CHARACTERISTICS AND RESISTANCE TO ANTIMICROBIALS OF STAPHYLOCOCCUS AUREUS AND STAPHYLOCOCCUS PSEUDINTERMEDIUS ISOLATED FROM PET ANIMALS AND THEIR OWNERS

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

VETERINARY ACADEMY

Jūratė Stankevičienė

CHARACTERISTICS AND RESISTANCE

TO ANTIMICROBIALS

OF STAPHYLOCOCCUS AUREUS AND

STAPHYLOCOCCUS PSEUDINTERMEDIUS

ISOLATED FROM PET ANIMALS AND

THEIR OWNERS

Doctoral Dissertation Agricultural Sciences, Veterinary (A 002)

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Dissertation has been prepared at the Department of Veterinary Patho-biology (former Infectious Diseases) of the Veterinary Academy of the Lithuanian University of Health Sciences during the period of 2011–2019.

Scientific Supervisor

Prof. Dr. Jūratė Šiugždaitė (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – A 002).

Dissertation is defended at the Veterinary Research Council of the Lithuanian University of Health Sciences:

Chairperson

Prof. Dr. Alius Pockevičius (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – A 002).

Members:

Assoc. Prof. Dr. Gintaras Daunoras (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – A 002);

Dr. Marius Virgailis (Lithuanian University of Health Sciences, Agricul-tural Sciences, Veterinary – A 002);

Prof. Dr. Algimantas Paulauskas (Vytautas Magnus University, Natural Sciences, Biology – N 010);

Dr. Olga Muter (University of Latvia, Natural Sciences, Biology – N 010).

Dissertation will be defended at the open session of the Veterinary Research Council of Lithuanian University of Health Sciences, Veterinary Academy, 2019 on 30 of August at 10 p.m. in the Dr. S. Jankauskas auditorium.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS

VETERINARIJOS AKADEMIJA

Jūratė Stankevičienė

STAPHYLOCOCCUS AUREUS IR

STAPHYLOCOCCUS PSEUDINTERMEDIUS,

IŠSKIRTŲ IŠ GYVŪNŲ AUGINTINIŲ IR

JŲ SAVININKŲ, CHARAKTERIZAVIMAS

IR ATSPARUMAS ANTIMIKROBINĖMS

MEDŽIAGOMS

Daktaro disertacija Žemės ūkio mokslai,

veterinarija (A 002)

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Disertacija rengta 2011–2019 metais Lietuvos sveikatos mokslų universi-tete, Veterinarijos akademijoje, Veterinarinės patobiologijos katedroje (bu-vusi Užkrečiamųjų ligų katedra), Mikrobiologijos laboratorijoje.

Mokslinė vadovė

prof. dr. Jūratė Šiugždaitė (Lietuvos sveikatos mokslų universitetas, že– mės ūkio mokslai, veterinarija – A 002).

Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterinarijos mokslo krypties taryboje:

Pirmininkas

prof. dr. Alius Pockevičius (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – A 002).

Nariai:

doc. dr. Gintaras Daunoras (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – A 002);

dr. Marius Virgailis (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – A 002);

prof. dr. Algimantas Paulauskas (Vytauto Didžiojo universitetas, gamtos mokslai, biologija – N 010);

dr. Olga Muter (Latvijos universitetas, gamtos mokslai, biologija – N 010).

Disertacija ginama viešame Veterinarijos mokslo krypties tarybos posėdyje 2019 m. rugpjūčio 30 d. 10 val. Lietuvos sveikatos mokslų universiteto Veterinarijos akademijos dr. S. Jankausko auditorijoje.

Disertacijos gynimo vietos adresas: Tilžės g. 18, LT-47181, Kaunas, Lietuva.

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ABBREVIATIONS

AML – amoxicillin AMP – ampicillin AUG – Augmentin Bp – base pair

BPA – Baird Parker agar C – cytosine

CFU/mL – colony-forming units per milliliter

CLSI – The Clinical and Laboratory Standards Institute CNS – coagulase-negative staphylococci

CPS – coagulase-positive staphylococci CI – confidence interval

DNA – deoxyribonucleic acid

EUCAST – The European Committee on Antimicrobial Susceptibility Testing

G – guanine

Luk – leucocidin toxins

M-PCR – multiplex polymerase chain reaction MBC – minimum bactericidal concentration MDR – multidrug resistant

MIC – minimal inhibitory concentration mol% – mole percent

MRSA – methicillin-resistant Staphylococcus aureus

MRSP – methicillin-resistant Staphylococcus pseudintermedius MSSA – methicillin-sensitive Staphylococcus aureus

ONPG – ortho-nitrophenyl-galactopyranoside OR – odds ratio

OX – oxacillin

PBP – penicillin binding protein PCR – polymerase chain reaction PFGE – pulsed field gel electrophoresis PG – penicillin G

PVL – Panton Valentine leucocidin PYR – L-pyrrolidonyl-β-naphtylamide rmp – revolutions per minute

RNA – ribonucleic acid

S. aureus – Staphylococcus aureus

S. delphini – Staphylococcus delphini S. hyicus – Staphylococcus hyicus

S. intermedius – Staphylococcus intermedius S. lutrae – Staphylococcus lutrae

S. pseudintermedius – Staphylococcus pseudintermedius

S. schleiferi subsp. coagulans – Staphylococcus schleiferi subspecies coagulans

SCCmec – Staphylococcal Cassette Chomosome mec SE – staphylococcal enterotoxins

SIG – Staphylococcus intermedius group spp – species

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INTRODUCTION

Dogs and cats probably are the most popular pet animals in Europe. The relationship between companion animals and humans has changed through the years. While in the past pets usually were maintained outside hou-seholds, today they are often kept inside houses. Close physical contact by touching, petting and licking occurs at high frequency on the basis of the current perception of household pets as actual family members. Companion animals and humans can act as reservoirs of resistant to many antimicrobials bacteria [71].

Staphylococci are Gram-positive spherical bacteria of enormous clinical and biotechnological relevance [94]. Staphylococcal infections are common to veterinary and human medicine. β-lactam antibiotics are among fre-quently prescribed antibiotics worldwide to treat staphylococcal infections. Antimicrobial resistance is changing over time and is generally rising steadily for those antimicrobials that are often used.

According to the published academic writings, the transfer of

Staphylo-coccus pseudintermedius between human and zoonotic hosts is possible.

There are several reports of serious MRSP-infections in humans [3, 4]. The situation is even more complicated, because the precise identification of

Staphylococcus pseudintermedius requires genetic methods that are rarely

used in routine diagnosis [15, 108]. It should be noted that this specie of bacteria until the year 2005 was identified and called as Staphylococcus

intermedius or even Staphylococcus aureus [106]. Consequently, the

importance of this bacterium as a pathogen appears to be underestimated [124].

The aim of the study is:

To determine the prevalence of Staphylococcus aureus and

Staphylo-coccus pseudintermedius colonization in companion animals and humans

and their resistance to antimicrobials.

Objectives of the study:

1. To isolate and identify Staphylococcus aureus and Staphylococcus

pseudintermedius in the populations of companion animals and their

owners using genotypic techniques.

2. To evaluate risk factors for pets and their owners for becoming carriers of Staphylococcus aureus and Staphylococcus pseudintermedius.

