiotics to investigate the potential for bacterial resistance development. It is important to investigate the antibiotic concentrations in various body compartments and how they fluctuate over time to highlight the impact on development of resistance by both pathogenic and commensal bacteria.

The persistence of resistance experiment showed that none of the E. coli isolates returned to their initial MICs post 144 hours of incubation in antibiotic free media. To better simulate in vivo conditions, the E. coli isolates would need to be incubated at sub-MIC cefovecin concentrations for a much longer period of time to see if MICs increased further, then passaged through antibiotic-free media for a longer period of time to see if the increase in MIC was persistent. If the increase in MIC is not reversible after removal of the antimicrobial selection pressure, this could lead to the development of pathogens and commensals increasingly resistant to cefovecin with potential cross-resistance to other antimicrobials as well.

This research also highlights the need for further in vivo studies involving short and long-acting cephalosporin antibiotics to investigate the rate of loss of bacterial resistance after total clearance of antibiotic from an animal. Once again this study has highlighted the need to investigate the antibiotic concentrations in various body compartments to determine how long they persist at sub-inhibitory concentrations.

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Third generation cephalosporin resistance and the interplay between pharmacokinetics, antibiotic half-life and bacterial resistance are of great significance in both the veterinary and human health fields. Veterinarians seek not only to limit the development of untreatable multi-drug resistant bacteria in animal populations but also to minimise the possibility of spreading third- generation cephalosporin-resistant bacteria to the human field, where this antibiotic class is crucial in treating life-threatening infections.

This research supports recommending that veterinary practitioners classify and use cefovecin as a secondary or tertiary antibiotic 135. Before its selection veterinarians should perform culture and sensitivity testing and confirm there are no first-line drugs that are suitable for treatment. Due to its long half-life cefovecin should also only be recommended for use in infections that require a long duration of treatment. This conclusion contrasts unfavourably with use recommendations in common formularies 136.

Treatment with cefovecin cannot be de-escalated after clinical or microbiological evidence that a bacterial infection is not present or has been eliminated or an alternative diagnosis has been made.

There is little debate that antibiotic resistance is a very relevant public health concern and there are numerous multi-drug resistant pathogens present in both human and animal medical fields at present. Infectious disease remains the second leading cause of human death worldwide137, with an estimated 25,000 deaths each year in the European Union alone caused by multi-drug resistant bacteria138. There have been reports that the world may be heading into a post-antibiotic era with minimal new antimicrobial drugs on the market. Reasons for a lack of new antimicrobials include high costs involved in research and development, shorter treatment durations with antimicrobials compared with drugs for chronic conditions, scientific challenges in finding new antimicrobials for gram negative infections and a lacking of robust stewardship programs to minimise the development of resistance to new antimicrobials and increase their lifespan139.

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Alexander Fleming, a Scottish bacteriologist who shared the Nobel Prize for the discovery of penicillin, predicted many of the current day antimicrobial predicaments. He prophesied in his 1945 Nobel lecture; "the time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily under-dose himself, and by exposing his microbes to non- lethal quantities of the drug make them resistant.”140

It is crucial to continue this research and use our current antimicrobials judiciously and in the most appropriate clinical manner to limit this spread of resistance, as new antimicrobials are not on the horizon and the race against antibiotic resistance has well and truly begun. Future steps in this research would include sequencing the E.coli to determine resistance mechanisms followed by building an in vitro dynamic pharmacokinetic pharmacodynamic model to simulate the continuously changing drug concentrations over a longer period of time. 141,142 Followed by an in vivo study in cats to assess effects on commensal bacteria post dosing with cefovecin, looking at a number of different bacteria and development of cross resistance to other antimicrobials.

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Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Robson, Sarah Anne

Title: The impact of pharmacokinetics on the emergence of in vitro bacterial resistance to cefovecin

Date: 2017

Persistent Link: http://hdl.handle.net/11343/192887

File Description: The impact of pharmacokinetics on the emergence on in vitro bacterial resistance to cefovecin.

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