How can P. aeruginosa infections be treated more efficiently?

A Comparison Between the Antibiotic Agents Gatifloxacin and Marbofloxacin

Diploma Thesis , 2017 , 60 Pages , Grade: 4,0

Medicine - Pharmacology, Pharmacy

This study is concerned with a comparison between the antibiotic agents marbofloxacin and gatifloxacin in fluoroquinolones and their efficiency in the treatment of Pseudomas aeruginosa.

Pseudomonas aeruginosa, a major cause of extremely dangerous nosocomial infection, has become problematic in both human and veterinary medicine due to it´s resistance to multiple drugs. Several experiments within research concerning development of bacterial resistance in P.aeruginosa have been reported. Imported resistance mechanism of P.aeruginosa is linked to mutations of DNA gyrase, the fluoroquinolone target, as well as overexpression of efflux pump. The worldwide, frequent use of different antibiotics contributes to a significant reduction in the bactericidal and bacteriostatic effect of different antibiotics. P.aeruginosa is represented as a ubiquitous organism present in environmental surroundings, and can be isolated from various living sources, including plants, animals, and humans.

The ability of P. aeruginosa to survive on minimal nutritional requirements and to tolerate a variety of physical conditions has allowed this organism to persist in both community and hospital settings. Within hospitals, P. aeruginosa can be isolated from a variety of sources such as respiratory therapy equipment, antiseptics, soap, sinks, mops and medicines. As resistance to antibiotic is a relevant topic within today’s global health care concerning both human and veterinary medicine, the curiosity for this expanding health problem resulted in a 9 days serial passage of P.aeruginosa in broth, investigating the susceptibility to marbofloxacin and gatifloxacin.

The aim in this study was, therefore, to demonstrate that 4th generation fluoroquinolones appears to be more beneficial and efficient in the treatment of P.aeruginosa infections, than 2nd generation fluoroquinolones. In other words, the aim is to prove that gatifloxacin is of better use than marbofloxacin, due its antibiotic activity.

Table of contents

ABBREVIATIONS

1. INTRODUCTION AND AIM OF THE STUDY

2. LITERATURE REVIEW
2.1 Pseudomonas infections and diseases
2.1.1 Human Medicine
2.1.1.1 Respiratory tract infections
2.1.1.2 Skin and soft-tissue infections
2.1.1.3 Urinary tract infections
2.1.1.4 Ocular infections
2.1.2 VETERINARY MEDICINE
2.1.2.1 External otitis
2.1.2.2 Urinary tract infections
2.1.2.3 Respiratory and other infections
2.2 Fluoroquinolones - 2nd and 4th Generation
2.2.1 Fluoroquinolones - Mechanism of action
2.2.2 Fluoroquinolone resistance in P.aeruginosa
2.2.3 Comparison of 2nd and 4th generation fluoroquinolones - Human medicine
2.2.4 Comparison of 2nd and 4th generation fluoroquinolones - Veterinary Medicine

3. MATERIALS AND METHODS

3.1 Bacteria involved in the study

3.2 Preparing stock solutions

3.3 Determination of MIC for marbofloxacin and gatifloxacin - Evaluation of the 9 days serial-passage

4. RESULTS
4.1 Results based on experimental data

5. DISCUSSION

6. SUMMARY

7. ACKNOWLEDGEMENTS

8. REFERENCES

Abbreviations

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1. INTRODUCTION AND AIM OF THE STUDY

Pseudomonas aeruginosa, a major cause of life-threatening nosocomial infection, has become problematic in both human and veterinary medicine due to multiple-drug resistance. Several experiments within research concerning development of bacterial resistance in P.aeruginosa have been reported. Imported resistance mechanism of P.aeruginosa is linked to mutations of DNA gyrase, the fluoroquinolone target, as well as overexpression of efflux pump. The worldwide, frequent use of different antibiotics contributes to a significant reduction in the bactericidal and bacteriostatic effect of different antibiotics. P.aeruginosa is represented as a ubiquitous organism present in environmental surroundings, and can be isolated from various living sources, including plants, animals, and humans. The ability of P. aeruginosa to survive on minimal nutritional requirements and to tolerate a variety of physical conditions has allowed this organism to persist in both community and hospital settings. Within hospitals, P. aeruginosa can be isolated from a variety of sources such as respiratory therapy equipment, antiseptics, soap, sinks, mops and medicines. As resistance to antibiotic is a relevant topic within todays global health care concerning both human and veterinary medicine, the curiosity for this expanding health problem resulted in a 9 days serial-passage of P.aeruginosa in broth, investigating the susceptibility to marbofloxacin and gatifloxacin.

Our aim was in this study was to demonstrate that 4th generation fluoroquinolones appears to be more beneficial and efficient in the treatment of P.aeruginosa infections, than 2nd generation fluoroquinolones. In other words, our aim was to prove that gatifloxacin is of better use than marbofloxacin, due its antibiotic activity. By performing a 9-days serial- passage of P.aeruginosa in broth we were able to determine the level of resistance by evaluating bacterial growth. It is important to increase awareness of the frequent use of antibiotic agents, as overuse can lead to development of resistant strains, thereby affecting in both human and veterinary health.

2. LITERATURE REVIEW

2.1 Pseudomonas infections and diseases

Pseudomonas aeruginosa is a worldwide multidrug-resistant bacterium and a major cause of life-threatening nosocomial infections.

2.1.1 Human Medicine

2.1.1.1 Respiratory tract infections

In a normal, healthy lung with functional immune defence, pathogens and other unwanted microorganisms entered by inhalation are usually removed and brushed away by ciliated epithelia that lines our airways, and prevent the colonization of bacteria. This natural defence provided by our body known as the mucociliary clearance (Figure 1). However, when a natural lung defence is impaired, bacteria will be able to invade and colonize in the airways, thereby affecting the lungs. Inflammation and infections may result reducing the health status of the individual. The majority of health problems caused by P.aeruginosa in humans is known to affect the respiratory tract. There is plenty of information published concerning respiratory infections in humans caused by P. aeruginosa, for instance in immune suppressed people like HIV-infected patients, Pseudomonas aeruginosa most commonly causes pneumonia or sinusitis. P.aeruginosa causes pneumonia in individuals with suppressed lung defence, and is claimed to affect the pathogenesis of the disease cystic fibrosis.The lung of patients with cystic fibrosis is susceptible to pseudomonas aeruginosa, leading to a worsening in the development of lung diseases in these patients, with its ability to switch off the expression of immunostimulatory agents, making it persistent in the lung. The bacterium also produces a lot of enzymes such as protease, elastase and phospholipase, that work for the microorganisms' benefits to survive in the lung. In cystic fibrosis patients having chronic airways inflammation, P.aeruginosa has shown to increase morbidity and mortality in these individuals. Observations show that isolates of P.aeruginosa from patients with cystic fibrosis have a characteristic mucoid colonial morphology. Apparently this morphology results in a worse prognosis than non-mucoid colonies isolated from P.aeruginosa. As mentioned, in individuals diagnosed with cystic fibrosis, the mucociliary clearance is greatly reduced (Gomez et.al, 2007).

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Figure 1. Host-pathogen interactions in chronic fibrosis patients. Underlying predisposing factors of the CF lung and specific characteristics of P. aeruginosa allow for colonization and persistent infection (Gomez et.al, 2007).

2.1.1.2 Skin and soft-tissue infections

In cases where P.aeruginosa causes infections in the skin and soft tissues, the outcome can be serious as this pathogen has invasive and toxigenic properties. The presence of P.aeruginosa on the human skin is not an abnormal finding as it can be found in the axillary and the anogenital region. Healthy people are not likely to develop Pseudomonas infection. Transmission of Pseudomonas infections from health care workers to patients in hospitals are common as a result of insufficient hand hygiene or disinfection. P.aeruginosa is considered to be an opportunistic pathogen, which can cause diseases in immunocompromised individuals. Patients with burns, HIV/AIDs, diabetes, malignancies or intravenous catheter have a higher risk of infection caused by P.aeruginosa. People who suffer from skin infections caused by P.aeruginosa show a variety of signs and symptoms, including deep abscesses, black or purple discoloration from scabs (after thermal burns), subcutaneous nodules, haemorrhagic or necrotic erythematous (red) lesions and cellulitis (Brian Wu, 2015).