3. To determine phylogenetic relationship between isolated Staphylococcus

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4. To evaluate antimicrobial resistance of Staphylococcus aureus and

Sta-phylococcus pseudintermedius

Practical significance and scientific novelty

The study was conducted to determine the prevalence of pathogenic staphylococci in pet animals and their owners. While Staphylococcus

pseudintermedius have been recently distinguished as a new pathogenic

species, the data on methicillin-resistant strains and resistance to other antimicrobial agents of these bacteria in this country are insufficient. As empirical treatment is often applied in veterinary clinics, antimicrobials are used to prevent secondary infection; it is useful to know which anti-microbials work best against pathogenic staphylococci.

It is popular to keep one or even a few pets in one household in this country. A close relationship is often observed between a pets and pet ow-ners. It is necessary to investigate the factors that influence the transmission of pathogenic bacteria between pets and their breeders in order to prevent the transmission of pathogenic and resistant bacteria between each other.

Colonization of Staphylococcus pseudintermedius strains was detected not only in pets but also in humans. It is considered as a zoonosis.

Staphylo-coccus aureus and StaphyloStaphylo-coccus pseudintermedius strains containing the blaZ gene which encodes resistance to β-lactam antibiotics were identified

during the study. Phylogenetic analysis of Staphylococcus aureus and

Staphylococcus pseudintermedius isolated from humans and their pets living

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1. LITERATURE REVIEW

1.1. History

The importance of staphylococci as pathogens for human and animals has been recognized for more than 100 years. The name Staphylococcus (staphyle, bunch of grapes) was introduced by Ogston (1883) for the group micrococci causing inflammation and suppuration. He was the first to dif-ferentiate two kinds of pyogenic cocci: one arranged in groups or masses was called “Staphylococcus” and another arranged in chains was named “Billroth’s Streptococcus” [42]. A formal description of the genus

Staphy-lococcus was provided by Rosenbach (1884). Rosenbach described the two

pigmented colony types of staphylococci and proposed the appropriate nomenclature: Staphylococcus aureus (yellow) and Staphylococcus albus (white) [9].

1.2. Taxonomy

The taxonomic designation of the Staphylococcus genus has been reor-ganized on several occasions, including historical placement in the family

Micrococcaceae and more recently, Bacillaceae [86]. DNA-ribosomal RNA

(rRNA) hybridization and comparative oligonucleotide analysis of 16S rRNA has demonstrated that staphylococci form a coherent group at the genus level. Staphylococci are differentiated from other close members of the family with its low G + C content of DNA ranging from 30 to 40 mol% [10]. The genus Staphylococcus belongs to the family Staphylococcaceae from 2010 by Schleifer and Bell [74].

A deeper look into the chemotaxonomic and genotypic properties of staphylococci led to the description of many new staphylococcal species [42]. According to current knowledge, including the newly described species, the Staphylococcus genus includes 52 species and 28 subspecies

[74].

Staphylococcus intermedius was described as being a new species

iso-lated from pigeons, dogs, mink, and horses by Hayek in 1976. The most of coagulase-positive staphylococci in healthy and diseased animals have been identified as being Staphylococcus intermedius strains for over 30 years [4].

However, recent research has demonstrated that isolates phenotypically identified as S. intermedius are indeed differentiated into three different but closely related bacterial species Staphylococcus intermedius,

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Staphylococ-12

cus delphini and Staphylococcus pseudintermedius, which belong to the Staphylococcus intermedius-group (SIG) [106].

S. pseudintermedius was isolated from a cat, a dog, a horse, and a parrot

in 2005 and has been described as a new coagulase-positive species of ani-mals [21].

The genus Staphylococcus is traditionally divided in two groups based on the bacteria’s ability to produce coagulase, an enzyme that causes blood clotting: the coagulase-positive staphylococci and the coagulase-negative staphylococci [32, 103]. The coagulase test usually correlates well with sta-phylococci pathogenicity. The coagulase-negative stasta-phylococci are com-mon commensals of the skin. The two major pathogenic staphylococci,

Sta-phylococcus aureus and StaSta-phylococcus pseudintermedius are

coagulase-positive [76]. Other coagulase-coagulase-positive staphylococci are S. intermedius, S.

schleiferi subsp. coagulans, S. hyicus, S. lutrae, S. delphini [106]. 1.3. Colonization of Staphylococcus aureus and Staphylococcus

pseudintermedius

The terms carriage and colonization are in many occasions used when humans and animals present staphylococci that multiply in the nares, throat or other superficial sites but without causing disease (18).

1.3.1. Staphylococcus aureus in canine and feline

There is not much data about S. aureus carriage among dogs and cats compared to a lot of information about this type of bacteria colonization in humans.

Published studies focus more on the study of methicillin-resistant strains. According to the literature, S. aureus colonizes 8.8–23.7% of dogs [14, 47, 52, 123]. Hanselman et al., in the study found that the anterior nares were the most common site of S. aureus colonization in dogs (68.4%) compared with rectal colonization (26.3%); however, the difference was not statis-tically significant (p=0.096) [47].

Carriage of S. aureus in 4.3% and 17% cats reported Hanselman et al. and Iverson et al. respectively [47, 52]. Bierowiec et al. based on the results obtained, found that the prevalence of S. aureus in domestic cats was 19.17%, while it’s prevalence in the feral cat population was only 8.3%; which was statistically significant. Also, samples were taken from four sites of body: nares, anus, skin and conjunctival sacs in this study. Researchers found that the nares turned out to be the most sensitive anatomical location to detect S. aureus colonization [13].

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1.3.2. Staphylococcus aureus in human

Many healthy people may carry S. aureus as a part of normal microflora in the nose, throat, perineum or skin. In the majority of cases, the bacterium does not cause disease [87].

According literature sources S. aureus nasal colonization of healthy hu-mans is about 18.5–27.7% [47, 123]. Approximately 10–35% of human beings are persistently colonized, 20–70% are intermittently colonized and a further 5–50% are non-colonized [46, 60, 127].

Despite an increased risk of infection, colonized individuals are more likely to survive a systemic S. aureus infection than non-colonized persons. This protective effect is hypothesized to be due to the immunological pri-ming effect of colonization [127, 128].

Based on the results obtained by Hanselman et al., younger age appro-ached significance on multivariable analysis, (p=0.052), with a mean age of 30 years for colonized individuals versus 34 years for non-colonized indi-viduals [47].

1.3.3. Staphylococcus pseudintermedius in dogs and cats

S. pseudintermedius as normal microflora usually is isolated from dogs.

It is a commensal of the skin and mucous membranes of canine and feline and possibly one of the most common bacterial pathogens treated by ve-terinarians. It primarily colonizes the anal mucosa and nares of healthy dogs and cats, but it can also be isolated from the mouth, forehead, and the in-guinal region [114]. Literature sources described the prevalence of S.

pseu-dintermedius in dogs approximately 14–78% [14, 47, 52, 123]. According to Hanselman’s study results rectal colonization 60.7% was significantly more common than nasal colonization 23.0% (p=0.001). S. pseudintermedius different rates colonization rates depending on the body-site. Highly va-riable nasal values have been detected in dogs (16–64%), with mouth (42– 74%) and perineum (28–72%) as the sites with higher S. pseudintermedius recovery rates [4]. According to published data, MRSP rates among healthy dogs are lower than 4.5%.

The prevalence of S. pseudintermedius colonization in cats was described lower than in dogs at 3–7% [47, 52].

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1.3.4. Staphylococcus pseudintermedius in human

S. pseudintermedius were isolated from 4.1% and 5.6% humans’ nasal

cavity [47, 123]. According researchers, high chances for S.

pseudinter-medius colonization observed for owners who allowed dogs to lick their

faces, to rest on the sofa, or to sleep in their bed, however, the data was not statistically significant. Dog owners who kept more than two dogs had a significantly higher chance of being colonized with S. pseudintermedius than those who kept 1–2 dogs (p=0.05) [123].

