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1 LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

VETERINARY ACADEMY Faculty of Veterinary Medicine

Gabriella Roslund

STAFILOKOKŲ IŠSKYRIMAS IŠ GYVŪNŲ AUGINTINIŲ KLINIKINĖS MEDŽIAGOS IR JAUTRUMO ANTIMIKROBINĖMS MEDŽIAGOMS NUSTATYMAS

ISOLATION OF STAPHYLOCOCCI FROM CLINICAL MATERIAL OF PETS AND ANTIMICROBIAL SUSCEPTIBILITY TESTING

MASTER THESIS

of Integrated Studies of Veterinary Medicine

Head of the work

Prof. dr. Jūratė Šiugždaitė

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2 THE WORK WAS DONE IN THE DEPARTMENT OF VETERINARY PATHOBIOLOGY

CONFIRMATION OF THE INDEPENDENCE OF DONE WORK

I confirm that the presented Master Thesis “ISOLATION OF STAPHYLOCOCCI FROM CLINICAL MATERIAL OF PETS AND ANTIMICROBIAL SUSCEPTIBILITY TESTING”

1. has been done by me;

2. has not been used in any other Lithuanian or foreign university;

3. I have not used any other sources not indicated in the work and I present the complete list of the used literature.

2017-12-15 Gabriella Roslund

(date) (author’s name, surname) (signature)

CONFIRMATION ABOUT RESPONSIBILITY FOR CORRECTNESS OF THE ENGLISH LANGUAGE IN THE DONE WORK

I confirm the correctness of the English language in the done work.

2017-12-15 Gabriella Roslund

(date) (author’s name, surname) (signature)

CONCLUSION OF THE SUPERVISOR REGARDING DEFENSE OF THE MASTER THESIS

2017-12-15 Jūratė Šiugždaitė

(date) (supervisor’s name, surname) (signature)

THE MASTER THESIS HAVE BEEN APPROVED IN THE DEPARTMENT/CLINIC

Saulius Petkevičius

(date of approbation) (name, surname of the manager of department/clinic)

(signature)

Reviewers of the Master Thesis

1) 2)

(name, surname) (signatures)

Evaluation of defense commission of the Master Thesis:

(date) (name, surname of the secretary of the defense commission)

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3

CONTENT

SUMMARY ... 4 SANTRAUKA ... 5 ABBREVIATIONS ... 6 INTRODUCTION ... 7 1. REVIEW OF LITERATURE ... 9

1.1. Main characteristics of the Staphylococcus genus ... 9

1.2. Common staphylococcal species in human and veterinary medicine ... 9

1.3. Taxonomical changes of the Staphylococcus intermedius group ... 10

1.4. Identification of the major pathogenic staphylococci in veterinary medicine ... 11

1.5. Pathogenicity and virulence factors of S. aureus and S. pseudintermedius ... 12

1.6. Antibiotic use and antimicrobial resistance ... 15

1.6.1. Beta-lactam antibiotics and beta-lactamase production ... 15

1.6.2. Methicillin and multi-drug resistance among staphylococci ... 16

2. RESEARCH METHODS AND MATERIAL ... 18

2.1. Data collection and preparation of samples ... 18

2.2. Antimicrobial susceptibility testing ... 19

2.3. Statistical analysis ... 20

3. RESEARCH RESULTS ... 21

3.1. Most frequently occurring staphylococcal species in canines and felines ... 21

3.2. Antimicrobial resistance patterns ... 21

3.3. Most common collection site in canines and felines ... 23

3.3.1. Results of the canine group ... 25

3.3.2. Results of the feline group ... 27

3.4. Investigation of antimicrobial susceptibility to select lactam antibiotics and beta-lactamase production ... 29 4. DISCUSSION OF RESULTS ... 31 CONCLUSIONS ... 35 RECOMMENDATIONS ... 36 ACKNOWLEDGEMENTS ... 37 REFERENCES ... 38 ANNEX 1 ... 43 ANNEX 2 ... 45

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4

SUMMARY

ISOLATION OF STAPHYLOCOCCI FROM CLINICAL MATERIAL OF PETS AND ANTIMICROBIAL SUSCEPTIBILITY TESTING

Gabriella Roslund Master Theses

The aim of this work was to investigate the occurrence and antibiotic susceptibility of

Staphylococcus species isolated from clinical material collected from pets, and the implications of

these findings.

A retrospective analysis of 78 Staphylococcus-positive samples from cats and dogs was made. The specimens were submitted between January 2016 and November 2017, and collected from the skin, outer ear, ocular conjunctiva and urogenital tract. 89.7% (70/78) were identified as S.

pseudintermedius, 7.7% (6/78) as S. aureus, and 2.6% (2/78) as S. epidermidis. Resistance among

isolates was highest to polymyxin B (91%) and sulfamethoxazole (82%). Resistance was lowest to enrofloxacin (14%) and gentamicin (16%). 77% of all staphylococcal isolates displayed resistance to at least one antimicrobial, and 30% were drug resistant. Antimicrobial resistance and multi-drug resistance were more common in S. aureus than in S. pseudintermedius.

A separate study involving clinical material from 28 Staphylococcus-positive dogs collected between April 2017 and October 2017 was made, where species identification, antimicrobial susceptibility against five common beta-lactams and beta-lactamase production were investigated using standard biochemical diagnostic methods and disc diffusion test. Specimens were collected from skin, outer ear, and the urogenital tract. All isolates were identified as S. pseudintermedius. Antimicrobial resistance was highest to ampicillin (96%), and penicillin G (93%). Sensitivity was highest to amoxicillin + clavulanic acid (79%). Beta-lactamase production was noted in 90% (10/11) of the isolates tested.

The staphylococci were most frequently isolated from the skin (61%) in canines and the ocular conjunctiva (43%) in felines. S. pseudintermedius (91%) and S. epidermidis (2.9%) were more

common in canines, whereas S. pseudintermedius (80%) and S. aureus (20%) were more common in felines. There was no correlation between age or the gender of the animals, and the staphylococcal species isolated. No collection site was more associated with a certain staphylococcal species or resistance to a particular type of antimicrobial agent. This indicates that most common infection site varies between canines and felines, but primary factors regarding susceptibility to infection does not include either age, gender or bacterial species. Furthermore, antimicrobial resistance could not be linked to site of infection in this study.

KEY WORDS: Staphylococcus pseudintermedius, Staphylococcus aureus, antimicrobial resistance, beta-lactam

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5

SANTRAUKA

STAFILOKOKŲ IŠSKYRIMAS IŠ GYVŪNŲ AUGINTINIŲ KLINIKINĖS MEDŽIAGOS IR JAUTRUMO ANTIMIKROBINĖMS MEDŽIAGOMS NUSTATYMAS

Gabriella Roslund Master Thesis

Darbo tikslas buvo ištirti stafilokokinių bakterijų rūšių, kurių kultūros buvo izoliuotos iš naminių gyvūnų klinikinių mėginių, pasiskirstymą pagal dažnumą bei nustatyti jų atsparumą antibiotikams ir padaryti bendras išvadas.

Buvo atlikta retrospektyvinė analizė įtraukiant 78 iš šunų ir kačių paimtus mėginius, kuriuose nustatytas stafilokokinių bakterijų augimas. Mėginiai surinkti iš odos, išorinės ausies, junginės bei urogenitalinio trakto laikotarpiu nuo 2016-ųjų sausio iki 2017-ųjų lapkričio. 89.7% (70/78) atvejų bakterijų rūšys buvo identifikuotos kaip S. pseudintermedius, 7.7% (6/78) kaip S. aureus, ir 2.6% (2/78) kaip S. epidermidis. Tarp tirtų kultūrų didžiausias atsparumas fiksuotas polimiksinui B (91%) ir sulfametoksazoliui (82%). Mažiausias atsparumas buvo enrolfloksacinui (14%) ir gentamicinui (16%). 77% stafilokokinių bakterijų kultūrų buvo atsparios bent vienam antibiotikui, o 30% atvejų buvo dauginis atsparumas. Antimikrobinis atsparumas ir dauginis atsparumas buvo būdingenis S.

aureus nei S. pseudintermedius kultūrose.