Skin infections caused by P. aeruginosa can appear differently according to severity and location of the infection:

- Ecthyma gangrenosum. These patients are often neutropaenic, presenting ulcerated, black, erythematous (red) lesions in the axillary, inguinal or anogenital area.
- External otitis, which is the most frequent Pseudomonas infection of the ear. People affected with external otitis show signs of pain, swelling and redness of the external ear and purulent discharge.
- Chronic leg ulcers. Foul-smelling, green superficial crust can often be seen as a result of Pseudomonas aeruginosa colonization.
- Thermal burn wounds. If an eschar is present on the skin, Pseudomonas aeruginosa may grow under the layer. These causes often result in bacteraemia, with a high risk of mortality.
- Chronic paronychia (inflammation of the nail, or around the nail region of the finger or toe) and onycholysis (disorder of the white nail plate, splitting of the nail bed). Greenish discoloration of the nail as a result of colonization of Pseudomonas aeruginosa (Wu, 2015).

2.1.1.3 Urinary tract infections

In humans, several bacterial families can cause urinary tract infections, which is a health problem affecting millions of people each year. If UTIs are not treated, the infection may extend and develop into an ascending infection, causing pyelonephritis. Infections of the urinary tract are the second most common type of infection in the body. One of the most common causes for urinary tract infections in humans are catheterization of the urinary tract, which is also called catheter associated urinary tract infection (CAUTI). This type of infection is responsible for more than 1 million cases in hospitals annually and does often involve other uropathogens than Escherichia coli, however, little is known about the pathogenesis of UTIs caused by pathogens like P. aeruginosa (Mittal et.al, 2009).

2.1.1.4 Ocular infections

Bacterial keratitis

Bacterial keratitis is a serious bacterial infection of the cornea, also referred to “corneal ulcer”. In severe cases, this infection may lead to loss of vision. Bacterial keratitis is most commonly caused by bacteria, but can also be caused by other pathogens such as fungi, mycobacteria, protozoa and viruses. It appears to be a high risk of infection for people using contact lens wear. Improper lens disinfection or wearing contact lenses overnight may increase the risk of getting infected. Foreign bodies, traumas like chemical and thermal trauma, contaminated eye solutions or reduced defence mechanism can be predisposing factors. In several cases of culture proven infections, 19-42% of the cases were associated with contact lens wear. In young individuals, trauma and contact lens wear are the most common causes of infection, while older individuals tend to be infected as a result of dry eyes, bullous keratopathy or surgical injuries (Bartomolei et.al, 2010).

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Figure 2. Pseudomonas keratitis. There is a large epithelial defect associated with a ring­like stromal infiltrate, which is “soupy” in appearance owing to stromal necrosis. The non- involved areas of the cornea have a characteristic “ground glass” appearance. A small hypopyon is present. Concerning the majority of cases where bacterial keratitis is concerned contact lens wearers (Weed et.al 2013).

As mentioned, microbial keratitis can be caused by a variety of pathogens such as viruses and fungi and protozoa. Bacterial keratitis, however, is most commonly caused by P.aeruginosa, which is associated with a more severe visual prognosis than what is caused by other bacterial pathogens (Weed et.al 2013).

Based on subjective experiences, onset and progression of ocular pain, redness, blurred vision, photophobia and tearing has been reported among patients infected with Pseudomonal keratitis. Corneal epithelial defect and stromal infiltration that can resemble a ring (figure 2), are clinical features that have been observed upon examination. Bacterial keratitis caused by pseudomonas aeruginosa is treated topically with antibiotics such as fluoroquinolones, cephalosporins, synthetic penicillins or aminoglycosides. (Weed et.al 2013).

Concerning the fact that the majority of cases with bacterial keratitis in contact lens wearers are associated with Pseudomonas aeruginosa, a study was conducted at the Iowa university of Health care. The purpose of this investigation was to determine the frequency and the level of visual loss in in bacterial keratitis caused by pseudomonas aeruginosa in patients who had no risk factors for infection except for contact lens wears, and had normal visual acuity. The study was performed based on the medical records, patients diagnosed with bacterial keratitis at the University of Iowa Hospitals and Clinics. Eyes of patients that had a history of contact lens use at the onset of the corneal infection, were examined. A total of 29 eyes from 28 patients, were treated for bacterial keratitits caused by Pseudomonas aeruginosa from july 2006, to june 2011. It turned out that 8 eyes from the 8 patients, being 6 men and 2 women, met the criterias of the investigation; all of the 8 patients were using contact lens wear at the time of the presentation, either occasionally, or regularly overnight wear (Weed et.al, 2013).

2.1.2 VETERINARY MEDICINE

2.1.2.1 External otitis

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Figure 3: Chronic otitis with Pseudomonas aeruginosa infection. Purulent discharge and the erosions. Apperance of ulcerative lesions of the anthelix and tragus around the ear canal opening. (Andrew Hillier 2005).

Otitis externa is a common disease facing veterinary dermatology, defined as an inflammation of the external ear canal. It may or may not involve the ear pinna. The infection can be acute or chronic, unilateral or bilateral. Chronic infections often lead to the middle ear, causing otitis media. Head-shaking, ulceration, swelling, unpleasant smell and erythema are clinical signs that may be seen in this condition (figure 3). Otitis externa can occur as a primary or secondary cause, depending on the invasive factor. In primary causes the infection occurs in a normal, healthy ear, while in secondary causes the infection enters an abnormal ear. Primary causes can cause otitis without any visible signs, and may not be discovered until secondary causes develop, and visible clinical signs appear. Primary causes can be allergy, autoimmune, fungal, viral, auto-immune, endocrinology, foreign bodies and epithelialization disorders. Secondary causes can be of bacterial or fungal origin, yeast- overgrowth or simply over-cleaning. The most frequent bacterial agents causing otitis externa include P.aeruginosa and Staphylococcus species. In dogs, P.aeruginosa is one of the most common bacteria isolated in cases of external otitis. While acute infections often respond to topical treatment, chronic infections require antibiotic therapy and proper cleaning in addition to topical treatment. Treating external otitis caused by P.aeruginosa is therefore facing a considerable challenge in clinical practice, both for the clinicians and clients (Moriello et.al 2016). Previous studies have reported a moderate to high levels of resistance to frequently used antimicrobials in P.aeruginosa isolates from otitis externa cases (Cole et.al, 1998).

Diagnostic procedures

Swab cytology of the otic exudate is required in in the case of otitis. In the presence of neutrophils, P.aeruginosa appear as a rod-shaped organisms (figure 4). (Hillier et.al, 2005)

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Figure 4: Cytology preparation of the exudate obtained from the dog shown in Figure 1. Note the rod-shaped Pseudomonas organisms that are visible both extracellularly and intracellularly within neutrophils (Diff-Quik stain, 31000 magnification) (Hillier 2005).

Antimicrobial therapy

Topical antibiotics

Topical antibiotic treatment for external otitis caused by P.aeruginosa exposes the bacteria to a high concentration of antibiotics, often more than 1000 times the MIC (minimum inhibitory concentration) of the organism. Certain strains of P.aeruginosa showing in vitro resistance can be killed by the application of a high concentration of antibiotics, thereby defeating the resistance mechanism of the bacteria. This explains why topical antibiotics must be used in all cases of external otitis caused by P.aeruginosa (table 1).

As a general guideline, 5-10 drops of antimicrobial solution, depending on the size of the dog, should be applied twice daily to the infected ear (Hillier, 2005).

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Systemic antibiotics

Application of systemic antibiotics for treatment of P.aeruginosa should only be considered in certain cases. In the presence of otitis media, swelling and hyperplasia of the ear canal epithelium, ulceration of the ear canal epithelium, or exhibiting adverse reactions to topical antimicrobial treatment are indications of where systemic antibiotic can be applied. In cases where owners fail or lack the ability to administer the medication topically, systemic antibiotics should be considered. There are few systemic antibiotics available having efficient activity against P.aeruginosa. Some of these agents must be administered intravenously or subcutaneously, being considered unsuitable for a medium or a long-term treatment.