1.4. Staphylococcus aureus and Staphylococcus pseudintermedius as pathogens

Infection is a condition in which staphylococci reaches a certain area of the body, multiplying in the tissues and causing clinical symptoms [27]. Pathogenic staphylococci can cause a variety of infections that can be clas-sified into three types: surface tissue damage (wound infections); toxicosis – food poisoning, scalded skin syndrome and toxic shock syndrome; systemic and life-threatening conditions, such as endocarditis, osteomyelitis, pneu-monia, cerebral abscesses, meningitis and bacteremia [12].

1.4.1. Diseases caused by Staphylococcus aureus and Staphylococcus

pseudintermedius in dogs and cats

S. aureus and S. pseudintermedius are commensal organisms, but are also

a cause of disease such as pyoderma and otitis externa in dogs [47]. Min et al. report describes rare eosinophilic dermatitis in a dog with suspected sepsis due to S. pseudintermedius infection [79]. Infection usually occurs when skin or mucosal barriers are affected by predisposing factors such as atopic dermatitis, medical and surgical procedures or immunosuppressive disorders [3].

There are several reports of toxic shock syndrome in dogs suspected of being caused by S. pseudintermedius strains [109].

1.4.2. Diseases caused by Staphylococcus aureus and Staphylococcus

pseudintermedius in human

S. aureus, a natural inhabitant of the human and animal body, is mostly

associated with community-acquired and nosocomial infections, which can be fatal in immunodeficient patients. Staphylococci are also responsible for food poisoning characterized by severe vomiting and cramping with or

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without diarrhea. S. aureus produces a large number of toxins and enzy-mes, of which the enterotoxins are most important cause of gastroenteritis (vomiting and diarrhea) and superantigen-associated illness. Strict hy-gienic practices are crucial in preventing staphylococcal food poisoning. Skin infection or systemic infection requires antibiotic therapy, while the foodborne intoxication does not require antibiotic therapy since the disease is caused by the toxin and it is mostly self-limiting [11].

In the majority of cases, the bacteria do not cause disease. However, da-mage to the skin or other injury may allow the bacteria to overcome the natural protective mechanisms of the body, leading to infection. Skin infec-tions including cellulitis, folliculitis, furuncles, impetigo and subcutaneous abscesses; infective endocarditis as well as osteoarticular, pleuropulmonary, and device-related infections are the most common types of disease caused by S. aureus [113, 115].

Longitudinal data from Denmark provide considerable insight into the impact of changes in access to health care interventions on Staphylococcus

aureus bacteremia incidence. Between 1957 and 1990, the incidence of Sta-phylococcus aureus bacteremia incidence increased from 3 per 100,000

person-years to 20 per 100,000 person-years [33].

Studies consistently demonstrate high rates in the first year of life, a low incidence through young adulthood, and a gradual rise in incidence with advancing age. Male gender is consistently associated with increased

Sta-phylococcus aureus bacteremia incidence, with male-to-female ratios of

∼1.5. The basis for this increased risk is not understood [115].

S. pseudintermedius can cause clinical infections in humans as well, but

these are rare and often misidentified as S. aureus [125]. Staphylococcus

pseudintermedius is a veterinary pathogen that has seldom been described as

an agent of human disease [108]. Van Hoovels et al. presented the first clinical report of S. pseudintermedius infection in human in 2006 years [118].

1.5. General characteristics and biochemical properties of Staphylococcus aureus and Staphylococcus pseudintermedius

Staphylococci differ in their ability to cause a risks to human and animal health, ranging from non-pathogenic to dangerous pathogens causing severe infections and being resistant to the treatment by most of the commonly applied antibiotics [23].

Staphylococci are Gram-positive cocci, approximately 1 µm in diameter, that generally occur in grape-like clusters, but can also be found in singles

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and pairs. They are non-motile, lack flagella and do not form spores, though they are able to survive in dormant state for years under unfavorable con-ditions. Most staphylococci are facultative anaerobes, catalase-positive and oxidase-negative. Two species, S. aureus subsp. anaerobius and S.

saccha-rolyticus, are anaerobic and catalase-negative [23]. General characterristics

of S. aureus and S. pseudintermedius are shown in Table 1.5.1.

Table 1.5.1. Biochemical characteristics of Staphylococcus aureus and

Staphylococcus pseudintermedius

Biochemical Test S. aureus S. pseudintermedius Reference

Catalase + + Devriese et al., 2005 Hemolysis + + Quinn et al., 2011 Slide coagulase + +/– Devriese et al., 2005 Tube coagulase + + Devriese et al., 2005 DNase + + Devriese et al., 2005 Mannitol fermentation + w Van Hoovels et al., 2006 Maltose fermentation + +/– Quinn et al., 2011 Acetoin + w Quinn et al., 2011 Sensitive to Polymyxin B – + Devriese et al., 2005 β-galactosidase – + Quinn et al., 2011 Pyrolidonyl arylamidase – + Van Hoovels et al., 2006 + positive; – negative; +/– 11–89% of strains positive, w – poor utilization.

Staphylococcal colonies are usually white, opaque and up to 4 mm in diameter. The colonies of bovine and human strains of S. aureus are golden yellow due to produced carotenoid pigment staphyloxanthin.

Four staphylococcal hemolysis types are recognized alpha, beta, gamma and delta. Individual hemolysins differ antigenically, biochemically and in their effects on the red blood cells of different animal species. Strains vary in their hemolysin-producing ability, and animal strains of S. aureus and S.

pseudintermedius usually produce both alpha-hemolysin and

beta-hemo-lysin [23].

The strains of S. aureus and S. pseudintermedius coagulate rabbit plasma, but S. pseudintermedius are clumping-factor-negative in the so-called slide or rapid coagulase test [21].

Staphylococcus pseudintermedius is positive in tests for acetoin, β-glu-cosidase, arginine dihydrolase, urease, nitrate reduction, pyrrolidonyl ary-lamidase and ONPG (β-galactosidase). It does not produce β-glucuronidase; it is susceptible to acriflavine and to novobiocin, and is resistant to defe-roxamine. Acid is produced from glycerol (weakly and delayed), ribose,

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ga-lactose, D-glucose, D-fructose, D-mannose, mannitol (weakly and delayed), Nacetylglucosamine, maltose, lactose, sucrose, trehalose and D-turanose (weakly and delayed). No acid is produced from erythritol, D-arabinose, L-arabinose, D-xylose, L-xylose, adonitol, methyl β-D-xyloside, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, methyl glucoside, methyl α-D-xyloside, amygdalin, arbutin, aesculin, salicin, cellobiose, melibiose, inulin, melezitose, D-raffinose, starch, glycogen, xylitol, D-lyxose, D-tagatose, D-fucose, L-fucose, L-arabitol, gluconate, 2-ketogluconate or 5-ketogluco-nate. The G+C content of the DNA is 38 mol% [31].