Atskirai nuo 2017-ųjų balandžio iki 2017-ųjų spalio tirta 28 šunis sudariusi grupė dėl stafilokokinių bakterijų buvimo, jų atsparumo penkiems laktaminiams antibiotikams ir beta-laktamazių produkcijos. Visos bakterijų kultūros buvo S. pseudintermedius, mėginiai paimti iš odos, išorinės ausies ir urogenitalinio trakto. Didžiausias atsparumas buvo ampicilinui (96%) ir penicilinui G (93%). Jautriausios bakterijos buvo amoksicilinui su klavulano rūgštimi (79%). Beta-laktamazių produkcija pastebėta 90% (10/11) bakterijų kultūrų.

Stafilokokinių bakterijų kultūros šunims dažniausiai buvo išskirtos iš odos (61%), o katėms iš junginės (43%). Šunims būdingesnės buvo S. pseudintermedius (91%) ir S. epidermidis (2.9%), tuo

tarpu katės S. pseudintermedius (80%) ir S. aureus (20%). Nebuvo rasta statistiškai reikšmingo ryšio tarp naminių gyvūnų amžiaus ir lyties su identifikuota stafilokokinių bakterijų rūšimi. Nerasta koreliacija ir tarp mėginio paėmimo vietos su išskirta bakterijų rūšimi bei jos atsparumu antibiotikams. Tai įrodo, kad dažniausia infekcijos vieta kačių ir šunų tarpe yra skirtinga, bet pirminiai faktoriai turintys įtakos infekcijos gydymo jautrumui nėra gyvūno amžius, lytis ar bakterijų rūšis. Be to šiame tyrime antimikrobinis atsparumas nėra priklausomas nuo mėginio paėmimo vietos.

RAKTAŽODIAI: Staphylococcus pseudintermedius, Staphylococcus aureus, atsparumas antimikrobinėms medžiagoms, beta-laktaminiai antibiotikai.

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6

ABBREVIATIONS

AMR – Antimicrobial Resistance

CLSI – Clinical and Laboratory Standards Institute CoNS – Coagulase Negative Staphylococci

CoPS – Coagulase Positive Staphylococci

EUCAST – European Committee on Antimicrobial Susceptibility Testing MDR – Multidrug Resistance

MRS – Methicillin-resistant Staphylococci

MRSA – Methicillin-resistant Staphylococcus aureus

MRSP – Methicillin-resistant Staphylococcus pseudintermedius MSS – Methicillin-susceptible Staphylococci

MSSA – Methicillin-susceptible Staphylococcus aureus

MSSP – Methicillin-susceptible Staphylococcus pseudintermedius PCR – Polymerase Chain Reaction

SE – Staphylococcal Enterotoxin

SIG – Staphylococcus Intermedius Group SSTI – Skin and Soft Tissue Infection

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7

INTRODUCTION

Several species in the Staphylococcus genus are considered among the most important pathogens in human and veterinary medicine. The Staphylococcus genus is part of the normal microflora in both humans and companion animals. They are also opportunistic pathogens and one of the main causes of skin, soft tissue, and body cavity infections, otitis externa, and post-operative wound infections in cats and dogs. [1,2]

In humans, Staphylococcus aureus is the main cause of bacteraemia, endocarditis, osteomyelitis, septic arthritis, pneumonia, as well as skin and soft tissue infections. [3]

Staphylococcus pseudintermedius is the most common species in cats and dogs especially, and

is the main cause of pyoderma in dogs. Some studies show that as many as 90% of all canines - both healthy and those with underlying skin disease may be colonized. In comparison, only about 30% of humans are colonized by S. aureus. [3,4]

The main threat of staphylococci in veterinary medicine is their ubiquitous nature, their capacity to cause disease and their ability to acquire resistance genes to one or several antimicrobial classes, making treatment a challenge for the clinician. [2] Clinically healthy animals may be carriers of drug-resistant and pathogenic bacteria, thus propagating the spread further by acting as reservoirs. [5]

Acquired resistance to many of the modern day antimicrobials makes this a particularly important and dangerous pathogen in both human and veterinary medicine. Emergence of both acquired and intrinsic methicillin resistant staphylococcal strains is steadily rising, rendering beta-lactam antimicrobials, such as penicillins, cephalosporins and carbapenems ineffective as treatment. Beta-lactamase producing species can be combated with addition of beta-lactamase inhibitors, but does not reconstitute susceptibility in the case of methicillin resistance. Methicillin resistance is furthermore often association with multidrug resistance. [1,4]

The world-wide spread of multi-resistant strains and inter-species transfer of bacteria is also a major threat to veterinary and human health care. Despite being primarily a human pathogen, S.

aureus seems to have the ability for interspecies colonization and adapt to new hosts, meaning that

animals can act as a potential reservoir for S. aureus and thus methicillin-resistant S. aureus (MRSA). [6] Similarly, species like S. pseudintermedius, which is not part of the human microflora can be transferred from pets to humans and cause infection, the spread being facilitated by the close proximity and co-habitation tradition that exist between humans and pets. In addition to that, recent global emergence of methicillin-resistant S. pseudintermedius (MRSP) in companion animals is a growing concern for the veterinary profession. [1,7]

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8 The aim of this work is to investigate the occurrence and antibiotic susceptibility of

Staphylococcus species isolated from clinical material collected from pets, and the implications of

these findings.

The objectives of the work:

1. To determine the Staphylococcus species occurring in canine and feline clinical material. 2. To determine the antimicrobial resistance of these staphylococci.

3. To determine what anatomical collection site these Staphylococcus species are most frequently isolated from, and determine any correlation between the animals’ gender, age, collection site and bacterial isolates and antimicrobial resistance.

4. To determine susceptibility to beta-lactam antimicrobials and to determine the production of beta-lactamase of culture isolates.

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9

1. REVIEW OF LITERATURE

1.1. Main characteristics of the Staphylococcus genus

More than 47 species in the Staphylococcus genus have been described. [8] The genus consists of spherical or ovoid bacteria which form grape-like clusters. They are Gram-positive, non-motile, facultative anaerobic, and are usually non-encapsulated, with the exception of S. aureus and a few other coagulase negative species. Production of coagulase and catalase are other common features, but varies according to species, and also within species. They are generally negative for oxidase. Colonies are smooth, 1-5 mm, the colour varies between white, cream, and yellow, with an opaque appearance. [9,10]

Staphylococci are commensal but opportunistic pathogens and are present on cutaneous and epithelial surfaces of all endothermic animals. The main habitat is the skin, anterior nares, mucous membranes, gastrointestinal and urogenital tracts. [11,12]

1.2. Common staphylococcal species in human and veterinary medicine

S. pseudintermedius is part of the normal microflora in dogs and cats, and colonize the

mucocutaneous areas, particularly skin, hair follicles, hair shafts and coat. The nares, oral cavity and perineum are most heavily colonized in healthy dogs. S. pseudintermedius is considered the most common isolate in companion animals, especially canines, both in healthy individuals and those with underlying skin disease, comprising up to 90% of all staphylococcal species found. Some studies suggest that up to 90% of both healthy and diseased dogs are carriers of this commensal bacterium. Other studies regarding carriage in healthy individuals marks carriage rate at 46-92%. [1,2,4]

However, in one study from the United Kingdom, the most commonly isolated species was S.

epidermidis (52%) in healthy canines, whereas S. pseudintermedius and S. aureus were detected in

44% and 8% of dogs, respectively. [13]

Co-colonization is not uncommon, as demonstrated by a study from Australia, [14] where the majority (85%) of healthy of dogs were colonized by S. pseudintermedius, and 13% were carriers of both S. pseudintermedius and S. aureus simultaneously.