Orally administered antibiotics for treating P.aeruginosa infections are related to the use of fluoroquinolones. Finding the suitable fluoroquinolone is based on bacterial culture and susceptibility testing results. According to theory, there are two formulas that can help to determine if the selected fluoroquinolone antibiotic is efficient, and whether resistance is likely to develop (Hillier, 2005).

1. The first formula stated that a maximum plasma concentration (Cmax)/MIC90) with a ratio of at least 8 is preferable. If the ratio is less than 8, resistant P.aeruginosa strains are likely to develop (Lode et.al, 1998).
2. The second formula stated that area under the curve (AUC)/MIC) with a ratio of at least 125, will most probably indicate clinical efficiacy (Nicolau et.al, 1995).

Finding and selecting a specific fluoroquinolone antimicrobial that meet these target ratios can be difficult. There is little or no information available explaining the tissue levels of fluoroquinolones in the ear epithelium of dogs with external otitis caused by P.aeruginosa. In spite of his, there are few guidelines available for clinical practitioners that can be helpful for selecting fluoroquinolones upon treatment (Table 2.)

Several studies concerning the susceptibility of clinical isolates of P.aeruginosa to different fluoroquinolones have been reported (Hillier, 2005)

Infected ears and chronic otitis externa

In this study, susceptibility testing of clinical isolates of P.aeruginosa to various fluoroquinolones were performed.

The different fluoroquinolones applied were ciprofloxacin, marbofloxacin and enrofloxacin. The study showed that 93,4% of isolates were susceptible to ciprofloxacin and marbofloxacin, while 71% of the isolates were susceptible to enrofloxacin. From the total number of 183 isolates, 106 came from infected ears (Seol et.al, 2002), A similar study showed that 89,9% of isolates were susceptible to marbofloxacin, and 42,1% of the isolates were susceptible to enrofloxacin. 19 of the isolates performed in this study came from ears diagnosed with chronic otitis externa (Barrasa et.al,2000).

Otitis media

A study was performed investigated the susceptibility testing of clinical isolates from the middle ear of dogs diagnosed with otitis media. 51% of 38 isolates were susceptible to enrofloxacin. P.aeruginosa to various fluoroquinolones were performed. Those isolates that were resistant to enrofloxacin by using the Kirby- Bayer method proved to be suceptible to MIC testing (Colombini et.al, 2000).

In a similar report, 12,5% of isolates from the external ear canal showed susceptibility to enrofloxacin, while 35% of isolates from the middle ear in dogs with chronic otitis externa showed susceptibility to the same antibiotic substance (Cole et.al, 1998).

Suppurative otitis

In another report, marbofloxacin alone was administered in orally once a day for 21 to 42 days in dogs with suppurative otitis. Out of the 54 dogs tested, 43,8% were infected with P.aeruginosa. Topical ear treatment was not applied. The study showed that 27,8% of the dogs were healed from the infection. 42,6% showed partial to complete healing, while 29,6% did not respond to therapy. It was claimed that the half-life of marbofloxacin is longer than other fluoroquinolones (Carlotti et.al, 1998).

Table 2. Treatment summary for Acute and Chronic Pseudomonas Otitis, indication ear cleaning and the use of topical and systemic antibiotics (Hillier 2005).

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2.1.2.2 Urinary tract infections

A urinary tract infection (UTI) is a condition where the ureter, urethra, kidney, bladder or any other part of the urinary tract is infected. (Balentine et.al, 2017). Unlike human patients, veterinary patients are often asymptomatic. Because of this, a UTI may be an incidental finding in veterinary medicine. In dogs, bacterial UTI is the most common form of the disease, however in cats, this condition is not common, but tend to affect older individuals as a consequence of age or diseases like diabetes mellitus, hyperthyroidism or renal failure. In ruminants, urinary tract infection most commonly occur due to catheterization or parturition in females, and occur as a cause of urolithiasis in males. In horses, urinary tract infections are not common, though it can occur in association with urethral damage, urolithiasis or bladder paralysis. If a urinary tract infection remains untreated, the consequences may lead to reduced urinary tract dysfunction, prostatitis, infertility, septicaemia, and pyelonephritis with scarring and eventually kidney failure. Studies have documented that Escherichia coli is the most common uropathogen in dogs and cats responsible for both acute recurrent UTI's. Other common uropathogens include Staphylococcus, Streptococcus, Pseudomonas species, Klebsiella and Proteus (Dowling, 2016).

Treatment of urinary tract infections require administration of antibiotics sometimes repeated medication is needed if the infection is recurrent. In cases of chronic urinary tract infections, the treatment may be limited, as bacteria might be resistant to the given antibiotic. Urinary culture is the most common way to diagnose a urinary tract infection. In cases of recurrent urinary tract infections or in cases where antibiotic therapy does not respond to the infection within 7 days, antibiotic sensitivity testing should be carried out. High concentration of antibiotics in the urine may indicate different conditions. Most antimicrobials undergo renal elimination, which involve processes such as secretion and reabsorption, which explains why antibiotics can be found in the urine. High concentration of antibiotics in the urine can be found in cases like uncomplicated cystitis, or in the case of complicated pyelonephritis. This is a significant feature, as the presence of antimicrobials in the urine is important for the elimination of the bacteria in the urine. Antibiotics prescribed by veterinarians to treat UTIs in animals should be easy for the owner to administer, not too expensive, and have few adverse effects. When the urine culture and sensitivity testing are made, the bacterial minimum inhibitory concentration (MIC) can be figured and the right antimicrobial drug can be found for treatment of the infection. Antibiotics like enrofloxacin, orbifloxacin, and marbofloxacin are fluoroquinolones used for treatment of UTIs in dogs. In North-America, pradofloxacin is only approved for treatment of skin infections in cats, while it is approved in Europe for treatment of UTI in both dogs and cats. The fluoroquinolones are bactericidal, amphoteric drugs. They possess acidic and basic properties but are very lipid soluble at physiologic pH, and have a high volume of distribution. They are the only orally administered antimicrobials effective against Pseudomonas. Therefore, fluoroquinolones should be reserved for UTIs that involve gram-negative bacteria, especially Pseudomonas, and for UTIs in intact male dogs and cats because of their excellent penetration into the prostate gland and activity in abscesses. They are concentration- dependent killers with a long post-administration effect, so once daily, a high-dose therapy for a relatively short duration of treatment is effective. Fluoroquinolones should be avoided for chronic, low-dose therapy, because this encourages emergence of resistant bacteria that are cross-resistant to other antimicrobial drugs as well. Cases that involve Pseudomonas should be carefully investigated for underlying pathology. Once Pseudomonas spp become resistant to the fluoroquinolones, there are no other convenient therapeutic options (Dowling, 2016).

2.1.2.3 Respiratory and other infections

Pneumonia is a condition described as an inflammation of the lungs or lower respiratory tract. Generally, this inflammation is a response of tissues or cells to irritation, injury or infection. In dogs, the most common form of pneumonia is caused by bacteria. There are several pathogens responsible for this infection, such as Bordetella Bronchiseptica, Streptococcus zooepidermicus, Pasteurella multocida, Klebsiella pneumoniae, E.Coli, Mycoplasma species and Pseudomonas aeruginosa. In many cases, pneumonia can develop as a secondary infection, provoked by an underlying cause such as a viral infection, irritants in the form of smoke, or other inhaled contaminants that predisposes to a bacterial infection. A bacterial pneumonia includes symptoms of high fever, cough, breathing difficulties, lethargy, and reduced exercise tolerance. Nasal discharge, weight loss, fast and loud breathing, dehydration and anorexia are other symptoms that may be present (Yuill, 2010).

Respiratory tract infections are generally a frequent cause of morbidity and mortality in cats. In spite of the fact that several bacteria have been isolated from the airway in healthy cats (Johnson et.al 2005), P.aeruginosa is an abnormal cause of respiratory tract infections (Schulz et.al, 2006). It has been documented that in experimental cat models, recurrent P.aeruginosa infections causes chronic bronchiolitis and pneumonia, having corresponding histological features to chronic P.aeruginosa infection to human CF (Thomassen et.al, 1984).