Staphylococcus aureus strains are positive for D-glucose, D-fructose, D-mannose, D-maltose, D-lactose, D-trehalose, D-mannitol, sucrose, N-ace-tyl-glucosamine, D-celiobiose, and D-turanose; meanwhile, no acid produc-tion was demonstrated by utilizaproduc-tion of D-ribose, xylitol, xylose, D-meli-biose, raffinose, L-arabinose, and α-methyl-D-glucoside. They are also po-sitive for catalase, coagulase, and benzidine reactions and are capable of nitrate reduction and acetylmethylcarbinol (acetoin) production. Results for DNase, clumping factor, urease, arginine dihydrolase, pyrrolidonyl arylami-dase, leucine arylamiarylami-dase, β-N-acetylglucosaminiarylami-dase, α-chymotrypsin, α-glucosidase, β-glucosidase, alkaline phosphatase, esterase C-4 and C-8, lipase (C-14), phosphatase acid, and naphthol-AS-BI-phosphohydrolase are positive. There is no production of oxidase, α-galactosidase, β-glucoro-nidase, β-galactosidase, valine arylamidase, cystine arylamidase, arginine arylamidase, trypsin, ornithine decarboxylase, α-mannosidase, and α-fuco-sidase. All strains are resistant to novobiocin [31].

1.6. Virulence factors of staphylococci

The pathogenicity of S. aureus is due to the production of certain en-zymes (coagulase, hyaluronidase, catalase, thermonuclease, etc.) and toxins. In addition, virulence is also associated with cell wall adhesion components (mucosal polysaccharide capsule, adhesins, protein A, teichoic acid, etc.). Virulence factors help bacteria to adapt to hostile environments, facilitate their survival and promote infection through cell invasion, the degradation of the immune system cells and tissues, facilitate the multiplication of the bacteria and are involved in the onset of clinical symptoms [41].

S. aureus produces a large number of toxins and enzymes, of which the

enterotoxins (24 serotypes of toxins are identified) are most important in the production of gastroenteritis (vomiting and diarrhea) and superantigen-associated illness. Enterotoxins are heat-stable and are produced when the temperature of food is at or above 46°C. Consumption of preformed toxins induces vomiting with or without diarrhea within 30 min–8 h (average

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3 h). The enterotoxin induces the release of 5-HT (5-hydroxytryptamine) from mast cells, which stimulates vagal nerves in the stomach lining and induces vomiting. Enterotoxins are also called superantigens, because they form a complex with MHC (major histocompatibility complex) class II molecules on the surface of antigen-presenting cells, activating and proli-ferating T cells to produce massive amounts of cytokines that contribute to fatal toxic shock syndrome. The genes for enterotoxin production are present in pathogenicity islands in the chromosome, in plasmids, in trans-posons, and in temperate bacteriophages. Toxin production is regulated by a two-component regulatory system called agrAC (accessory gene regu-lator) [11].

Similar to S. aureus, S. pseudintermedius produces a variety of virulence factors, including enzymes (coagulase, thermonuclease proteases, etc), to-xins (cytototo-xins, exfoliative toxin and enterototo-xins) and surface proteins (clumping factor and protein A, etc) [130]. Both bacterial species have been shown to form biofilm [34].

The toxins described to date in both bacterial species can be classified into three main groups: cytotoxins (leukocidins, haemolysins), exfoliatins and pyrogenic toxin superantigens (PTSAgs). This latter group includes the toxic shock syndrome toxin (TSST) and enterotoxins.

Leukocidins. One of the most virulent toxins is the Panton-Valentine leukocidin (PVL) (a pore-forming toxin) encoded by the genes lukS-PV and

lukF-PV. This cytotoxin is composed of two protein sub-units, LukS-PV and LukF-PV, which act together assembling in the membrane of host defense

cells, in particular, white blood cells, monocytes, and macrophages, induc-ing the formation of pores, alterinduc-ing the permeability and thus destroyinduc-ing the cell. PVL produce leukocyte destruction causing necrotising pneumonia – an aggressive condition that often kill patients – and skin and soft tissue infections (SSTIs) [38, 68]. Only 2–3% of S. aureus strains produce this toxin. Rarely, PVL is responsible for other S. aureus infections such as osteomyelitis, septicemia or endocarditis. The leukotoxin LukE/LukD (luKE/D genes) produces dermonecrosis in rabbits but has a weak leu-kotoxic activity and no hemolytic activity. Ruminant polymorphonuclear leukocytes are highly sensitive to the leukotoxin LukM/LukF-PV (encoded by lukM), and its presence has been associated with cases of bovine mastitis [55].

Similar to the PVL, S. pseudintermedius produces a bicomponent leu-kotoxin Luk-I, which is encoded by two cotranscribed genes (lukS-I and

lukF-I), and has shown to be leukotoxic for polymorphonuclear cells but

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Haemolysins. Four hemolysins are described: alpha, beta, delta and gamma. The vast majority of S. aureus cells produce any of these haemo-lysins. Its broad distribution is due, in part, to their location in very stable regions of the chromosomal DNA [55]. The best studied haemolysin is α-haemolysin; it has dermonecrotic and neurotoxic activity [22], being lethal for a variety of eucariotic cells of different animal species. Susceptibility to this toxin depends on the animal species (rabbit cells are 1000 times more susceptible than human cells) and the cell type (erythrocytes are more sus-ceptible than fibroblasts). The β-haemolysin has sphingomyelinase activity with a high affinity for sphingomyelin. It is then active against a variety of cells including erythrocytes, leukocytes, fibroblasts and macrophages [85]. The δ-haemolysin acts as a surfactant that alters the cell membranes by a detergent-like action. It has a broad spectrum of cytolytic activity, affecting erythrocytes and other mammalian cells, and also to various subcellular structures protoplasts, spheroplasts and lysosomes [119]. The γ-haemolysin lyses red blood cells and other mammalian cells, and is also a bi-component toxin [55].

S. pseudintermedius produces α-hemolysin and β-hemolysin and causes

hemolysis of rabbit erythrocytes and hot-cold hemolysis of sheep eryth-rocytes [4].

Exfoliatins. There are three exfoliative toxins or epidermolytic toxins first detected in humans, EtA, EtB and EtD and other of animal origin EtC, first described in horses [2, 107]. The exfoliatins are proteases which act by cutting peptide bonds between the extracellular domains of desmoglein [89], a transmembrane protein that forms part of desmosomes that bind the epithelial cells. These toxins are responsible for the staphylococcal scalded skin syndrome disease that usually affects children and is characterized by loss of superficial layers of skin, dehydration and secondary infections by other microorganisms.

Exfoliative toxin targets a cell–cell adhesion molecule in canine epidermis and might be involved in a broad spectrum of canine pyoderma. PCR analysis revealed that the orf gene, which encodes an exfoliative toxin, was present in 23.2% of S. pseudintermedius strains from dogs with super-ficial pyoderma exhibiting various clinical phenotypes, while the occurrence in S. pseudintermedius strains from healthy dogs was 6.1% [53].

Pyrogenic toxin super antigens (PTSAgs). The bacterial PTSAgs are exocellular proteins with the ability to stimulate non-specifically a large number of T cells in the host, resulting in cytokine production to toxic levels [91]. PTSAgs enclose the Toxic Shock Syndrome Toxin (TSST) and 18 staphylococcal enterotoxins.

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The TSST, causative agent of the Toxic Shock Syndrome (TSS), pro-duces high fever, headache, disorientation, vomiting, diarrhea and rashes. New animal variants were described subsequently, the ovine TSST and bovine TSST [64]. Eighteen staphylococcal enterotoxins have been de-scribed to date in addition to some variants of SEC, SEG, SEH and SEI. These toxins are heat stable and resistant to the digestive enzymes respon-sible for food poisoning caused by S. aureus. Other diseases that have been associated with this type of PTSAgs include arthritis, atopic dermatitis, in-flammatory bowel disease, collagen vascular disease, Kawasaki disease and autoimmune disease [63].