Animals can be either persistent or intermittent carriers, suggesting the possibility of decolonization, both by natural means and by antimicrobial treatment. [1,4]

S. aureus and S. epidermidis are the most common species to cause infection in humans. [2] S. aureus is commensal in humans, with 30% of the population being carriers. Primary site of

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10 colonization is the epithelium of the nares and the skin. It is a serious human pathogen, being the main cause of bacteraemia, infective endocarditis, infections associated with indwelling medical devices, skin and soft tissue infection, respiratory and urinary tract infection. [3,15]

Compared to nasal carriage of S. aureus in humans, where a decline has been noted over the past 80 years due to societal changes and improvement of personal hygiene, there is a higher carriage rate and greater genetic diversity of S. pseudintermedius in dogs. This is likely due to different social interactions in canines and greater exposure rate and interchange of bacteria between canine populations in general. [4]

In the case with canines, studies show that they are typically not colonized by S. aureus, as they are not a part of the natural microbial flora, and that carriage and infection with this bacterium is typically the result of human transfer. The same cannot be said about cats, since some studies suggest that they might be natural hosts of S. aureus. [16]

Most pathogenic staphylococci produce coagulase and these coagulase-positive staphylococci (CoPS) are thought to be the predominant commensal bacteria in domestic animals. [2,17] The most common infections attributed to CoPS in dogs and cats are primarily S. pseudintermedius, S. aureus subsp. aureus and anaerobius and S. schleiferi subsp. coagulans. [6,18]

Coagulase negative staphylococci (CoNS), such as S. epidermidis, lack many of the virulence factors found in more dangerous species and pose lower risk of infection. On the other hand they are often methicillin resistant, and may even be multi-resistant. [17] CoNS were previously considered nonpathogenic to companion animals, but recent research show that some CoNS, such as S. schleiferi subsp. schleiferi and S. lugdunensis possess virulent and pathogenic potential, and especially S.

schleiferi infections are increasing in frequency in both human and veterinary medicine. [18,19]

Limitations in laboratory diagnostic methods may lead to misidentification of these species, and further studies are required to properly define the role CoNS could play in as potential pathogens in veterinary medicine. [20]

1.3. Taxonomical changes of the Staphylococcus intermedius group

Staphylococcus intermedius was described in 1976, but exhibited such diverse phenotypic and

genotypic properties that the existence of more than one species was suspected. Indeed, new molecular techniques have lead to a re-classification of this species in recent years. Three species, all coagulase positive, have been differentiated from the original clone into three clusters: S. delphini, S.

intermedius, and S. pseudintermedius, comprising the Staphylococcus intermedius group (SIG). S. pseudintermedius itself was described in 2005 as a new species, and has been determined to be the

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11 identified as S. intermedius probably were S. pseudintermedius. The species in the SIG group are difficult to distinguish by standard diagnostic methods, and it is recommended that strains found on dogs are classified as S. pseudintermedius, if no further molecular diagnostic methods have been employed. Even though there has been a change in taxonomy there is low risk of misidentification in canine clinical material, and it does not have such a major impact for clinical practice. [4,21]

1.4. Identification of the major pathogenic staphylococci in veterinary

medicine

Presumptive identification is usually made on the basis of growth on appropriate media, appearance of colonies, Gram-staining and catalase production and slide coagulase and latex agglutination test.

Traditionally, routine method of identification of S. aureus has relied on tube coagulase tests for detection of free coagulase, or rapid identification utilizing detection of clumping factor and protein A. S. pseudintermedius, as well as other pathogenic staphylococci also display coagulase production, so further identification methods are needed. Other common tests include catalase production (staphylococci are positive), modified oxidase test (staphylococci are negative), and staphylococcal production of DNAse and thermostable nuclease. [4,10]

Colony morphology of S. aureus and S. pseudintermedius differs in colour and patterns of haemolysis on sheep or bovine blood agar, and can usually be distinguished macroscopically by a trained eye. S. pseudintermedius colonies are medium sized and non-pigmented, whereas S. aureus has variable colour, often with golden pigment. The zone of β-haemolysis on sheep or bovine agar is usually large and the zone of α-haemolysis is small. CoNS colonies are smaller and usually display no haemolysis. [4]

No standardized methods or rapid detection of S. pseudintermedius from clinical samples have been developed. Observation of growth on various agar media and additional biochemical tests are needed for identification. One study found that S. pseudintermedius show lecithinase production on Baird-Parker agar, thus making it an effective medium for identification and recovery. [14]

There is lack of phenotypic tests and readily available commercial test kits regarding differentiation of species within the SIG. Some characteristics, such as S. pseudintermedius being mannitol negative and trehalose positive, can help to distinguish it from other the members of the SIG, and some chromogenic agar has shown success in this regard as well, but diagnostic laboratories classify SIG isolates in dogs as S. pseudintermedius, since they do not appear to be carriers of the other species in the SIG. Molecular methods are needed for correct species identification, however.

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12 Further phenotypic identification by biochemical methods can be done to distinguish between

S. aureus and S. pseudintermedius, such as biochemical reactions with pyrrolidonyl arylamidase (both

are negative), β-Galactosidase (S. pseudintermedius positive, S. aureus negative), acetoin production (S. pseudintermedius negative, S. aureus positive), polymyxin B (S. pseudintermedius sensitive, S.

aureus resistant), acid production from D-mannitol (11-89% of S. pseudintermedius are positive but

show a delayed reaction, S. aureus is positive).

For accurate speciation of coagulase-positive staphylococci genotypic methods are recommended. Multiplex PCR is a sensitive and specific method, and other available systems are PCR-RFLP, and proteomic mass spectrometry such as MALDI-TOF MS, which is both cost-effective and highly accurate. [4]

1.5. Pathogenicity and virulence factors of S. aureus and S. pseudintermedius

S. aureus possesses various virulence factors that all contribute to disease; tissue colonization

and destruction, and immune evasion, just to mention a few. Toxins and enzymes convert host tissues into nutrients for bacterial growth and have immunomodulatory effects. Bacterial surface proteins aid in adhesion to epithelial cells in the nasal mucosa and other tissues. Capsular polysaccharide, teichoic acids and protein A interfere with opsonization and phagocytosis by host immune system. Coagulase shields the bacterium from phagocytic cells and production of catalase facilitates survival within them. Research has shown that a previous infection with S. aureus does not induce protective immunity. It is thought that activation of T cells and B cells is disrupted by expression of superantigen proteins. [1,15]

A lot is still unknown about the pathogenesis of S. pseudintermedius, and most virulence factors have not been clearly categorized. Some virulence factors are similar to S. aureus; such as production of enzymes such as coagulase, thermonucleases, DNase, and proteases. S. pseudintermedius possesses cytotoxins, such as haemolysin, similar to α- and β-toxins in S. aureus, with sphingomyelinase activity. Another cytotoxin is leukotoxin, which has toxic activity on granulocytes. Exfoliative toxins are also produced. The genes encoding the exfoliative toxin (SIET and EXI) have been found in S. pseudintermedius isolates recovered from canines with pyoderma, wound infection and otitis externa. Staphylococcal enterotoxins (SE), or superantigens, may play a role in disease progression of pyoderma. S. pseudintermedius has been reported to produce SEA, SEB, SEC and two unique SE’s; SECcanine and SE-int, but their role in the pathogenesis is unclear as of yet. [22]