A previous study reports the interspecies transmission of the LES strains of P.aeruginosa from an adult human patient with CF to a cat. As chronic infections with P.aeruginosa are common in cystic fibrosis patients, certain strains of P.aeruginosa are more transmissible and virulent than others. In this study, the LES strain was used in the interspecies transmission from human to an animal, as cross infections have earlier been reported between patients with CF and healthy non-CF individuals. It was also mentioned that the risk of transmission from human to animals are unknown. The transmission was carried out between a 5-year old neutered male Egyptian Mau cat and his owner, a 54-year old male diagnosed with cystic fibrosis. The cat was only living indoor and had been living closely with his owner since the age of 13 weeks. The cat had a history of sneezing and sporadic serous nasal discharge over the last 6 weeks, including antibiotic treatment for two weeks without any improvement. Growth of P.aeruginosa was proved after culture of retrograde deep nasal flushing conducted under general anaesthesia. Diagnostic tests carried out for calicivirus, feline herpesvirus-1 and fungal pathogens were all negative. Tube array genomic fingerprinting (Wiehlmann et.al,2007) and PCR diagnostic tests (Smart et.al 2006) both confirmed several P.aeruginosa isolates matching the LES (Liverpool Epidemic Strain), a strain that had chronically infected his owner with cystic fibrosis, for more than 10 years. Following the discussion, it was affirmed that the chronic respiratory infection caused by P.aeruginosa in the cat was a consequence of transmission from the owner, via infected sputum or spread by droplets. In spite of the fact that cross infection of bacteria like MRSA, viruses and fungi may occur between human and animals (Weese et.al,2006), this was the first report made to be performing interspecies cross reaction of a distinctly transmissible P.aeruginosa strain (Mohan et.al, 2008)

Dermatitis

Pseudomonas aeruginosa is a frequent pathogen in dogs diagnosed with chronic otitis externa and otitis media, but is rarely isolated from skin infections. This bacterial organism may be isolated from the skin of dogs with chronic, deep pyoderma, where it is typically associated with infection with other pathogens such as Staphylococcus intermedins and Escherichia coli. The isolation of P.aeruginosa alone from pyoderma lesions is not a frequent procedure and the authors are aware of only a single case report in the literature that describes the clinical signs of so called pseudomonal pyoderma in dogs. In a recent study, P.aeruginosa was isolated from 42 of 561 skin samples submitted to a diagnostic laboratory during a 6-year period, and it was the only organism isolated from 14 of these 42 samples. However, the clinical signs of pyoderma caused by P.aeruginosa were not reported in this study (Hillier, 2006)

2.2 Fluoroquinolones - 2nd and 4th Generation

The development of the fluoroquinolones can be traced back to the discovery of nalidixic acid in 1962, and its first introduction of clinical use in 1967, which marks the beginning of the development of fluoroquinolones. Nalidixic acid was the first quinolone to be discovered, and served mainly for the purpose of treating uncomplicated UTIs (urinary tract infections) for decades until the fluoroquinolones developed in the 1970s and 1980s (Emmerson et.al, 2003).

The newer fluoroquinolones possess a broad spectrum bactericidal activity, outstanding oral bioavailability, high standard tissue penetration commendatory safety and tolerability profiles. The recent developed 4th generation fluoroquinolones represents a broader antimicrobial spectrum, including excellent activity against anaerobic bacteria. The 1st generation quinolones such as nalidixic acid, do only reach minimal serum levels. The 2nd generation quinolones, such as ciprofloxacin, possess a high gram-negative and systemic activity, while the 3rd generation drugs such as levofloxacin, have an increased activity against gram-positive bacteria and atypical pathogens. All quinolones can be distinguished according to their pharmacokinetic properties (King et.al,2001).

Generally, the fluoroquinolones are represented as broad-spectrum antibiotics with exceptional activity against gram-negative bacteria, especially P.aeruginosa. Tissue and fluid concentration frequently overreach the serum drug concentrations, making fluoroquinolones convenient in treatment of infections such as pneumonia (Garey et.al, 1999). Few side effects are rare in the use of fluoroquinolones, as they are usually well tolerated (Norrby et.al, 1993)

Antimicrobial activity

After their introduction in the 1970s and 1980s, ciprofloxacin came to be the most frequently used antibiotic drug in the world (Norrby et.al, 1993). The first fluoroquinolones were the only orally administered antibiotics available against serious gram-negative infections including P.aeruginosa, and were therefore used widely.

The overuse of fluoroquinolones is at risk because of their broad spectrum and oral bioavailability, and this has become a concern according to professionals in the medical field (King et.al,2000).

Bacterial resistance

Resistance to fluoroquinolones in both gram-positive and gram-negative bacteria have previously been reported. (Acar et.al, 1997). The reason for this is assumed to be a result of modification in the DNA gyrase, reduced membrane permeability or development in the efflux mechanisms. These bacterial mutations have been related to the development of an increased MIC to ciprofloxacin in isolates of Staphylococcus aureus, P.aeruginosa and Enterobacteriacea species (Acar et.al,1997). Cross-reaction of fluoroquinolones may also occur, depending on the minimal inhibitory concentration that varies from agent to agent. As for this, when determining the efficiency of specific agents, the pharmacokinetic properties and bacterial susceptibility of each fluoroquinolone must be taken into consideration. (Wolfson et.al, 1989).

2nd and 4th generation fluoroquinolones

A study made in ophthalmology compared the 4th generation fluoroquinolones gatifloxacin and moxifloxacin, to 2nd and 3rd generation fluoroquinolones in order to determine differences in susceptibility patterns and potency. Gatifloxacin and moxifloxacin were compared to levofloxacin of 3rd generation, and to ciprofloxacin and ofloxacin of 2nd generation fluoroquinolones.

By using an in vitro model, results showed that S.aureus isolates were resistant to ciprofloxacin and ofloxacin, but appeared to be most susceptible to moxifloxacin. Coagulase negative Staphylococci were resistant to ciprofloxacin and ofloxacin, but appeared to be most susceptible to moxifloxacin and gatifloxacin.

Streptococcus viridans represented a higher susceptibility to gatifloxacin, moxifloxacin and levofloxacin, than that of ciprofloxacin and ofloxacin.

Least, Streptococcus pneumoniae were least susceptible to ofloxacin compared to all the other fluoroquinolones.

Following the discussion, this in vitro study concluded that 4th generation of fluoroquinolones appears to be supreme compared to the 2nd and 3rd generation fluoroquinolones as they cover bacterial resistance, were more potent to gram-positive bacteria than the 2nd and 3rd generation fluoroquinolones, and were equally potent to gram­negative bacteria as the 2nd and 3rd generation fluoroquinolones (Mather et.al, 2001)

2.2.1 Fluoroquinolones - Mechanism of action

The fluoroquinolones exert their antibiotic activity by inhibiting bacterial replication. This mechanism is achieved by blocking their DNA replication pathway. The DNA represents the core material of the cell, responsible for the proper function of the cell. In the process of protein synthesis and DNA replication, the double stranded DNA undergo unwinding, resulting in a single stranded DNA. By unwinding, the complementary base pairing will occur, and the synthesis of mRNA will continue. The unwinding of the double stranded DNA is completed by the enzymes called DNA gyrase or topoisomerase. The DNA gyrase is a type of topoisomerase II enzyme that is responsible for the unwinding of the double stranded DNA by presenting negative supercoils. The fluoroquinolones are characteristic in that they inhibit the DNA gyrase by binding to the A-subunit of the enzyme, making the bacteria unable to synthesize proteins or replicate (Mehta, 2011).

Concerning the development of antibacterial drugs, there are five bacterial targets that have been utilized: Cell wall synthesis, protein synthesis, ribonucleic acid synthesis, deoxyribonucleic acid (DNA) and intermediary metabolism (table 3). The Fluoroquinolones are simply unique as they are the only direct inhibitors of DNA synthesis. This is done by binding to the enzyme-DNA complex, as they stabilize the DNA strand breaks which is produced by DNA gyrase and topoisomerase IV, representing the the dual targets of fluoroquinolones. A multiple complex consisting of a drug, an enzyme and DNA acts by blocking the progress of the replication fork. The cytotoxicity of fluoroquinolones can be divided into a process of two steps. The first step involves the transformation of the topoisomerase-fluoroquinolone-DNA complex into an irreversible form, while the second step involves the induction of a double-strand break by denaturation of the topoisomerase (Hooper, 2009).