The S. pseudintermedius enterotoxin genes SEC_canineand SE-INT have been described to cause pyoderma, and chronic otitis [34]. The presence of the aforementioned S. aureus enterotoxins genes in S. pseudintermedius isolates is rare.

Staphyloxanthin. A yellowish-orange carotenoid pigment, is one of the important virulence factors of S. aureus [69]. Lennette et al. reported that 90% of S. aureus strains from human infections are pigmented [67]. The carotenoid pigment of the pathogen provides integrity to its cell membrane [80]. Staphyloxanthin has been associated with enhancing bacterial survival in harsh environments and during infections [36, 54]. The membrane pig-ment promotes resistance to reactive oxygen species such as O2 2, H2O2 and

HOCl generated by host neutrophils [65]. Loss of pigmentation translates to a significant decrease in S. aureus virulence in murine skin abscess or systemic infection models [69].

1.7. Identification of staphylococci 1.7.1. Phenotypic identification

In order to determine the prevalence of staphylococci, samples are taken from the body sites, where bacteria usually colonize: oral or nasal cavity, rectum and skin. In the case of disease, samples are taken from an affected site(s): for example, from the ear canal, damaged skin by dermatitis, abscesses, mastitic milk, etc. Clinical substances are commonly taken and put in to special sterile transport media or sterile containers, following the basic principles of aseptic sampling techniques. It is important to collect specimens as aseptically as possible, otherwise the relevant pathogen may be overgrown by numerous contaminating bacteria. The laboratory should be informed if treatment has commenced [76].

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Isolation of coagulase-positive staphylococci on Phenol Red Mannitol Agar supplemented with 7.5% NaCl was studied by Chapman [16]. The resulting Mannitol Salt Agar Base is recommended for the isolation of coagulase-positive staphylococci from cosmetics, milk, food and other spe-cimens [50, 84, 105]. MSA (Mannitol salt agar) contains an additional indi-cator for monitoring mannitol fermentation, which makes it a differential media also. Of the bacteria which can grow in the presence of high NaCl, some are halophilic (requiring a certain concentration of salt to grow) while other are haloduric (do not use the salt, but can tolerate it). Staphylococcus spp. is not halophilic, but rather haloduric, in that it can live in or endure high NaCl concentrations. The high salt content in MSA inhibits other common skin microorganisms, therefore, this medium is used to isolate S.

aureus and S. pseudintermedius [100].

Colonies of S. pseudintermedius on sheep blood agar are non-pigmented and surrounded by double zone hemolysis. The outer band, which is incompletely hemolytic, turns into complete hemolysis after being put at +4°C (hot–cold hemolysis), and is typical of staphylococcal β-hemolysin (a sphingomyelinase) [21]. On blood agar S. aureus appear as glistening, smooth, entire, raised, translucent colonies that often have a golden pig-ment. The colonies are 2–3 mm in diameter after 24 h incubation and most strains show β-hemolysis surrounding the colonies. Microscopically, staphylococci form characteristic “grape like clusters” [129].

The catalase test can be used to differentiate staphylococci from other Gram-positive cocci. A positive catalase test results from the presence of cytochrome oxidase enzymes found in staphylococci and micrococci, but not streptococci or enterococci [129].

The ability to clot plasma continues to be the most widely used and ac-cepted criterion for the identification of pathogenic staphylococci associated with acute infections [84]. Slide and tube coagulase tests are used to detect if investigative staphylococci produce bound and free coagulase. A sus-pension of staphylococci is mixed with rabbit plasma either on a slide or in a small tube. The fibrinogen in rabbit plasma is converted to fibrin by coagulase. The slide test detects the presence of a bound coagulase or clumping factor on the bacterial surface. A positive reaction is indicated by clumping of bacteria within 1 to 2 minutes. The tube test detects both free coagulase (staphylocoagulase), which is secreted by the bacteria into the plasma, and bound coagulase. It is the definite test for coagulase production and a positive reaction is indicated by clot formation in the tube following incubation at +37°C for 24 h [97].

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Commercially available kits, which detect capsular polysaccharides and cell wall components including clumping factor and protein A, are useful for the presumptive identification of S. aureus [97].

Coagulase-positive staphylococci can be distinguished by the sensitivity of polymyxin B. S. aureus is characterized by polymyxin B resistance, defined as a Kirby-Bauer zone of inhibition less than 10 mm using a 30 μg Genotypic identification disk. Species of the Staphylococcus intermedius group are polymorphic B-sensitive [129].

The production of acetoin is determined by the Voges-Proskauer test, which helps to distinguish S. aureus from S. pseudintermedius, since S.

aureus produces acetoin and the species of SIG do not [106, 118].

DNase production are characteristic for coagulase-positive staphylococci can be detected with DNase solid agar [129]. S. aureus and S.

pseudin-termedius are DNase positive, distinguishing them from coagulase-negative

species [118].

Bacterial species differ in their ability to ferment carbohydrates, and some sugar fermentation is the most commonly defined biochemical charac-teristic. Adding to minimal media as the only source of carbohydrates, fermentation results in acidification, which can be detected colorfully by the pH indicator [129].

Although S. aureus can be easily identified by biochemical methods, S.

intermedius group of species (S. intermedius, S. pseudintermedius and S. delphi) cannot be separated biochemically. The biochemical identification

of S. intermedius group species, generally with knowledge about the host, allows us to determine the variety of isolated species. For example, SIG isolated from dogs are considered to be S. pseudintermedius. Molecular studies are needed to accurately differentiate species of S. intermedius group [3, 21, 106, 124].

In routine diagnostic bacteriology, S. aureus has long been differentiated from other species by means of a single test, most often coagulase or clumping-factor production. However, S. pseudintermedius and other patho-genic staphylococci are also coagulase-positive. Extensive phenotypic testing or molecular identiﬁcation methods are needed in order to identify these strains adequately [21]. Sasaki and others devised a multiplex-PCR (M-PCR) method for species identification of coagulase-positive staphylo-cocci by targeting the nuc gene locus [106].

PCR is a highly sensitive technique that is more labor and cost efficient than16srRNA sequencing. It is more accurate as biochemical tests as a means of identification as biochemical testing often requires interpretations of color change, and may also be subject to intraspecies variability. PCR, however, is dichotomous, providing either a positive or negative result. PCR

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technique involves strategic targeting and amplification segments of genes chosen for high fidelity and species specificity. This allows for design of unique, complementary DNA primers that anneal to DNA flanking the DNA sequence (amplicon) of interest. PCR requires annealing of the primer to the sample (template) DNA, and extension of the complementary DNA [110].

The heat stable enzyme, Taq polymerase, is necessary to synthesize the amplicons during the extension phase in the presence of free nucleotides. PCR is performed in a thermocycler unit, that repeatedly providing tempe-rature cycles optimal for the three phases of PCR: 1) denaturation of DNA, 2) primer annealing to the DNA, 3) extension to produce amplicons of unique length. When repeated, this ultimately results in exponential produc-tion of millions of amplicons. Amplicons are resolved by gel electropho-resis, with size dependent migration distance through the gel, producing species predictive patterns and identifiable fragment sizes [110].

Heat-stable nuclease, thermonuclease, is encoded by the nuc gene. Am-plification of the nuc gene has proven highly sensitive and specific in multiplex PCR assays in the rapid identification of human MRSA isolates, coupled with amplification of mecA [18] S. pseudintermedius from the dog, cat, camel and human have been successfully identified with nuc PCR as well [6].