The presence of protein adhesins on the bacterial cell surface provides the ability to bind to fibrinogen, cytokeratin and fibronectin. It also possesses a protein which is capable of binding to

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13 immunoglobulin, a feature similar to staphylococcal protein A. Cell-wall-anchored proteins aids in bacterial attachment to host epithelial cells and facilitates colonization. [2,4]

Staphylococci have the ability to form biofilms, a process where bacteria congregate, attach to a living (surgical site, wound) or non-living surface (indwelling medical devices, such as surgical implants, catheters, and sutures) and form a film-like matrix to evade antimicrobials or the host’s immune system. Bacteria can potentially detach from this biofilm several days, weeks or even years after initial attachment, disseminate through the body and cause clinical infection, and then re-attach at another site. [23]

It has long been known that S. aureus is capable of biofilm formation, but recent studies have also demonstrated this same ability in S. pseudintermedius, both in animal-associated and human strains, indicating pathogenic and zoonotic potential. [24]

Once a biofilm has formed treatment options are limited. A wound may be debrided, but oftentimes surgical implants must be removed if a biofilm is detected. Since antimicrobial therapy is of limited use, stressing the importance of hygienic measures to prevent infection and aseptic techniques during surgical procedures. [23]

Acquired and intrinsic resistance to many of the modern day antibiotics makes this a particularly important and dangerous pathogen in both human and veterinary medicine. Methicillin-resistance of

S. aureus mediated by the mecA gene has long been an issue in human medicine, and in 2006

methicillin resistant S. pseudintermedius (MRSP) emerged as a significant health threat in the veterinary field. [1]

Despite being primarily a human pathogen, S. aureus seems to have the ability for interspecies colonization and adapt to new hosts, meaning that animals can act as a potential reservoir for S. aureus and thus methicillin-resistant S. aureus (MRSA), and vice versa. [1,6]

Though the emergence of MRSP is recent, more and more reports show its ability to colonize humans. It has been shown to cause skin and soft tissue infection (SSTI), and less commonly bacteraemia and infection involving indwelling medical devices and prosthetics in humans, so it remains a potential zoonotic risk. But it is still rather uncommon and carriage is usually transient due to the fact that S. pseudintermedius is not part of the normal commensal microflora in humans. [25,26] Interspecies carriage and transfer of pathogenic strains have been described between humans, dogs, and cats, but little research involving other species regarding this topic has been done. Fig. 1 illustrates the process of dissemination of resistant bacteria and the transfer between humans and animals. One of the first cases to describe an infection with S. pseudintermedius in a human was reported in 2006. It involved an infection of an indwelling medical device, and phenotypic and

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14 genotypic testing revealed S. pseudintermedius to be the infectious agent. Though this might have been the first officially confirmed case, it does not exclude the possibility that humans might have been infected with S. pseudintermedius prior to this report. Some strains of S. pseudintermedius have similar pathogenicity and virulence as S. aureus, and therefore might risk being mislabeled as such. [27]

In a study with 28 dogs with pyoderma, 15 dogs were found to have MRSP, and MRSP were also isolated from two owners at the time, where both isolates shared the same genotype and susceptibility pattern. [25] Another study has shown that colonization with MRSP could be more common in humans than MSSP, with possible indication that it is more adapted to survive in a human host. MRSP colonization among veterinary dermatologists working with small animals seem to be higher than the rest of the human population. The MRSP isolates had a higher prevalence of antibiotic resistance when compared with MRSA isolates, and had the typical multidrug-resistance of MRSP in pets. [7]

Fig. 1. Exchange and transfer of resistant bacteria between humans and animals. Thickness of

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15

1.6. Antibiotic use and antimicrobial resistance

Novel antibiotic classes with new mechanisms of action have not been developed since 1987, leaving the veterinary profession with limited options regarding multi-drug resistant staphylococci. [29]

As first-line empirical use of systemic antibiotic therapy it is recommended to employ amoxicillin–clavulanic acid, cefalexin or clindamycin and then treat according to culture and sensitivity results. [30]

1.6.1. Beta-lactam antibiotics and beta-lactamase production

Infections with staphylococci are commonly treated with antimicrobial agents which inhibit the bacterial cell wall synthesis. The main examples are the beta-lactams (penicillins, cephalosporins, carbapenems). The semisynthetic penicillins, such as methicillin, also have the same mechanism of action – they bind to the penicillin binding protein (PBP2a), a key enzyme involved in the synthesis of the cell wall. [31]

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16 Many staphylococci show resistance to beta-lactams by producing an enzyme – beta-lactamase – that hydrolyze and inactive penicillins, mediated by the blaZ gene. This is combated by adding beta-lactamase inhibitors, such as clavulanic acid, to the beta-lactam antibiotics. [21,31]

1.6.2. Methicillin and multi-drug resistance among staphylococci

Methicillin-resistance is controlled by the mecA gene that encodes production of a modified penicillin binding protein, PBP2a, which the beta-lactams fail to inhibit. The mecA gene is located on the staphylococcal chromosomal cassette, also known as SCCmec. It is a mobile genetic element which can be transferred between different staphylococcal species, though the types of the SCCmec elements in MRSP differ slightly from those in MRSA. [1,18,21]

Since MRSP per definition is resistant to all beta-lactam antimicrobials, including penicillins, cephalosporins, carbapenems, and amoxicillin-clavulanic acid, they would therefore be ineffective as systemic treatment. Most MRSP are also multidrug resistant, ruling out fluoroquinolones, macrolides, trimethoprim-sulfonmides and lincosamides as options. Some drugs that were developed and approved 50 years ago, such as chloramphenicol, tetracyclines, aminoglycosides, and vancomycin, might be an option. [33]

In some cases MRSP isolates are not susceptible to any antimicrobials authorized for veterinary use, forcing veterinarians to look at treatment used in human medicine. [1]

There are new antimicrobials used against MRSA in human medicine, such as linezolid, ceftaroline, tigecycline and daptomycin. Although they may not be considered for use in veterinary medicine, it can be benificial to keep the properties of these substances in mind. [33]

For example, MRSA strains related to hospital-acquired infections are classified as HA-MRSA isolates. They carry SCCmec types I, II, or III (sometimes even types IV or V) and are generally multidrug resistant. Community-acquired MRSA strains on the other hand are classified as CA-MRSA isolates, which carry SCCmec type IV or V and are resistant to beta-lactams, but susceptible to many other antibiotic drugs. [34] Community-acquired staphylococcal bacteraemia is thought to occur when the bacterium enters the skin through minor lesions, whereas hospital-acquired staphylococcal bloodstream infections gain entry most commonly via intravascular catheters. [35]

Dogs are more commonly colonized by MRSP than cats. Fewer studies have been made about carriage in cats, and it is not clear whether S. pseudintermedius or S. aureus is the main colonizing CoPS species. The prevalence rate of MRSP has been studied in various dog populations in different countries. Community-acquired MRSP is prevalent with rates of 0%–4.5% in dogs, and in patients suffering from skin disease the prevalence is 0%–7%. MRSP was found in 4% of healthy cats,

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17 whereas no MRSP was found in cats with inflammatory skin disease. Although, these numbers can vary extensively depending on country and region. [1]

Methicillin-resistance in staphylococci does not always equal multidrug resistance, but most MRSP isolates have shown resistance to a majority of the veterinary drugs in use today. The most prevalent European clone of MRSP is normally resistant to β-lactam antibiotics, macrolides, tetracyclines, aminoglycosides, fluoroquinolones, lincosamides, chloramphenicol, trimethoprim, and susceptible only to amikacin, fusidic acid, vancomycin, rifampicin, linezolid, and teicoplanin, but not all of these latter drugs are licensed for systemic use in companion animals. [36]

This multidrug resistance is mediated by different resistance genes, involving altered target molecules, membrane-associated efflux, enzyme inactivation, altered cell membrane permeability and protective protein production. [31]

Also, antimicrobial susceptibility patterns differ between countries, and therefore it is recommended that veterinarians look at data according to their location. [37]

One study showed that differences exist in genotypes and susceptibility patterns among S.