Table 3. Different bacterial targets of antimicrobial agents (Hooper, 2001)

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Based on previous genetic studies of nalidixic acid-resistance of Escerichia coli mutants, DNA gyrase was the first quinolone target to be identified. E.coli proved mutations in the gyrA and gyrB genes, encoding the two subunits of the enzyme (Nakamura et.al, 1989). The identification of topoisomerase IV as a second fluoroquinolone target was disovered in studies where quinolone resistance mutation was proved inparC in Staphylococcus aureus (Ferrero et.al 1995).

An important feature that differentiates some fluoroquinolones is their ability to keep possession of their bactericidal activity towards resistant mutants. Moxifloxacin is an example in which loss of bactericidal activity is not seen (figure 5.) in grIAgyrA double mutants. Other fluoroquinolones like ciprofloxacin, represents loss of bactericidal activity of against strains of S.aureus with dual parC and gyrA mutations in the exposure to concentration 4-fold above the MIC, which represents a concentration where killing occurs in both wild-type and single grlA single mutants (Ng et.al, 1996).

Abbildung in dieser Leseprobe nicht enthalten

Figure 5. Representing the bactericidal activity of moxifloxacin and ciprofloxacin against S.aureus EN1252 grlA (topoisomerase IV) and gyrA mutants. The diamonds indicate no drug control, the triangles represent ciprofloxacin at 4 times its MIC, and the squares represent moxifloxacin at 4 times its MIC (Hooper, 2009).

2.2.2 Fluoroquinolone resistance in P.aeruginosa

P.aeruginosa exerts the highest level of resistance to fluoroquinolones, which limits their usefulness. Through imported resistance mechanism related to genetic elements and combination of imported and chromosomally encoded resistance mechanisms, a multidrug- resistant phenotype might occur in strains of P.aeruginosa. An increase of several chromosomal changes over time and single mutations might result in overexpression of a multidrug resistance mechanism such as the efflux pump (figure 6.) Resistance of fluoroquinolones in P.aeruginosa is assumed to be expressed through chromosomal genes and mutations of the DNA gyrase (gyrA and gyrB) and/or and topoisomerase IV (parC and parE), including the overexpression of multidrug efflux pumps (Lister et.al, 2009).

Abbildung in dieser Leseprobe nicht enthalten

Figure 6. Mutational resistance to fluoroquinolones and carbapenems involving chromosomally encoded resistance mechanisms of P.aeruginosa (Lister et.al 2009)

According to different studies, there has been investigations concerning resistance of fluoroquinolones in P.aeruginosa. Following a study from Japan, it was claimed that resistance in pseudomonas aeruginosa relates to the expression of the efflux pump-genes and mutation in DNA gyrase (topoisomerase II) or topoisomerase IV. Cells with a double­mutation in GyrA and mexR encoding DNA gyrase and repressor for the MexAB-OprM operon, was constructed. The mutant did show 1,024 times higher fluoroquinolone resistance than the cells lacking the MexAB-OprM. Cells with a single mutation in gyrA and producing a wild-type level of the MexAB-OprM efflux pump showed 128 times higher resistance than cells laking the MexAB-OprM. On the other hand, cells with a single mutation in GyrA or mexR caused only 4 and 64 times higher resistance. This article manifested the interplay between the MexAB-OprM efflux pump and the target mutation in fluoroquinolone resistance (Nakajima et al,2002).

Following another investigation carried out in Denmark, there was an investigation focusing on the molecular mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. In this article, it was written that fluoroquinolones are the only available antibiotics for oral treatment of Pseudomonas aeruginosa infections in most countries (Drlica et.al,1997) In this study, 20 quinolone-resistant P. aeruginosa isolates from cystic fibrosis lungs was analyzed for gyrA, parC, nfxB, and mexR genes and the expression of membrane proteins mexT-related OprN and nfxB-related OprJ associated with fluoroquinolone resistance. The aim of this study was to determine molecular mechanisms for quinolone resistance of repeated isolates of P. aeruginosa from CF patients. Quinolone resistance genes were analysed in 20. P.aeruginosa isolates collected in 1994 and 1997 from six cystic fibrosis patients, with long time exposure to ciprofloxacin treatment. Five of the total patients had the same type of strain in their lungs in 1994 and 1997, as referred to a previously claim of that cystic fibrosis patients possess the same strain over several years (Ojeniyi et.al,1993). The average number of mutations was higher in isolates from 1997 than in those from 1994, and the PFGE analysis showed that a patient encoded CF89, had different strains in 1994 and 1997. A suggestion after observing the results, was the that efflux resistance mechanisms are more common in isolates from CF patients than in strains from urine and wounds from non-cystic fibrosis patients, in which mutations of gyrA and parC dominate. Methods used to perform this experiment was PCR, antibiotic sensitivity testing, nucleotide sequencing and PGFE. (Jalal et.al,2000)

2.2.3 Comparison of 2nd and 4th generation fluoroquinolones - Human medicine

A comparison of gatifloxacin and ciprofloxacin was investigated in the treatment of bacterial keratitis, where the purpose was to compare the bacteriologic and the clinical efficacy of gatifloxacin and ciprofloxacin. In this study, 104 eyes among 104 patients diagnosed with bacterial keratitis were treated with gatifloxacin 0,3% eyedrops or ciprofloxacin 0,3% eyedrops. Bacterial keratitis is an infection of the eye, affecting the cornea. The cornea is the transparent window covering the iris and the pupil, which is simply the colored part of the eye. If the infection occurs due to the presence of a certain bacteria, it is called bacterial keratitis. The most common bacterial agents responsible for causing keratitis are P.aeruginosa and Staphylococcus aureus. Results of this study showed that gatifloxacin was more efficient in healing of ulcers than ciprofloxacin. In other words, gatifloxacin demonstrates a significantly better action than ciprofloxacin. Considering the fact that bacterial keratitis is a worldwide illness, it was written that gatifloxacin should be a better suggestion and a better alternative for treatment of bacterial keratits, than ciprofloxacin (Parmar et.al, 2006).

2.2.4 Comparison of 2nd and 4th generation fluoroquinolones - Veterinary Medicine

The efficiacy of the fourth-generation fluoroquinolone gatifloxacin was demonstrated in a study where the comparison of gatifloxacin 0,3% to ciprofloxacin 0,3% was made, in preventing Streptococcus pneumoniae keratitis in a rabbit LASIK (laser in situ keratomileusis) model. In this study, three groups of albino rabbits were tested, divided into group A and B and C. All groups consisted of 4 rabbits, so 12 rabbits in total were tested. Eight eyes were tested in each group, given different fluroquinolone antibiotic treatment. Group A was given gatifloxacin 0,3%, Group B was given ciprofloxacin 0,3 %, Group C served as controls.

Both group A and B received one drop of antibiotic treatment 20 minutes before the lamellar flap was created. This treatment was given four times a day for 3 days. Immediately after the flap formation, all corneas of the eyes were inoculated with 0,1 Ml of 4x 10 organisms/Ml of S. pneumoniae. All corneas were examined and cultured on day 3.

Based on the results, group A given topical gatifloxacin showed no infiltrates. On the third day, only one of eight corneas had a positive culture. Group B given topical ciprofloxacin had seven infiltrates, one perforation, and six of eight corneas had positive cultures. Group C, which served as control had eight infiltrates, and all eight corneas had positive cultures. With these outcomes, it was concluded that topical gatifloxacin 0,3%, as a fourth-generation fluoroquinolone is superior to topical ciprofloxacin 0,3% in prevention of prophylaxis against a clinical isolate of S.pneumoniae in a rabbit LASIK model (Donnenfeld et.al, 2006).

In a study from 2007 in the UK, the comparative activity of pradofloxacin was compared with other fluoroquinolones, against anaerobic bacteria isolated from dogs and cats. Pradofloxacin, which is a 3rd generation fluoroquinolones, is being exclusively developed for use in veterinary medicine. Pradofloxacin has similar structure to moxifloxacin, making it potentially active against gram-positive bacteria and anaerobes.