1.7.2. DNA sequencing

DNA sequencing is the process of determining the sequence of nuc-leotide bases (As, Ts, Cs, and Gs) in a piece of DNA. Today, with the right equipment and materials, sequencing a short piece of DNA is relatively straightforward.

Sanger sequencing, also known as the chain termination method, is one well-established method. This technique is for DNA sequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication [39, 58].

Classical Sanger sequencing requires a single-stranded DNA template, a DNA polymerase, a DNA primer, normal deoxynucleoside triphosphates (dNTPs), and modified nucleotides (ddNTPs) that terminate DNA strand elongation. These ddNTPs lack a 3′-OH group that is required for the formation of a phosphodiester bond between two nucleotides, causing the extension of the DNA strand to stop when a ddNTP is added. The DNA sample is divided into four separate sequencing reactions, containing all four of the standard dNTPs (dATP, dGTP, dCTP, and dTTP), the DNA polymerase, and only one of the four ddNTPs (ddATP, ddGTP, ddCTP, or

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ddTTP) for each reaction. After rounds of template DNA extension, the DNA fragments that are formed are denatured and separated by size using gel electrophoresis, with each of the four reactions in one of four separated lanes. The DNA bands can then be visualized by UV light or auto-radio-graphy, and the DNA sequence can be directly read off the gel image or the X-ray film. The ddNTPs may also be radioactively or fluorescently labeled for detection in automated sequencing machines. The four reactions can be incorporated into one reaction run, and the DNA sequence can be read from radioactive or fluorescent labels [131].

Methodology. Sanger sequencing is a targeted sequencing technique that uses oligonucleotide primers to seek out specific DNA regions. Sanger sequencing begins with denaturation of the double-stranded DNA. The single-stranded DNA is then annealed to oligonucleotide primers and elon-gated using a mixture of deoxynucleotide triphosphates (dNTPs), which provide the needed arginine (A), cytosine (C), tyrosine (T), and guanine (G) nucleotides to build the new double-stranded structure. In addition, a small quantity of chain-terminating dideoxynucleotide triphosphates (ddNTPs) for each nucleotide is included. The sequence will continue to extend with dNTPs until a ddNTP attaches. As the dNTPs and ddNTPs have an equal chance of attaching to the sequence, each sequence will terminate at varying lengths.

Each ddNTP (ddATP, ddGTP, ddCTP, ddTTP) also includes a fluores-cent marker. When a ddNTP is attached to the elongating sequence, the base will fluoresce based on the associated nucleotide. By convention, A is in-dicated by green fluorescence, T by red, G by black, and C by blue. A laser within the automated machine used to read the sequence detects a fluo-rescent intensity that is translated into a “peak.” When a heterozygous va-riant occurs within a sequence, loci will be captured by two fluorescent dyes of equal intensity. When a homozygous variant is present, the expected fluorescent color is replaced completely by the new base pair’s color.

Benefits. Sanger sequencing is a robust testing strategy able to determine whether a point mutation or small deletion/duplication is present. It has been widely used for several decades in many settings, including defining the mutational spectrum of a tumor as well as identifying a constitutional va-riant in diagnostic testing. Primers can be created to cover several regions (amplicons) to cover any size region of interest [39].

Limitations. Sanger sequencing gives high-quality sequence for relati-vely long stretches of DNA (up to about 900 base pairs). It’s typically used to sequence individual pieces of DNA, such as bacterial plasmids or DNA copied in PCR. However, Sanger sequencing is expensive and inefficient for larger-scale projects, such as the sequencing of an entire genome or

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meta-genome (the “collective meta-genome” of a microbial community). For tasks such as these, new, large-scale sequencing techniques are faster and less expen-sive [58].

1.8. Resistance to antimicrobials

The discovery of antibiotics was one of the most important milestones in the history not only of medicine but also of humanity, given that they drastically reduced mortality rates during the first years of its introduction in the clinic, in the early 1940s. However, soon after its introduction, bacteria resistant to those antimicrobials began to evolve and spread. These micro-organisms began to disseminate in hospitals; however, the hospital environment is not an isolated ecological niche but an atmosphere exposed to constant flow of genetic exchange with the environment. Thus, resistant bacteria were gradually appearing and spreading within the community, in animals and even in food [29].

The discovery and development of new families of antibiotics was very quick in the first decades after the discovery of penicillin, but this rate has stopped, and very few molecules with new activities or new families of antibiotics have been incorporated to the arsenal in recent decades. Worry-ingly, the deployment of any novel antibiotic has been followed by the evolution of clinically significant resistance to that antibiotic in as little as a few years [17].

The European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) proposed that the isolates were multidrug-resistant (MDR) if they were resistant to at least one antimicrobial in three or more antimicrobial classes. However, it should be emphasized that not only methicillin-resistant staphylococcal strains may be MDR [75].

During the last decade, various MDR bacteria such MRSA and MRSP have spread among dogs and cats on a worldwide basis [83]. Hospitalization and antimicrobial treatment, especially with broad-spectrum drugs such as cephalosporins and fluoroquinolones, are major risk factors associated with carriage and infection with MDR bacteria in animals [124]. The prevalence of MDR bacteria in the pet population varies considerably between countries. The reason for this geographical variation is unclear, but it is likely related to local variations in patterns of antimicrobial use [121].

β-lactams are bactericidal agents that inhibit the synthesis of the bacterial cell wall by blocking the final step of peptidoglycan synthesis (transpep-tidation). These antimicrobials covalently associate to PBPs (penicillin-binding proteins), which are the enzymes (transpeptidases, transglucosilases

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and carboxypeptidases) involved in binding the different peptidoglycan components.

In the 1940s, penicillin was introduced for the treatment of staphylo-coccal infections and only a year later S. aureus strains resistant to penicillin began to appear. Penicillin resistance is mediated by the blaZ gene, with β-lactam ring of penicillin’s by hydrolytic cleavage.

Due to the emergence of these enzymes, new antimicrobials were sought to treat infections caused by S. aureus, including methicillin, a semi-synthe-tic penicillin that is able to ihibate β-lactamases. In 1961, two years after the introduction of this drug, MRSA emerged. Since then, a widespread distri-bution of these strains occurred in many hospitals, and MRSA is nowadays one of the main pathogens that cause nosocomial infections worldwide [104].

Mechanisms of Antibiotic Resistance. Antimicrobial resistance limits the ability of clinicians to select appropriate antimicrobials for the treatment of bacterial infections. Resistance can be caused by a variety of mecha-nisms: the presence of an enzyme that inactivates the antimicrobial agent; the presence of an alternative enzyme for the enzyme that is inhibited by the antimicrobial agent; a mutation in the antimicrobial agent’s target, which reduces the binding of the antimicrobial agent; transcriptional or post-translational modification of the antimicrobial agent’s target, which reduces binding of the antimicrobial agent; reduced uptake of the antimicrobial agent; active efflux of the antimicrobial agent; and over production of the target of the antimicrobial agent [30].

Mechanisms of methicillin resistance. Methicillin is a β-lactam anti-microbial which binds penicillin-binding proteins in the cell envelope and prevents cross-linking of the peptidoglycan chains in the cell wall. S. aureus renders methicillin ineffective by the production of an alternative PBP (PBP2a), which has reduced affinity for β-lactams [117]. Molecular targets for MRSA detection resistance to β-lactam antibiotics is due to acquisition of the exogenous gene, mecA that is incorporated into a large segment of DNA called Staphylococcal Chromosomal Cassette (SCC) mec that was first described by Katayama and co-workers in 2000 and which encodes for the penicillin-binding protein 2a (PBP2a) [57].