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18

2. RESEARCH METHODS AND MATERIAL

2.1. Data collection and preparation of samples

This microbiological research was conducted at the Department of Veterinary Pathobiology at the Veterinary Academy at the Lithuanian University of Health Sciences in Kaunas, Lithuania. Retrospective analysis was conducted of staphylococcal species isolated from canine and feline clinical material submitted between January 2016 and November 2017 to the microbiology laboratory. The samples investigated in this study were obtained from a select few veterinary clinics in the Kaunas region and were submitted to the laboratory for routine diagnostic testing. A total of 78 samples were analyzed; 68 from canine specimen and 10 from feline. When it was mentioned in the laboratory report form, variables such as gender, age, collection site, and antimicrobial agents used for the susceptibility test were included in the analysis. Data that did not mention exact staphylococcal species was excluded.

A separate analysis of clinical sample material from 28 dogs was conducted, and staphylococcal species identification was made, as well as antimicrobial susceptibility test with 5 common beta-lactams and beta-lactamase production test were performed at the microbiology laboratory.

Swabs were taken by the treating clinician from skin lesions and cutaneous wounds, outer ear, conjunctiva of the eye, vaginal or preputial swabs or in the form of a urine sample. The bacteriological swabs were placed in Transwab® Amies gel or charcoal medium (Medical Wire, UK) and sent to the laboratory, where it was immediately inoculated on appropriate culture media, such as nutrient agar and blood agar (sheep blood 5%), and Rose-Bengal agar, E.M.B. Levine agar, and Pseudomonas Agar Base (Liofilchem®, Italy), to exclude contamination with other bacteria. If the sample was not immediately inoculated upon arrival, it was stored at 4°C until processing. If bacteriological growth occurred 24 or 48 hours after incubation at 35-37°C, a smear was made, stained by Gram’s method and examined microscopically to ensure that the morphology was compatible with staphylococcal species, and that isolation of a pure culture had been made.

Isolates displaying staphylococcal characteristics, such as raised, smooth 1-3 mm colonies of white, cream or yellow colour; microscopically Gram-positive cocci arranged in clusters; and haemolytic pattern (α-, and/or β-haemolysis in S. aureus and S. pseudintermedius, and non-haemolysis in S. epidermidis) on blood agar were subcultured to Baird-Parker medium and Mannitol Salt Agar (Liofilchem®, Italy) and incubated at 35-37°C for 18-24 hours. Growth of yellow colonies on Mannitol Salt Agar surrounded by yellow zones confirmed fermentation of mannitol, a feature of

S. aureus. Colony growth but non-yellowing of the medium was presumed to be non-fermenting

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Baird-19 Parker medium was evaluated for lechitinase production (clear zone around colonies) and lipase activity (opaque zone). Presence of clear and opaque zones was indicative of S. aureus. Presence of black colonies with lechithinase production or was indicative of S. pseudintermedius, no zones were indicative of S. epidermidis.

Additional biochemical tests were conducted, such as latex agglutination test with identification of bound coagulase and protein A (Microgen® Staph), where a visible clumping reaction was used to separate coagulase positive staphylococci (S. aureus, S. pseudintermedius) from coagulase negative ones (S. epidermidis). Catalase activity was investigated, where production of bubbles was considered positive.

Further species biochemical identification test was made with MicrogenTM STAPH-ID Identification and Microgen Identification System Software (MID-60).

Additional beta-lactamase production was measured in 11 of S. pseudintermedius isolates by stick test containing nitrocefin (Liofilchem®, Italy), which is sensitive to hydrolysis by all known beta-lactamases produced by Gram positive bacteria. A colour change to pink/red was considered a positive result, even in cases with very weak colour change.

2.2. Antimicrobial susceptibility testing

The susceptibility test was performed using the Kirby-Bauer disc diffusion method. [38] An inoculum preparation was made by transferring isolated colonies grown on a non-selective medium with a sterile cotton tip to a tube containing 0.9% sodium chloride solution (Liofilchem®, Italy). The tube was then agitated until the turbidity was equal to or slightly greater than 0.5 McFarland units. A sterile cotton-wool was dipped into the suspension and inoculated on a Mueller-Hinton based medium (Liofilchem®, Italy). Commercial filter paper discs (Liofilchem®, Italy) containing a pre-determined amount of antimicrobials were selected and placed on the growth medium.

A total of 15 different antimicrobials were tested. The penicillins were represented by amoxicillin (30 µg), amoxicillin + clavulanic acid (20+10 µg), ampicillin (10 µg), oxacillin (1 µg), and penicillin G (10 µg). Aminoglycosides were represented by gentamicin (10 µg). Cephalosporins were represented by cephalexin (30 µg), and cefovecin (30 µg). Fluoroquinolones were represented by enrofloxacin (5 µg), and norfloxacin (10 µg). Tetracyclines were represented by doxycycline (30 µg). Sulfonamides and potentiated sulfonamides were represented by sulfamethoxazole (50 µg) and sulfamethoxazole + trimethoprim (25 µg). Polymyxin B (100 µg) and fusidic acid (30 µg) were also tested.

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20 For the separate study the beta-lactams ampicillin (10 µg), amoxicillin + clavulanic acid (20+10 µg), cefovecin (30 µg), oxacillin (10 µg), and penicillin G (10 µg) were chosen for susceptibility testing.

Following placement of the antimicrobial discs, the plates were incubated at 35-37°C for 18-24 hours. They were evaluated for bacterial growth and the diameter of each zone of inhibition was measured in millimeters, and the isolates were categorized as either sensitive, intermediately sensitive or resistant to each antimicrobial drug. Where applicable, standards regarding break-off points set by the European Committee on Antimicrobial Susceptibility Testing (EUCAST Breakpoint Tables v. 7.1) were used, otherwise the recommendation of breakoff points according to the manufacturer of the antimicrobial discs were followed. [39]

2.3. Statistical analysis

The program Microsoft Office Excel 2016 was used to calculate the statistical results of the research. Comparison of data was performed using the Chi-square test and Fisher’s exact probability test, when expected observations was <5, using exact p-values. Results were considered statistically significant if p<0.05.

Analysis was made of the canine and feline group as a whole, and then separated into a canine group and a feline group, with separate analysis of differences within each group. Age was divided into three groups; <1 year, 1-6 years and ≥7 years, to better analyze any trends associated with age and bacterial colonization and antimicrobial resistance. Statistical analysis and comparison were made with regards to age, gender, anatomical collection site and species of bacteria and antimicrobial susceptibility patterns.

Statistical analysis was made for all staphylococcal species as a whole (n=3), and individual bacterial species comparison was made between S. pseudintermedius and S. aureus. Data regarding the third species S. epidermidis were too sparse for meaningful statistical analyses, so it was not used as a dependent variable.

Three of the samples (n=3) were taken from clinically healthy animals per owner request. In all other cases it was assumed that a disease process prompted the treating veterinarian to take a sample with subsequent submission for microbiological testing.

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21

3. RESEARCH RESULTS

3.1. Most frequently occurring staphylococcal species in canines and felines

A total number of 78 clinical samples from retrospective case data were collected and analyzed. 68 of the submitted samples were from canine clinical material, and 10 from feline. The staphylococcal species identified from all samples were S. pseudintermedius, 89.7% (70/78), S.

aureus, 7.7% (6/78), and S. epidermidis, 2.6% (2/78). Enumeration of all staphylococcal species

found in cats and dogs can be found in Table 1 in Annex 2.

