LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY Faculty of Veterinary Medicine. Gabriella Roslund

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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 KAUNAS

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 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 Gabriella Roslund (date) (author s name, surname) (signature) CONCLUSION OF THE SUPERVISOR REGARDING DEFENSE OF THE MASTER THESIS Jūratė Šiugždaitė (date) (supervisor s name, surname) (signature) THE MASTER THESIS HAVE BEEN APPROVED IN THE DEPARTMENT/CLINIC (date of approbation) Saulius Petkevičius (name, surname of the manager of department/clinic) (signature) Reviewers of the Master Thesis 1) 2) (name, surname) Evaluation of defense commission of the Master Thesis: (signatures) (date) (name, surname of the secretary of the defense commission) (signature) 2

3 CONTENT SUMMARY... 4 SANTRAUKA... 5 ABBREVIATIONS... 6 INTRODUCTION REVIEW OF LITERATURE Main characteristics of the Staphylococcus genus Common staphylococcal species in human and veterinary medicine Taxonomical changes of the Staphylococcus intermedius group Identification of the major pathogenic staphylococci in veterinary medicine Pathogenicity and virulence factors of S. aureus and S. pseudintermedius Antibiotic use and antimicrobial resistance Beta-lactam antibiotics and beta-lactamase production Methicillin and multi-drug resistance among staphylococci RESEARCH METHODS AND MATERIAL Data collection and preparation of samples Antimicrobial susceptibility testing Statistical analysis RESEARCH RESULTS Most frequently occurring staphylococcal species in canines and felines Antimicrobial resistance patterns Most common collection site in canines and felines Results of the canine group Results of the feline group Investigation of antimicrobial susceptibility to select beta-lactam antibiotics and betalactamase production DISCUSSION OF RESULTS CONCLUSIONS RECOMMENDATIONS ACKNOWLEDGEMENTS REFERENCES ANNEX ANNEX

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 multi-drug resistant. Antimicrobial resistance and multidrug 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 4

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 beta-laktaminiams antibiotikams ir betalaktamazių 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. 5

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 6

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 drugresistant 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 betalactam 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] 7

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. 8

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 9

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 most common isolate in dogs, in particular. It is assumed that most isolates that were previously 10

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. 11

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 12

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 It involved an infection of an indwelling medical device, and phenotypic and 13

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 the arrow represents the likelihood of spread. By S. Schwarz, A. Loeffler and K. Kadlec. [28] 14

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] 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] Fig. 2. Antibiotic targets and mechanisms of resistance. By G. Wright. [32] 15

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 betalactamase inhibitors, such as clavulanic acid, to the beta-lactam antibiotics. [21,31] 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, 16

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. pseudintermedius strains taken from different areas of the body from the same dog. [4] 17

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 betalactams 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 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 nonhaemolysis in S. epidermidis) on blood agar were subcultured to Baird-Parker medium and Mannitol Salt Agar (Liofilchem, Italy) and incubated at C for 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 staphylococci, such as S. pseudintermedius and S. epidermidis. Growth of black colonies on Baird- 18

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 Microgen TM 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 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. 19

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 C for 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. 20

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 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 55.6 (35/63) Cephalosporins Amoxicillin + clavulanic acid Cephalexin 46.5 (33/71) 24.1 (13/54) Cefovecin 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 a n = number of resistant isolates, N = total number tested 82.1 (23/28) 70.8 (17/24) 21

22 RESISTANCE, IN % 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. S. aureus S. pseudintermedius ANTIMICROBIALS 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. 22

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 % 70% 71% 60% 50% 50% 40% 30% 20% 10% 8% 25% 21% 25% 0% < 1 y 1-6 y 7 y Dogs Cats 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). 23

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. 80% 60% 61% 40% 23% 20% 8% 8% 0% Skin Eye Ear Urogenital tract Fig. 5. Microorganism isolation and collection site in dogs 45% 40% 35% 30% 25% 20% 15% 10% 5% 0% 29% 43% 14% 14% Skin Eye Ear Urogenital tract Fig. 6. Microorganism isolation and collection site in cats 24

25 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

26 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 59% 51% 18% 19% 18% 16% 10% 20% 19% 88% 85% 79% 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 > 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). 26

27 RESISTANCE, IN % S. pseudintermedius S. aureus 100% 80% 60% 40% 20% 0% 67% 61% 50% 55% 50% 33% 33% 33% 33% 25% 15% 18% 15% 13% 14% 13% 6% 88% 88% 81% 50% ANTIMICROBIALS 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 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. 27

28 50% 50% 40% 38% 30% 20% 12% 10% 0% Resistant Sensitive Intermediate Fig. 9. Proportion of resistant, sensitive and intermediately sensitive staphylococci in male cats 100% 86% 80% 60% 40% 20% 0% 14% 0% Resistant Sensitive Intermediate 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. 28

29 100% 83% 100% 100% 80% 67% 67% 67% 60% 40% 20% 0% 50% 25% 25% 22% 11% 50% 50% 33% 17% 0% 0% 0% 50% 50% 50% 50% 40% 40% 33% 20% 0% 0% 0% 0% 0% 0% 0% Resistant Sensitive Intermediate 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) 29

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) ,6 21,4 Amoxicillin + clavulanic acid 96,4 92,9 71,4 67,9 32,1 14,3 14,3 3,6 7,1 Ampicillin Cefovecin Oxacillin Penicillin Sensitive Intermediate Resistant 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. 30