1.8.1. Antimicrobial resistance of Staphylococcus pseudintermedius

Among S. pseudintermedius, resistance is emerging to the β-lactams, with resistance to penicillin reported in over 70% of S. pseudintermedius isolates colonizing healthy dogs and ~95% of clinical isolates [5, 56, 132]. Researchers found that 15–20% S. pseudintermedius strains were resistant

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to tetracycline. Resistance to trimethoprim, sulfamethoxazole, chloram-phenicol, and gentamicin was less common [5, 96].

A recent review found that between 1980 and 2013 there was increasing resistance among methicillin susceptible S. pseudintermedius (MSSP) not only to penicillin and ampicillin but also to other classes antimicrobials including the fluoroquinolones, aminoglycosides, and chloramphenicol [81].

From 2004 through 2013, the incidence of canine MRSP infections in-creased sevenfold at a veterinary diagnostic lab in Utrecht, the Netherlands [24]. Colonization of 0–30% of healthy dogs with MRSP has been reported, and this bacteria has also been isolated from cats and people [89, 94–97].

Priyantha et al. reported that 6.8% isolates of S. pseudintermedius from dogs were MDR, higher than in 2008 where only 1 dog carried MDR S.

pseudintermedius. Other studies have reported that among 3–27.5% were

MDR [20, 35]. In contrast to the literature, MDR was more frequently iden-tified among MSSP than MRSP. The most common resistance profile among MRSP was simply β-lactam resistance. MDR among MRSP is a serious threat to the ability of veterinarians to treat their patients [96].

In 2009, a community associated urinary tract infection caused by MRSP resistant to the β-lactams, macrolides, fluoroquinolones, aminoglycosides, trimethoprimsulfamethoxazole, chloramphenicol and rifampin was reported in an otherwise healthy, neutered male Pug dog [96].

Methicillin-resistant S. pseudintermedius (MRSP) has recently appeared to be a major problem in veterinary medicine, which can also have impli-cations for human health, since the genes encoding resistance to antimic-robials are highly mobile and can be transmitted between different staphylo-coccal species including S. aureus [101, 126]. According to the literature sources, the transfer of S. pseudintermedius between human and zoonotic hosts is possible, there are several reports of serious MRSP-infections in humans [108, 111]. The situation is even more complicated, because the precise identification of S. pseudintermedius requires genetic methods that are rarely used in routine diagnosis [15, 108]. It should be noted that this specie of bacteria until the year 2005 was identified and called as S.

inter-medius or even S. aureus [106]. Consequently, the importance of this

bacte-rium as a pathogen appears to be underestimated [124].

Exposure to fluoroquinolones and β-lactams in the past and long-term treatment with β-lactam drugs was identified as a risk factor for MRSP infection [134]. Another study found that prior hospitalization and antibiotic exposure within the previous 6 months were risk factors [88]. The risk factors for colonization of MRSP in dogs have been studied to a limited extent; positive correlations between the number of veterinary visits, hos-pitalization, administration of glucocorticoids and topical antimicrobials, or

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being a breeding female were found [44, 66]. In addition, Lloyd highlighted close contact with carriers or infected animals, ongoing invasive infections or surgical procedures as risk factors for MRSP colonization [70].

1.8.2. Antimicrobial resistance of Staphylococcus aureus

In a study performed in the Greater London area among healthy and veterinary-treated dogs, cats, and horses, it was shown that healthy animals carried MRSA in a percentage of 0.66%, 0.46%, and 0%, respectively, whereas the prevalence was higher among treated animals (3.23%, 2.16%, and 1.97%) [73].

Kottler et al. screened healthy pets for MRSA colonization, whereas their households were assigned into three groups: veterinary personnel, health care workers, and those without any contact with the health care system. No differences were found in MRSA carriage among the human groups (MRSA in total 5.63%), whereas MRSA prevalence among pets was 3.41% [61].

Higher MRSA rates were detected in a large study performed in Ger-many during 2010–2012 among dogs, cats, and horses with wound infec-tions. MRSA accounted 3.6%, 5.7%, and 9.4%, respectively. MRSA geno-typing showed that the infection source for dogs and cats is humans, since the identified clones (CC22 and CC5) cause wound infections predomi-nately in the country [120, 122].

Morris et al., in USA, studying household contacts and their pets (dogs and cats) previously diagnosed with SSTIs, found that 11.6% of the pets were MRSA-positive [82].

Nasal staphylococcal colonization was performed in a veterinary clinic in Rio de Janeiro among 130 companion animals. Only one cat was found MRSA positive. The strain was classified as a PVL-negative ST30-SCCmecIV carrying enterotoxins and phenol-soluble modulins α3 genes, is already characterized as the human clone causing SSTIs [98].

Studies performed in different countries have shown that among com-panion animals in close contact with humans, cats and dogs carry MRSA lineages of human origin, whereas horses and pets living in farms carry MRSA of animal origin [92].

Boost and others have found that S. aureus is the most resistant to pe-nicillin (82%), erythromycin (25%) and tetracycline (17%). Staphylococcal strains isolated from the dog were most resistant to penicillin (62%), tetracycline (29%), and fusidic acid (22%). Canine S. aureus isolates tended to be more resistant than human isolates, with significant differences in frequency of resistance to several antibiotics [14].

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1.8.3. Antimicrobial susceptibility testing

The performance of antimicrobial susceptibility testing by the clinical microbiology laboratory is important to confirm susceptibility to chosen empirical antimicrobial agents, or to detect resistance in individual bacterial isolates [99]. A number of laboratory methods have been developed to eva-luate an antibiotic against selected microbes in vitro. Antimicrobial suscep-tibility testing follows a meticulously standardized protocol in the current diagnostic laboratory [37].

Standardized methods were initially introduced by Bauer et al. in 1966, and have subsequently developed into our modern methods [78]. Standar-dization of protocols is critical to ensure that results are reproducible and reliable, two main international standards are recognized: The Clinical and Laboratory Standards Institute (CLSI), and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (CLSI, 2016; EUCAST, 2017).

Disk diffusion method – Kirby-Bauer technique. Disk diffusion is one of the oldest approaches to antimicrobial susceptibility testing and remains one of the most widely used antimicrobial susceptibility testing methods in routine clinical laboratories. It is suitable for testing the majority of bacterial pathogens, including the more common fastidious bacteria, is versatile in the range of antimicrobial agents that can be tested and requires no special equipment (EUCAST, 2017).

The test is performed by applying a bacterial inoculum of approximately (1÷2)×108 CFU/mL to the surface of a large (150 mm diameter)

Mueller-Hinton agar plate. Up to 12 commercially-prepared, fixed concentration paper antimicrobial disks are placed on the inoculated agar surface Plates are incubated for 16–24 h at 35°C prior to determination of results. The zones of growth inhibition around each of the antibiotic disks are measured to the nearest millimeter. The diameter of the zone is related to the suscep-tibility of the isolate and to the diffusion rate of the drug through the agar medium. The zone diameters of each drug are interpreted using the criteria published by the EUCAST, CLSI (formerly the NCCLS).

The advantages of the disk method are the test simplicity that does not require any special equipment, the provision of categorical results easily interpreted by all clinicians, and flexibility in selection of disks for testing. It is the least costly of all susceptibility methods (approximately 2–4 EUR per test). The disadvantages of the disk test are the lack of mechanization or automation. Although not all fastidious or slow growing bacteria can be accurately tested by this method, the disk test has been standardized for testing streptococci, Haemophilus influenzae, and N. meningitidis through

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use of specialized media, incubation conditions, and specific zone size in-terpretive criteria [99].