3.2. Antimicrobial resistance patterns

The antimicrobial resistance for all staphylococcal isolates (n=78) is summarized in Table 1. The highest level of resistance was seen against polymyxin B. Isolates were also highly resistant to the sulfonamide group. Low level of resistance was noted with fluoroquinolones and aminoglycosides, especially enrofloxacin and gentamicin, respectively.

Table 1. Antimicrobial resistance profile of all Staphylococcus isolates

Group Antimicrobial Resistance, percent (n/N)a

Beta-lactams:

Penicillins Amoxicillin

Amoxicillin + clavulanic acid

55.6 (35/63) 46.5 (33/71) Cephalosporins Cephalexin Cefovecin 24.1 (13/54) 19.1 (9/47) Aminoglycosides Gentamicin 16 (8/50) Tetracyclines Doxycycline 18.4 (9/49) Fluoroquinolones Enrofloxacin Norfloxacin 14.3 (8/56) 23.1 (6/26)

Fusadines Fusidic acid 25 (7/28)

Polymyxins Polymyxin B 90.9 (10/11)

Sulfonamides & potentiated sulfonamides Sulfamethoxazole Sulfamethoxazole + trimethoprim 82.1 (23/28) 70.8 (17/24)

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22 For the purpose of this study, comparison of resistance to antimicrobials between species will be made only with S. aureus and S. pseudintermedius. Too few isolates of S. epidermidis (n=2) were found in this study to make an adequate comparison and they were therefore excluded. None, or too few isolates of S. aureus (n=6) were tested for norfloxacin, polymyxin B and sulfamethoxazole + trimethoprim to be included in the comparison.

S. pseudintermedius (n=70) displayed highest resistance against polymyxin B (10/11), and the

potentiated sulfonamides; sulfamethoxazole + trimethoprim (15/21). Low levels of resistance were noted against gentamicin (3/49). S. aureus on the other hand displayed highest resistance against cefovecin (3/5) and fusidic acid (2/3). Lowest resistance was against amoxicillin + clavulanic acid (1/5) and enrofloxacin (1/5).

The resistance pattern of the two main staphylococcal species is depicted in fig. 3, and the full antimicrobial susceptibility pattern can be found in Table 1 in Annex 1.

Fig. 3. Comparison of resistance to various antimicrobials between S. aureus and S.

pseudintermedius. The asterisk (*) indicates that S. aureus was not tested for norfloxacin, polymyxin B, or sulfamethoxazole + trimethoprim

Out of the 78 isolates, 76.9% (60/78) exhibited antimicrobial resistance (AMR) to at least one drug, whereas 29.5% (23/78) were multidrug resistant (MDR). Multidrug resistance is defined here as resistance to at least one microbial agent in three or more antimicrobial groups. AMR and MDR were the highest in S. aureus; 83.3% (5/6) and 50% (3/6). AMR and MDR in S. pseudintermedius were also relatively high; 77.1% (54/70) and 27.1% (19/70), respectively. One isolate even displayed resistance to five out of the six antimicrobial groups tested.

0 20 40 60 80 100 40 20 25 50 60 33 20 67 33 56 50 13 20 15 16 6 20 13 91 88 71 R ES IS TA N C E, IN % ANTIMICROBIALS S. aureus S. pseudintermedius

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23

3.3. Most common collection site in canines and felines

In 71 cases the collection site of the animal was known (dogs, n=64 and cats, n=7), and consequently grouped into four categories; skin, ear, eye, and urogenital tract (see Table 2 below). The term urogenital tract in this case refers to clinical material collected from either urine, vagina, or the prepuce. The relation between collection site and staphylococcal species isolation was not considered statistically significant (p=0.62).

All three staphylococcal species were mainly isolated from the skin. The urogenital tract was the least colonized area.

Table 2. Microorganism isolation and collection site

Microorganism isolation

Collection site (%)

Eye Skin Ear Urogenital tract

S. pseudintermedius 8 (11.9) 38 (56.7) 15 (23.9) 5 (7.5)

S. aureus - 3 (75) - 1 (25)

S. epidermidis - 2 (100) - -

Total: 8 Total: 42 Total: 15 Total: 6

Age of the animals was known in 52 cases (dogs, n=46, cats, n=6). For the purpose of this study, age groups were divided into < 1 years, 1-6 years, ≥ 7 years. Age distribution in the dogs and cats can be seen in fig. 3.

Fig. 4. Age distribution among the dogs and cats

There was no statistically significant relation between age of the animals and the staphylococcal species isolated (p=0.41). The age of the animal was not a factor regarding carriage of resistant bacteria (p=0.38). 8% 71% 21% 25% 50% 25% 0% 10% 20% 30% 40% 50% 60% 70% 80% < 1 y 1-6 y ≥ 7 y Dogs Cats

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24 Similarly, no significant relation could be detected between males (n=37) and females (n=26) and carriage of certain staphylococcal species (p=0.82). Similarly, the relation between the gender of the animal and proportion of bacterial resistance was not regarded as statistically significant. (p=0.95).

Microorganism isolation and collection site were significantly different between cats and dogs (p=0.039). In the canine group (n=64) the majority of the bacterial isolates were found on skin (see fig. 5), whereas in the feline group (n=7) the majority was isolated from the eye (see fig. 6). The fewest numbers of staphylococcal bacteria were isolated from the eye and urogenital tract in dogs, and from the ear and urogenital tract in cats. The differences are depicted graphically in fig. 5 and 6.

Fig. 5. Microorganism isolation and collection site in dogs

Fig. 6. Microorganism isolation and collection site in cats 0%

20% 40% 60% 80%

Skin Eye Ear Urogenital tract 61% 8% 23% 8% 0% 5% 10% 15% 20% 25% 30% 35% 40% 45%

Skin Eye Ear Urogenital tract 29%

43%

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25 3.3.1. Results of the canine group

A total of 28 different canine breeds were represented in this study, with the most common ones being Yorkshire terrier (n=7), West Highland white terrier (n=6), German shepherd (n=4) and French bulldog (n=4). There were 27 females and 33 males.

In 64 cases the collection site was known, and the most common anatomical sites colonized by staphylococcal species were skin, 61% (39/64), ear, 23.4% (15/64), eye, 7.8% (5/64), and urogenital tract, 7.8% (5/64). In four cases the collection site was unknown.

Three of the samples were vaginal swabs taken from healthy breeding bitches, as per owner request. The rest of the 65 samples sent in for testing were assumed to be associated with a disease process.

Age was not a statistically significant factor with regards to staphylococcal species isolation (p=0.72). Similarly, gender did not play a significant role when it came to bacterial colonization (p=0.67).

S. pseudintermedius was identified in 91.2% (62/68) of the samples, followed by S. aureus,

5.9%, (4/68), and lastly S. epidermidis, 2.9%, (2/68). Collection site in the canine group was not considered a statistically significant factor with regards to staphylococcal species isolation (p=0.54). A full depiction of microorganism isolation and collection site in the canine group can be found in Table 2 in Annex 2.

The antimicrobial resistance among all isolates in dogs (n=68) was greatest to polymyxin B (7/8). Fairly high resistance was also noted against the amoxicillin (32/54) and amoxicillin + clavulanic acid (31/61). The lowest level of resistance was against enrofloxacin (5/42). The resistance pattern of all staphylococci isolated in canines can be seen in fig. 7.

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26

Fig. 7. Resistance pattern among all staphylococcal isolates in canines

Fig. 8 shows comparison between of resistance between S. pseudintermedius and S. aureus. Resistance to sulfamethoxazole, polymyxin B and norfloxacin is only shown for S. pseudintermedius, since too few of the S. aureus isolates were tested for those antimicrobial drugs for adequate comparison.