141 anaerobic strains were isolated from dogs and cats, originating from oral infections, abscesses and wounds infections, including faecal flora. In total, the isolated strains came from 94 dogs and 47 cats. None of the animals had been receiving antimicrobial treatment during the last 3 months prior to the sampling. Some of the strains were of Actinomyces spp, clostridium spp, fusobacterium spp, and Bacteroides spp. The MIC of all the test compunds, including pradofloxacin, marbofloxacin, enrofloxacin, difloxacin and ibafloxacin were determined (Figure 7). The results of this study showed that pradofloxacin exhibited the greatest antibacterial activity followed by marbofloxacin, enrofloxacin, difloxacin and ibafloxacin. The MIC (minimum inhibitory concentration) for pradofloxacin was 0.25 mg/L compared to 1 mg/L for marbofloxacin, 2 mg/L for enrofloxacin and difloxacin and 4 mg/L for ibafloxacin (Silley et.al, 2007).

Abbildung in dieser Leseprobe nicht enthalten

Figure 7. Showing the MIC distribution of anaerobic bacteria from dogs and cats (n = 141) for (a) pradofloxacin, (b) marbofloxacin, (c) enrofloxacin, (d) difloxacin and (e) ibafloxacin (Silley et.al 2007).

In a study made in Germany, the activity of pradofloxacin and marbofloxacin was compared against isolates of Staphylococcus intermedins, and against a reference strain of Staphylococcus aureus, using an in vitro pharmacokinetic-pharmacodynamic model. Staphylococcus intermedius is the primary cause of the canine pyoderma, causing a suppurative inflammation in the skin of dogs. In the canine serum, the time course of free drug concentration was modelled, resulting in a daily standard oral dose of 3 mg pradofloxacin/kg, and 2 mg marbofloxacin/kg. The least susceptible strain was tested applying experimentally high doses, of this 6mg of pradofloxacin/kg and 16 mg of marbofloxacin/kg. Results made clear that in concentrations associated with standard doses, pradofloxacin proved faster and more sustainable killing of all strains, than marbofloxacin. At experimentally high doses, pradofloxacin was greater than marbofloxacin in performing immediate killing. It was therefore concluded that pradofloxacin is likely to be a drug of efficient therapy in canine staphylococcal infections. (Korber-Irrgang et.al, 2006)

3. MATERIALS AND METHODS

3.1 Bacteria involved in the study

In this study, we followed the activity of bacterial growth from strains P. aeruginosa.

Our aim of investigation was to compare the resistance of 2nd and 4th generation fluoroquinolones in strains of P. aeruginosa isolated from dogs (table 4). The primary substances, gatifloxacin, a 4th generation fluoroquinolone and marbofloxacin, a 2nd generation fluoroquinolone was selected as the experimental targets.

A 9-days long serial-passage was conducted to test the sensitivity and the development of resistance of Pseudomonas aeruginosa.

Table 4. Showing the different bacterial strains used to test antibiotic sensitivity in the experiment.

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3.2 Preparing stock solutions

Marbofloxacin and Gatifloxacin stock solution at concentration of 1600 mg/ml was prepared with dissolving 16.1 mg of marbofloxacin (Menovo Pharmaceutical Co. Ltd., Zhejiang, China, active substance content 99.3%) in 10 ml of sterile distilled water. Marbofloxacin and Gatifloxacin stock solution was sterilized with filtration through a 0.22 pm membrane filter and stored at -80 °C.

Every 96 well microplate was inoculated with bacteria from an overnight culture. 120 microliters of MHB (Mueller-Hinton broth) were prepared in all wells. 15 microliters of antibiotics were added from a previously prepared antibiotic working solution (Table 5). 15 microliters of 1:1000 diluted bacterial culture was inoculated.

Table 5. Working solutions of marbofloxacin and gatifloxacin in the study Source: Author's own work

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The following concentration was used every day of the experiment:

- 120 pl from broth
- 15 pl from bacteria
- 15 pl from antibiotics

A 10-fold dilution of the antibiotics from the workings solutions were prepared, resulting in two separate bacterial evaluation cards of marbofloxacin and gatifloxacin (Table 6. and 7).

Table 6. Bacterial evaluation card of gatifloxacin Source: Author's own work

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Table 7. Bacterial evaluation card of Marbofloxacin Source: Author's own work

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3.3 Determination of MIC for marbofloxacin and gatifloxacin - Evaluation of the 9 days serial-passage

Passaging of bacteria at sub-inhibitory concentrations of fluoroquinolone antibiotics can increase the incidence of two-step mutations as the organisms are not inhibited nor killed by a certain concentration of the substance. An 9-day serial passage was conducted to determine the ability of P. aeruginosa strains to develop high level resistance against marbofloxacin and gatifloxacin. Our results may supply information about the risk of inappropriate application on the development of resistance in these important veterinary pathogens.

The first step was to determine the MICs for marbofloxacin in the selected strains (Table 8). A two-fold dilution was prepared from the stock solution in 96-well microplates. In this case however, much lower bacterial concentration (lower CFU count, 10⁵ CFU/ml) was used. The final concentrations of marbofloxacin and gatifloxacin were set in each line of a new 96-well microplate to 32.0 pg/ml, 16.0 pg/ml, 8.0 pg/ml, 4.0 pg/ml, 2.0 pg/ml, 1.0 pg/ml, 0.5 pg/ml, 0.25 pg/ml, 0.125 pg/ml, 0.0625 pg/ml with the exertion of working solutions. Bacteria were inoculated in 15 pl inoculum size using a bacterium-suspension (10⁶ CFU/ml). This resulted in a final bacterial density of 10⁵ CFU/ml. Final concentrations of marbofloxacin in the microplates are shown in Figure 8. Positive control wells contained only Mueller-Hinton broth and were inoculated with the certain bacterial strain. Negative control wells contained Mueller-Hinton broth without inoculation. Incubation lasted for 24 hours at 37⁰ C.

After the determination of the MICs at each individual strain the next step was to inoculate bacteria to a different microplate containing the same concentrations of marbofloxacin and gatifloxacin. For each strain, the highest concentration of the antibacterial was chosen where bacterial growth occurred. From this well bacteria were passaged to another plate with all of the concentrations present.

Table 8. Final concentrations of marbofloxacin and gatifloxacin in 96-well microplates with the application of positive and negative controls (concentration values in pg/ml, each well corresponds to 150pl volume) - P.aeruginosa.

Source: Author's own work

Abbildung in dieser Leseprobe nicht enthalten

By using this method, each bacterium was individually observed each day, and their colonies, that were grown in the presence of marbofloxacin and gatifloxacin were passaged each day to another plate. Each day the bacterium was inoculated to a new plate from that well, that contained the highest concentration of the antibiotic.

This method resembles the effect of sub inhibitory concentrations present in biological systems, and helps understanding and interpreting the risk of drug usage on the development of bacterial resistance.

4. RESULTS

4.1 Results based on experimental data

For each day of our experiment, the activity of bacterial growth was marked in the tables of bacterial evaluation. The MIC (minimal inhibitory concentration) was recorded each day (Table 9 and 10) including the strains representing the highest bacterial growth. The MIC of the two primary target substances throughout the time of investigations did not behave identically, as gatifloxacin in general showed a lower MIC value than marbofloxacin (table 11, 12, 13 and 14).

On the first day of bacterial growth evaluation, gatifloxacin had an MIC of 2 pl/ml for all except strain 30 and 31 of P.aeruginosa, while the MIC of marbofloxacin had a higher MIC for all strains, except for strain 21, 22, 24 and 27, which was equal MIC to that of gatifloxacin. The same feature was observed on day 4 and 5, as the MIC for marbofloxacin in strain 20 was 32 pl/ml both days, while the MIC for gatifloxacin was again, much lower, as the MIC for the same strain was 4 pl/ml both days. As a result of the developing resistance in strains of P. aeruginosa, the MIC increased, and we therefore had to increase the concentration of gatifloxacin and marbofloxacin in the last four days. Following the last day of the serial-passage, gatifloxacin behaved superior to marbofloxacin as the MIC was higher than that of gatifloxacin. The explanation of the high MIC of marbofloxacin throughout the serial-passage might have indicated a development of resistant strains in P.aeruginosa.