Broth dilution tests. Dilution method for determining the minimal inhi-bitory concentration (MIC) and minimum bactericidal concentration (MBC) of an antimicrobial material for a test bacterium.

One of the earliest antimicrobial susceptibility testing methods was the macro-broth or tube-dilution method. This procedure involved preparing two-fold dilutions of antibiotics (for example: 1, 2, 4, 8, and 16 mg/mL) in a liquid growth medium dispensed in test tubes. The antibiotic-containing tubes are inoculated with a standardized bacterial suspension of (1÷5)×105 CFU/mL. Following overnight incubation at 35°C, the tubes were examined for visible bacterial growth as evidenced by turbidity. The lowest concen-tration of antibiotic that prevented growth represents the minimal inhibitory concentration (MIC). The precision of this method was considered to be plus or minus 1 two-fold concentration, due in large part to the practice of manually preparing serial dilutions of the antibiotics. MBC is determined from the broth dilution of MIC tests by sub-culturing to agar plates that do not contain the test agent. The MBC is identified by determining the lowest concentration of antibacterial agent that reduces the viability of the initial bacterial inoculum by a pre-determined reduction such as ≥99.9%. The MBC is complementary to the MIC; whereas the MIC test demonstrates the lowest level of antimicrobial agent that greatly inhibits growth, the MBC demonstrates the lowest level of antimicrobial agent resulting in microbial death.

The advantage of this technique was the generation of a quantitative result (the MIC). The principal disadvantages of the macro-dilution method were the tedious, manual task of preparing the antimicrobial solutions for each test, the possibility of errors in preparation of the antibiotic solutions, and the relatively large amount of reagents and space required for each test [99].

Antimicrobial gradient method. The antimicrobial gradient diffusion method uses the principle of establishment of an antimicrobial concentration gradient in an agar medium as a means of determining susceptibility. The E-test is a commercial version. It employs thin plastic E-test strips that are impregnated on the underside with a dried antimicrobial concentration gradient and are marked on the upper surface with a concentration scale. As many as 5 or 6 strips may be placed in a radial fashion on the surface of an appropriate 150-mm agar plate that has been inoculated with a standardized organism suspension like that used for a disk diffusion test. After over-night incubation, the tests are read by viewing the strips from the top of the plate.

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The MIC is determined by the intersection of the lower part of the ellipse shaped growth inhibition area with the test strip [99].

E-test strips cost approximately 1.6–2.5 EUR each and can represent an expensive approach if more than a few drugs are tested. This method is best suited to situations in which an MIC for only 1 or 2 drugs is needed or when a fastidious organism requiring enriched medium or special incubation atmosphere is to be tested. Generally, E-test results have correlated well with MICs generated by broth or agar dilution methods. However, there are some systematic biases toward higher or lower MICs determined by the E-test when testing certain organism-antimicrobial agent combinations. This can represent a potential shortcoming when standard MIC interpretive criteria derived from broth dilution testing are applied to E-test MICs that may not be identical.

Automated instrument systems. Use of instrumentation can standardize the reading of end points and often produce susceptibility test results in a shorter period than manual readings because sensitive optical detection systems allow detection of subtle changes in bacterial growth [99].

1.9. Interspecies transmission of staphylococci

There is increasing concern about the rapid emergence and spread of MDR bacteria among household pets in recent years. Various genetic similarities have been observed between MDR isolates from human infec-tions and from household pets. This implicates a zoonotic risk, which is further supported by recent studies indicating contact with pets as a risk factor for human infections with resistant bacteria, and by several case reports suggesting household transmission of resistant strains between pets and their owners [19].

Zoonotic transmission from infected or colonized pets to people can occur by direct contact or indirectly through environmental contamination of households, veterinary clinics and public spaces. It should be noted that human-to-pet transmission may also occur. The risk that pets acquire MRSA from people is particularly high, since the MRSA types found in dogs and cats often correspond to widespread clones in the local human population [121].

Significant public health concerns exist because of the possible risk of animal-to-human transmission of resistant clones and/or resistance genes. MRSA colonization (and perhaps infection) is a recognized occupational risk in veterinary staff and various studies have identified the same MRSA strains in people and pets sharing the same household [126]. Although the most common MRSA clones infecting or colonizing pets (e.g. ST22)

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occurred in people a long time before their emergence in pets, and are likely to originate from man, pets may serve as infection sources for MRSA infection or (re)colonization of human patients [72]. Considering that S.

pseudintermedius has a canine origin and is not a commensal in people, the

relatively high MRSP carriage rates (up to 8%) among owners of infected dogs and veterinary personnel provide indirect evidence of zoonotic transmission [123]. MRSP infections have been reported in dog owners and their frequency may be underestimated due to diagnostic problems regard-ing identification of S. pseudintermedius, and consequently MRSP, in hu-man clinical microbiology laboratories [93]. The occurrence of MDR bacteria in household pets has induced veterinary use of critically important antimicrobials authorized for human use only (e.g. carbapenems and gly-copeptides) [126]; which may further aggravate the problem. In addition to the risks of zoonotic transmission, untreatable MDR infections in household pets have negative emotional and social effects on the owners and their families [8].

Prevention. Considering that hospitalization and antimicrobial treatment are the main risk factors for colonization and infection with MDR bacteria, hospital infection control and rational antimicrobial use are essential mea-sures to prevent further spread of MDR bacteria in household pets and, ultimately, to reduce the risk of zoonotic transmission to people. Veteri-narians play an important role in educating the owners of patients infected with MDR bacteria to follow best hygiene practices for prevention of zoo-notic transmission. Both veterinarians and physicians should raise aware-ness about the risks of zoonotic infection, especially among risk groups (i.e. young, old, pregnant and immunocompromised people).

Veterinary use of clinical important antimicrobials licensed for human use only must be reduced to an absolute minimum and regulated by legis-lation. Use of broad-spectrum antimicrobials licensed for veterinary use (e.g. cephalosporins and fluoroquinolones) should be controlled by imple-mentation of antimicrobial stewardship programs at both the national and the clinic level [45]. Development of new narrow-spectrum, veterinary-specific antimicrobial products, including anti-infective biological agents such as phage and bacteriocins, is urgently needed for treatment of MDR infections in household pets [19].

CHARACTERISTICS AND RESISTANCE TO ANTIMICROBIALS OF STAPHYLOCOCCUS AUREUS AND STAPHYLOCOCCUS PSEUDINTERMEDIUS ISOLATED FROM PET ANIMALS AND THEIR OWNERS

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

VETERINARY ACADEMY

Jūratė Stankevičienė

CHARACTERISTICS AND RESISTANCE

TO ANTIMICROBIALS

OF STAPHYLOCOCCUS AUREUS AND

STAPHYLOCOCCUS PSEUDINTERMEDIUS

ISOLATED FROM PET ANIMALS AND

THEIR OWNERS

LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS

VETERINARIJOS AKADEMIJA

Jūratė Stankevičienė

STAPHYLOCOCCUS AUREUS IR

STAPHYLOCOCCUS PSEUDINTERMEDIUS,

IŠSKIRTŲ IŠ GYVŪNŲ AUGINTINIŲ IR

JŲ SAVININKŲ, CHARAKTERIZAVIMAS

IR ATSPARUMAS ANTIMIKROBINĖMS

MEDŽIAGOMS

CONTENTS

ABBREVIATIONS

INTRODUCTION

1. LITERATURE REVIEW