No statistically significant difference regarding susceptibility pattern to antimicrobials and collection site was noted - all p-values for all antibiotics were > 0.05. A weak correlation (p=0.052) was noted with amoxicillin + clavulanic acid resistance and collection site.

Resistance among S. pseudintermedius was highest to polymyxin B (7/8) and sulfamethoxazole (21/24) and lowest to enrofloxacin (3/47). S. aureus displayed highest resistance to fusidic acid (2/3) and lowest resistance to amoxicillin + clavulanic acid (1/4).

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 59% 51% 18% 19% 18% 16% 10% 20% 19% 88% 85% 79%

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27

Fig. 8. Comparison of resistance between S. aureus and S. pseudintermedius. The asterisk (*)

indicates that S. aureus was not tested for norfloxacin, polymyxin B, or sulfamethoxazole.

3.3.2. Results of the feline group

The collection site was known in seven cases, and were distributed as such: eye (n=3), skin (n=2), ear (n=1) and urine (n=1). This differs from the canine group, where skin was the most common collection site (see fig. 5 and 6 above for a comparison). The specific breed of cat was not always mentioned in the report form sent to the microbiology laboratory, but the known breeds were listed as Bengal (n=2), Scottish fold (n=1), British short hair (n=1), Persian (n=1) and mixed breed (n=1).

In seven cases the gender of the patient was known; four males and three females. Carriage of resistant and sensitive bacteria in male cats was significantly higher than in females (p=0.0046). Half of the bacterial population in male cats displayed resistance against one antimicrobial drug. The proportion of intermediately sensitive bacteria were the same in both genders, but the staphylococci in female cats displayed very high sensitivity to antimicrobials, and no resistance.

0% 20% 40% 60% 80% 100% 61% 55% 15% 18% 15% 13% 6% 14% 13% 88% 88% 81% 50% 25% 33% 33% 50% 33% 33% 67% 50% RE SIST AN CE , IN % ANTIMICROBIALS S. pseudintermedius S. aureus

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28

Fig. 9. Proportion of resistant, sensitive and intermediately sensitive staphylococci in male cats

Fig. 10. Proportion of resistant, sensitive and intermediately sensitive staphylococci in female cats

S. pseudintermedius was isolated in 80% (8/10) of the samples, and S. aureus in 20% (2/10).

There was no significant relation between collection site and antimicrobial resistance. (p=0.14). The antimicrobial susceptibility pattern for all staphylococcal isolates is depicted in fig. 11. All isolates were resistant to polymyxin B and all were sensitive to enrofloxacin.

0% 10% 20% 30% 40% 50%

Resistant Sensitive Intermediate

50% 38% 12% 0% 20% 40% 60% 80% 100%

Resistant Sensitive Intermediate 0%

86%

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29

Fig 11. Antimicrobial susceptibility pattern for all staphylococcal isolates in felines

3.4. Investigation of antimicrobial susceptibility to select beta-lactam antibiotics

and beta-lactamase production

A separate study was made to determine the antimicrobial resistance against a select few common beta-lactams. The antibiotics chosen to represent beta-lactams were ampicillin, amoxicillin + clavulanic acid, cefovecin, oxacillin, and penicillin G.

A total of 28 samples collected from clinical material from canines were analyzed. 71.4% (20/28) of samples were taken from the skin, 14.3% (4/28) from the vagina, and 14.3% (4/28) from the ear. All isolates were identified as S. pseudintermedius.

Table 3. Antimicrobial susceptibility pattern of S. pseudintermedius isolated from clinical material

Antimicrobial No of isolates (%) S I R Amoxicillin + clavulanic acid 22 (78.6) 0 6 (21.4) Ampicillin 1 (3.6) 0 27 (96.4) Cefovecin 21 (75) 3 (10.7) 4 (14.3) Oxacillin 19 (67.9) 0 9 (32.1) Penicillin G 2 (7.1) 0 26 (92.9) 0% 20% 40% 60% 80% 100% 25% 11% 0% 50% 33% 20% 0% 67% 100% 50% 50% 50% 67% 83% 50% 67% 40% 100% 33% 0% 50% 50% 25% 22% 17% 0% 0% 40% 0% 0% 0% 0% 0%

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30 The susceptibility pattern of all S. pseudintermedius isolates is depicted graphically in fig. 12. Almost all bacterial isolates displayed resistance against ampicillin and penicillin G. Sensitivity was highest to amoxicillin + clavulanic acid. No statistically significant difference was detected between the collection site and resistance to a particular type of antimicrobial (p>0.05).

Fig. 12. Antibiotic susceptibility pattern of S. pseudintermedius against beta-lactam antimicrobials

Worth noting is that two of the clinical samples were vaginal swabs of healthy breeding bitches, and the isolates were resistant to all beta-lactams except amoxicillin + clavulanic acid.

10/11 (90.9%) samples tested positive for beta-lactamase production.

0 10 20 30 40 50 60 70 80 90 100 Amoxicillin + clavulanic acid

Ampicillin Cefovecin Oxacillin Penicillin 78,6 3,6 71,4 67,9 7,1 14,3 21,4 96,4 14,3 32,1 92,9 %

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31

4. DISCUSSION OF RESULTS

The Staphylococcus species in this study were all isolated from the skin, ear, eye and urogenital tract, confirming what is known about their common colonization sites in animals and ability to cause frequently encountered pathologies such as pyoderma, wound infection, otitis externa and urinary tract infection. [40]

As confirmed by many other studies, the most frequently isolated staphylococcal species in companion animals is S. pseudintermedius, in skin and outer ear especially. Other, albeit less common isolates, such as S. aureus, S. schleiferi, and coagulase-negative species such as S. epidermidis and S.

felis (in cats) in companion animals have also been identified. [6,40]

CoNS are not the most common species implicated in disease in companion animals, but there is concern about their possible virulent potential, increasing antimicrobial resistance and potential to further spread the genes encoding for resistance. [41] Co-colonization by CoNS and CoPS might increase the potential for horizontal gene transfer. [20] While the two isolates of S. epidermidis in this study were too few to draw a definite conclusion about their pathogenicity, their presence in companion animals should not be completely discarded.

AMR and MDR was common among the staphylococci, most likely a result of previous and current extensive antibiotic use in both human and veterinary medicine, which has selected for and favored resistant bacteria. [28]

A Portuguese study categorizing trends of antimicrobial resistance in clinical staphylococci noted that 79% of isolates were AMR, and 35% of isolates were MDR. Resistance to ampicillin and penicillin was most common in methicillin-susceptible isolates, which correlates to my findings. The study also showed that resistance to beta-lactams and fluoroquinolones has increased over a 16-year period. [40] This highlights the importance of choosing the correct antimicrobial group for treatment. Staphylococcal isolates displayed an overall high level of resistance to polymyxin B in this study. Although mainly used against Gram negative bacteria, it has effect on Gram positive bacteria by disrupting the bacterial cell wall. One study from 2012 demonstrated that polymyxin B could be beneficial as a topical agent in cases of otitis externa caused by MRSA and MRSP. [42] But the high resistance of staphylococci in this study to polymyxin B indicates that another treatment choice would be more prudent.

The retrospective study demonstrated that resistance to amoxicillin-clavulanic acid was relatively high in the canine group. A similar number was also noted in an Australian study [43], where 43% of S. pseudintermedius isolates in dogs, and 54% of isolates in cats were resistant to this

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32 antimicrobial drug. This is disquieting, since amoxicillin-clavulanate is recommended as first-line empirical antibiotic therapy. [30]

A wide variety of antimicrobial resistance genes have been detected in S. pseudintermedius, suggesting a remarkable ability to acquire new genetic material. The mecA and blaZ genes are resposible for resistance to beta-lactam, and the mutation capacity or movement of resistance genes might make a previously susceptible isolate resistant against certain types of drugs, such as amoxicillin with clavulanic acid. [21] I am not familiar in detail with the antimicrobial usage of veterinarians in the Kaunas region, but if the use of amoxicillin + cavulanic acid is extensive, that might be the reason behind the high percentage of resistance.