Table 9. An overview of the minimum inhibitory concentration (MIC) values for gatifloxacin following 9 days of serial-passage of P.aeruginosa strains Source: Author's own work

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Table 10. An overview of the minimum inhibitory concentration (MIC) for marbofloxacin, following 9 days of the serial-passage of P.aeruginosa strains Source: Author's own work

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Table 11. MIC of marbofloxacin day 1-5 of the 9 days' serial-passage of P.aeruginosa Source. Author's own work

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Table 12. MIC of gatifloxacin from the first 5 days of a 9 days serial-passage of P. aeruginosa Source: Author´s own work

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Table 13. MIC of marbofloxacin from day the 4 last days of a 9-days serial passage of Pseudomonas aeruginosa Source: Author´s own work

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Table 14. MIC value of gatifloxacin from the last 4 days of a 9-days serial-passage of P.aeruginosa. Source: Author´s own work

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5. DISCUSSION

It was hypothesized that gatifloxacin, a 4thnd generation fluoroquinolone is superior to marbofloxacin, a 2nd generation fluoroquinolone, in treatment of P.aeruginosa infections. Following a 9 days serial-passage of P.aeruginosa in broth, the average MIC (minimum inhibiting concentration) of gatifloxacin resulted in a lower MIC than marbofloxacin. To highlight our findings, day 6 of the experiment could be taken as an example as the MIC differ between gatifloxacin and marbofloxacin (Table 15 and 16) The MIC of gatifloxacin in strain 20, 22, 22 and 30 of P.aeruginosa was considerably lower than that of marbofloxacin.

Table 15. and 16. Comparison of the MICs of marbofloxacin and gatifloxacin on day 6

Source: Author's own work.

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These results allowed us to refer to the studies mentioned above, comparing the activity of multiple fluoroquinolones.

One of the studies mentioned above, compared topical antibiotics using gatifloxacin 0,3% eyedrops to ciprofloxacin 0,3% eyedrops in the treatment of bacterial keratitis in humans. As the application of gatifloxacin resulted in better healing of the ulcers, gatifloxacin exerts a better efficiency for treatment of bacterial keratitis than ciprofloxacin. This indicated an approach in our primary hypothesis. (Parmar et.al, 2006).

Another study above compares the antibiotic activity of pradofloxacin and marbofloxacin against isolates of Staphylococcus aureus and Staphylococcus intermedius. The outcome proved pradofloxacin as a superior antimicrobial agent compared to marbofloxacin. These results aid in our hypothesis of gatifloxacin being superior to marbofloxacin (Körber-Irrgang et.al, 2006).

Yet another study above compared the activity of gatifloxacin to ciprofloxacin against isolates of Streptococcus pneumoniae keratitis in a rabbit LASIK model. Again, gatifloxacin surpassed ciprofloxacin in antibiotic activity showing no infiltrates of the cornea, while the application of ciprofloxacin generated both infiltrates and perforation of the cornea, including positive cultures of S.pneumoniae (Donnenfeld et.al, 2006).

These studies amplified our hypothesis, by comparing the results to our own experimental data.

6. SUMMARY

As several bacteria possess the ability of developing resistance to multiple classes of antibiotics, treatment of infectious diseases becomes more demanding every year. This is evidently concerning infections caused by the opportunistic pathogen P.aeruginosa .

During the work of this thesis, I gained broad and useful information about the impact of P.aeruginosa, a ubiquitous, gram-negative, rod-shaped bacteria causing infections in plants, animals and humans, with an ability to survive under minimal nutritional requirements (Lister et.al, 2009). The investigation of P.aeruginosa was highly interesting due to the fact that both humans and animals are affected, and is therefore affecting public health issues. Among several illnesses, this pathogen causes urinary tract infections, respiratory infections, ear infections, skin infections and eye infections. Due to all these illnesses, antibiotic treatment is essential, often in the form of fluoroquinolone application. In this topic, we wanted to determine the most efficient medical treatment for P.aeruginosa infections by comparing fluoroquinolones.

Previous research material show that fluroquinolones are the most efficient and used antibacterial agent against infections caused by P.aeruginosa. Though, there are differences in fluoroquinolone activity as these are divided into different generations having different bacterial targets. Our purpose of investigation was to prove that antibtiotic agents from 4th generation of fluoroquinolones prove to be more efficient to treatment of P.aeruginosa than 2nd generation fluroquinolones. Our primary target substance from the 4th generation fluoroquinolones was gatifloxacin, while the primary target substance from the 2nd generation of fluoroquinolones was marbofloxacin. Gatifloxacin is used for medical treatment in both human and veterinary medicine, while marbofloxacin is exclusively used for treatment in veterinary medicine. The content of this thesis was mainly based on scientific and literature material, including our own investigation of a 9-day serial passage of P.aeruginosa in broth. In spite of the fact that multiple studies has proved that P.aeruginosa is resistant to fluroquinolones, our aim was to find out which generation of fluoroquinolones that generates the most efficient antibacterial activity against P.aeruginosa infections. We used 7 different strains of P.aeruginosa to test the sensitivity against our primary substance targets. Following our investigation, the MIC values of gatifloxacin was in general lower than marbofloxacin, which may present a developing resistance in the strains of P.aeruginosa. The background for this may be linked to the imported By comparing our results with other studies made, we concluded that all of the P.aeruginosa isolates investigated showed high susceptibility to gatifloxacin, while several strains proved to be moderately susceptible or resistant to marbofloxacin. This passage of bacteria containing broth with the fluoroquinolone alone resulted in rapid development of resistance to marbofloxacin, while gatifloxacin exerted stronger properties in antimicrobial activity, and is therefore superior to marbofloxacin in the treatment of P.aeruginosa infections, which is what we hypothesized. Experimental findings like these are important to take into consideration, as the risk of drug usage promotes development of bacterial resistance, thereby affecting the health of humans and animals on a global level.

OSSZEFOGALAS

Szamos bakterium rendelkezik rezisztenciaval kulonbozo osztalyu antibiotikumokkal szemben, ezaltal evrol evre nehezedik a fertozo betegsegek kezelese. Ez nyilvanvaloan vonatkozik az opportunista patogen P.aeruginosa altal okozott fertozesekre is egyarant.

A szakdolgozat elkeszitese alatt atfogo es hasznos informaciokra tettem szert a mindenutt jelen levo, gram-negativ rud alaku P.aeruginosa baktertim globalis egeszsegugyi hatasaval kapcsolatban, ami a tulelesehez szukseges minimalis taplalkozasi kovetelmenyek ellenere fertozeseket okoz novenyekben, allatokban es emberekben. Rendkivul erdekes volt a Pseudomonas aeruginosa kutatasa, mivel mind az emberek es az allatok is erintettek es amely egyarant erint kozegeszsegugyi kerdeseket is. Tobb betegseg kozott, ez a patogen hugyuti, legzoszervi, ful, bor es szemfertozeseket okoz. Ezen betegsegek miatt, az antibiotikumos kezeles elengedhetetlen, gyakran fluorokinolonok hasznalata formajaban. Jelen tanulmanyban szerettuk volna meghatarozni a leghatekonyabb orvosi kezeleset a P. aeruginosa fertozesnek, osszehasonl^tva a fluorokinolonoknak nevezett antibiotikum csoportot.