In the separate study focusing on beta-lactams only bacteria showed high sensitivity to amoxicillin-clavulanate. This figure is similar to a majority of studies [44,45,46], where susceptibility is usually high.

One study recommends that “strains that are oxacillin-resistant and mecA-positive or PBP 2a-producing should be reported as being resistant to all penicillins, cephalosporins, carbapenems and cephems regardless of the in vitro susceptibility test results obtained with these agents”. [47] I am reluctant to name the isolates that were resistant to oxacillin in this study as MRSP or resistant to all beta-lactams without further molecular testing for the presence of the mecA gene.

The oxacillin zone diameter breakpoint has been a source for discussion and differs between human and veterinary laboratories depending on what staphylococcal isolate is being tested, with subsequent risk of naming a resistant isolate as sensitive. Now, a S. pseudintermedius-specific breakpoint is recommended. MIC break off points and zone of inhibition are being constantly revised and new guidelines are regularly updated by EUCAST and CLSI. It is important for clinical microbiologists to stay up to date, to avoid labelling an isolate as sensitive to certain antimicrobials, leading to incorrect treatment suggestion and risking treatment failure.

But there is no doubt that methicillin-resistance is growing, and studies regarding MRSP carriage rates among dogs and cats can reach numbers around 30-45%. [40,48,49]

Rapid detection of carriers of MRS is important, especially since colonization often persists after treatment resolution of MRSP. The treatment course itself can lead to bacterial acquisition of methicillin-resistance. [49]

Few studies on carriage of MRSP in companion animals in Lithuania have been made. One of the first studies aiming to characterize S. pseudintermedius in diseased dogs in Lithuania reported 29.4% of S. pseudintermedius strains as MRSP, confirmed by molecular methods. [50] The same study also reported a high resistance to penicillin G (96.4%), which is similar to the findings in this study. Another Lithuanian study found the MRSP carriage rates in dogs and cats to be 4.6% and 10%,

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33 respectively, [51] suggesting some discrepancy and fluctuation within the pet population, and regional differences.

PCR and additional molecular methods were not employed in this particular study, which would have been useful to confirm presence of resistance genes, such as mecA and blaZ. In a clinical setting this might not be of great significance, since PCR is not routinely performed for every bacteriological sample, and exact species identification down to taxonomical level is not necessarily required – unless there is suspicion of MRS or multi-resistance. For clinicians the most important factor is the antimicrobial susceptibility, which enable them to choose the correct antimicrobial treatment, and subsequently reduce the burden of unnecessary and ineffective antimicrobial use.

A higher proportion of cats in this study were carriers of S. aureus compared to dogs, which may be attributed to them being a more natural host of this bacterial species. [16] A significant difference was noted between cats and dogs and site of infection (p = 0.039) in this study. Here, the conjunctiva was deemed the most common site of infection, whereas a South African study defined the skin and outer ear as the most common infection sites in cats. [52] The low sample number of cats (n=10) compared to dogs (n=68) might have skewed the results in this study. Fewer studies regarding colonization sites and staphylococcal infection in cats have been made compared to dogs [1] so this could be an area of future research.

No significant relation was detected between gender and age and carriage of certain staphylococcal species. A Lithuanian study characterizing CoPS noted that colonization of S. aureus in female dogs was significantly higher compared to male dogs. [53] This is supported by an Australian study, where especially intact bitches were more frequently colonized with S. aureus, suggesting that hormonal factors may play a role. [14] Comparison of age and gender between different studies is sometimes not feasible, since the definition of age groups vary. Some define age in two groups; juveniles and adults (where the age break-off point varies), other studies have three defined age groups, making comparison difficult. Further, and more targeted research is required to pursue a true correlation between these factors.

In this study it was noted that male cats seem to carry a higher proportion of antimicrobial resistant staphylococcal species compared to female cats (p = 0.0046). These findings are the opposite of the results of a study from Portugal, where variables such as gender and age were not considered risk factors for carriage of resistant bacteria. [40] None of the female cats in this study were carriers of resistant bacteria, which is very likely a coincidence. The low sample size of especially female and male cats in this study might have impacted the precision of the results and may not be representative for the population as a whole.

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34 Since part of this research is a retrospective study, the results should be interpreted judiciously. Some data about the animal, such as gender, anatomical collection site, age, and clinical history were lacking, making it difficult in some regards to create a uniform group for analysis. History of disease and previous antimicrobial or medical treatment were not included in the anamnesis, so therefore the resistance pattern was interpreted in isolation. Studies show that resistance pattern change after antimicrobial treatment. One study [54] suggests that antimicrobial therapy within 1 month prior to sampling is associated with antimicrobial and multidrug resistance. Prior treatment with beta-lactams or fluoroquinolones is associated with resistance to these same classes and other antibiotic classes. The longer the treatment period with beta-lactams, the higher the proportion of MDR.

This study found that beta-lactamase producing S. pseudintermedius are common. Beta-lactamase production is one of the protective mechanisms of bacteria as a response to extensive use of lactams, and it is widespread among staphylococci. [21] The high number of positive beta-lactamase producers explains the high level of resistance to penicillin G and ampicillin in this study, and the sensitivity to beta-lactamase inhibitors.

Studies have shown that antimicrobial susceptibility patterns differ between countries, and veterinarians are recommended to look at data according to their location. [37] Although further research on a larger scale is needed, the received results of this study provide a useful preliminary indication of the bacterial infection and antimicrobial resistance patterns of S. aureus and S.

(35)

35

CONCLUSIONS

1. The staphylococcal species identified in canine and feline clinical material were S.

pseudintermedius (89.7%), S. aureus (7.7%), and S. epidermidis (2.6%).

S. pseudintermedius (91%) and S. epidermidis (2.9%) were more common in canines, whereas S. pseudintermedius (80%) and S. aureus (20%) were more common in felines.

2. Resistance among all staphylococcal isolates was highest to polymyxin B (91%). 77% of all staphylococcal isolates displayed resistance to at least one antimicrobial class, and 30% were multidrug resistant. S. aureus displayed higher antimicrobial resistance and multidrug resistance than S. pseudintermedius.

3. The staphylococcal species were most frequently isolated from the skin in canines and the ocular conjunctiva in felines (p=0.039).

There was no correlation between age or the gender of the animals, and the staphylococcal species isolated.

The anatomical site was not associated with resistance of bacteria to a particular antimicrobial agent.

4. Highest level of resistance to beta-lactams was noted against ampicillin (96%). Lowest level of resistance was against amoxicillin + clavulanic acid (21%). 90% of Staphylococcus

(36)

36

RECOMMENDATIONS

Judging by the high level of resistance of staphylococci, it would be prudent to always employ culture and sensitivity testing when a bacterial infection is suspected and confirmed. Antimicrobial therapy should be based on susceptibility of the infective agent to avoid treatment failure. Since new antimicrobial classes are unlikely to be introduced to the market in the near future, judicial use of current antimicrobials is needed to preserve their activity and usefulness, and to prevent selecting for multidrug-resistant bacteria.

(37)

37

ACKNOWLEDGEMENTS

My sincerest thanks and gratitude to prof. dr. Jūratė Šiugždaitė and her assistant Laurencija Strolienė, who helped me throughout this process. Without their guidance and help with the microbiological procedures and analysis it would have been a much more difficult task to complete this thesis.

Thank you to the Lithuanian University of Health Sciences and the Veterinary Academy for opening your doors to foreign students with veterinary hopes and dreams.

And last, but not least – my eternal gratitude to my family and friends, without their undying support and love I would not be where I am today.

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