A korabbi kutatasi anyag azt mutatja, hogy a fluorokinolonok a leghatekonyabb antibiotikumok es a leghasznalatosabb antibakterialis hatoanyagok a P.aerugionosa fertozesekkel szemben. Habar kulonbsegek figyelhetok meg a fluorokinolonok aktivitasban, mivel kulonbozo generaciokra vannak osztva, kulonbozo bakteriumokat celozva. A mi kutatasi celunk annak bizony^tasa volt, hogy a negyedik generacioju fluorokinolonok hatasosabbak a P.aeruginosa kezeleseben a masodik generacioju fluorokinolonknal. Az elsodleges celpontunk a negyedik generacios fluorokinolon - gatifloxacin es a masodik generacios fluorokinolon - marbofloxacin volt. A gatifloxacint human es allatorvosi kezelesekhez is hasznaljak, m^g a marbofloxacin kizarolagosan csak allatorvosi kezelesben fordul elo. A szakdolgozat tartalma fokent tudomanyos es irodalmi anyagra epul, tartalmazva a sajat tanulmanyunkat, a P.aeruginosa 9 napos atoltasat. Annak ellenere, hogy tobb vizsgalat igazolta a P.aeruginosa rezisztenciajat a fluorokinolonokkal szemben - celunk az volt, hogy megtudjuk melyik generacioju fluorokinolon fejt ki leghatekonyabb antibacterialis hatast a P.aeruginosa fertozesek ellen. 7 kulonbozo P.aeruginosa torzset hasznaltunk a valasztott antibiotikumok, a gatifloxacin es marbofloxacin erzekenysegenek tesztelesere. A vizsgalatunkat kovetoen, a MIC ertekei a gatifloxacinnak alacsonyabbak voltak a marbofloxacin ertekeinel, amely a P.aeruginosa torzsek fejlodo rezisztenciajat feltetelezi. Ennek a hattere kapcsolatban allhat a P.aeruginosa importalt fluorokinolon rezisztencia mechanizmusaval, tartalmazva mutacios valtozasokat a fluorokinolon altal celzott DNS giraz es/ vagy topoizomeraz IV, egy/ vagy tulexpresszalasa az ugynevezett multidrog „efflux pumpaknak” (Lister et.al, 2009).

Osszehasonl^tva az eredmenyeinket mas vizsgalatokkal, arra a kovetkeztetesre jutottunk, hogy a gatifloxacin erosebb antimikrobialis hatast fejt ki, ezaltal felsobbrendu a marbofloxacinnal a P. aeruginosa fertozesek kezeleseben, ami egyezik a hipotezisunkkel. Fontos a Mserleti eredmenyeket figyelembe venni mivel a gyogyszer hasznalatanak a kockazata el6seg^ti a bakterialis rezisztencia kiepuleset, ami hatassal van az emberek es allatok egeszsegere globalis szinten.

Appendices

Table 1. Growth of P.aeruginosa in the presence of marbofloxacin and gatifloxacin on day 1.

Source: Author's own work

Abbildung in dieser Leseprobe nicht enthalten

Table 2. Growth of P.aeruginosa in the prescence of marbofloxacin and gatifloxacin on day 2

Source: Author´s own work

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Table 3. Persistent growth of Pseudomonas aeruginosa in the presence of marbofloxacin and gatifoxacin on day 3.

Source: Author´s own work

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Table 4. Growth of P.aeruginosa in the presence of marbofloxacin and gatifloxacin on day 4. Note the increase in MIC

Source: Author´s own work

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Table 5. Growth of P.aeruginosa in the prescence of marbofloxacin and gatifloxacin on day 5. Note the increase in MIC

Source: Author´s own work

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Table 6. Growth of P.aeruginosa in the presence of marbofloxacin and gatifloxacin on day 6. Note the increased concentration as a result of increased MIC

Source: Author's own work

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Table 7. Growth of P.aeruginosa in the presence of marbofloxacin and gatifloxacin on day 7. Note the increased concentration as a result of increased MIC

Source: Author's own work

Abbildung in dieser Leseprobe nicht enthalten

Table 8. Growth of P.aeruginosa in the prescence of marbofloxacin and gatifloxacin on day 8.

Note the increased concentration as a result of increased MIC.

Source: Author's own work

Abbildung in dieser Leseprobe nicht enthalten

Abbildung in dieser Leseprobe nicht enthalten

Table 9. Increased MIC of marbofloxacin and gatifloxacin in the presence of P.aeruginosa on day 9.

Source: Author's own work

Abbildung in dieser Leseprobe nicht enthalten

7. ACKNOWLEDGEMENTS

Writing this thesis has been fascinating and extremely rewarding. The author greatly appreciates the Department of Pharmacology and Toxicology and the supervisor, Dr.Jerzsele Akos for consultation to decide a great topic on the diploma thesis. The author gratefully the supervisor for his timely advices, friendly demeanour and brilliant supervision.

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Figures

Figure 1. Gomez M.I. Prince A. 2007. Host - pathogen interactions in CF. Underlying predisposing factors of the CF lung and specific characteristics of P. aeruginosa allow for colonization and persistent infection. Opportunistic lung infections in lung disease: Pseudomonas infections in cystic fibrosis.

Figure 2. Weed M.C., Rogers G.M., Kitzmann A.S., Goins K.M., Wagoner M.D. 2013. Pseudomonas keratitis in human. Vision Loss After Contact Lens-Related Pseudomonas Keratitis.

Figure 3. Hillier A. 2005. Chronic otitis with Pseudomonas aeruginosa infection. Purulent discharge and the erosive and ulcerative lesions of the anthelix and tragus around the ear canal opening.

Figure 4. Hillier A.2005. Cytology preparation of ear exudate from dog in otits externa caused by Pseudomonas Aeruginosa. Rod-shaped Pseudomonas organisms that are visible both extracellularly and intracellularly within neutrophils (Diff-Quik stain, 31000 magnification).

Figure 5. Hooper DC. 2001. Bactericidal activity of moxifloxacin and ciprofloxacin against Staphylococcus aureus EN1252a grlA gyrA. Diamonds, no drug control; triangles, ciprofloxacin at 4 times its MIC; squares, moxifloxacin at 4 times its MIC.

Figure 6. Wolter DJ, Lister PD, Hanson N.D. 2009. Mutational resistance to fluoroquinolones and carbapenems involving chromosomally encoded mechanisms expressed by P. aeruginosa. Antibacterial-Resistant Pseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms. Clinical Microbiology Reviews.

Figure 7. Silley P., Heinrich A.B.S., Pridmore A. MIC distribution of anaerobic bacteria from dogs and cats (n = 141) for (a) pradofloxacin, (b) marbofloxacin, (c) enrofloxacin, (d) difloxacin and (e) ibafloxacin. Comparative activity of pradofloxacin against anaerobic bacteria isolated from dogs and cats. Journal of Antimicrobial Chemotherapy, Volume 60, Issue 5, 1 November 2007.

Tables

Table 1. Hillier A. 2005. Antibiotics with potential activity against Pseudomonas Aeruginosa. 2005. College of Veterinary Medicine, The Ohio State University Columbus, Ohio.

Table 2. Hillier A. 2005. Treatment summary of acute and chronic Pseudomonas otitis. College of Veterinary Medicine, The Ohio State University Columbus, Ohio

Table 3. Hooper D.C. 2001. Bacterial targets of antimicrobial agents. Mechanism of Action of Antimicrobials: Focus on fluoroquinolones. Clinical Infectious Diseases, Volume 32, Issue Supplement 1.

Table 5. Showing the different bacterial strains from isolates of dog ear, used to test antibiotic sensitivity in the experiment.

Table 6. Working solution from the performed experiment containg marbofloxacin, gatifloxacin and different strains of pseudomonas.

Table 7. Bacterial evaluation card of marbofloxacin

Table 8. Bacterial evaluation card of gatifloxacin

Table 9. MICs of marbofloxacin through a 9 days serial-passage of P.aeruginosa

Table 10. MICs of gatifloxacin through a 9 days serial-passage of P.aeruginosa

Table 11. MIC concentration of Marbofloxacin from day 1-5 of the 9 days serial-passage of P.aeruginosa. Custom made graph from Excel.

Table 12. MIC concentration of Gatifloxacin from day 1-5 of the 9 days serial-passage of P.aeruginosa. Custom made graph from Excel.

Table 13. MIC concentration of Marbofloxacin from day 6-9 of the 9 days serial-passage of P.aeruginosa. Custom made graph from Excel.

Table 14. MIC concentration of Gatifloxacin from day 6-9 of the 9 days serial-passage of P.aeruginosa. Custom made graph from Excel.

Table 15. and 16. Comparison of the MICs of marbofloxacin and gatifloxacin on day 6.

• College: University of Veterinary Medicine Budapest
• Course: Veterinary Medicine
• Grade: 4,0
• Author: Helena Maria Gunnulfsen (Author)
• Publication Year: 2017
• Pages: 60
• Catalog Number: V453407
• ISBN (eBook): 9783668877009
• ISBN (Book): 9783668877016
• Language: English
• Tags: pharmacology, antibioticresistance, pseudomonasaeruginosa, science, pharmaceuticalscience, fluoroquinolones, research, pathology, publichealth, onehealth, antibiotics, pathogen, laboratory, modeofaction