A. Ü.Veteriner Fakültesi Bakteriyoloji Vt Salgınlar Kürsüsü Prof. Dr. Hasan Başka]a
ST APHYLOCOCCt tN AMMALS. CHARACTERİsTtCS, DtST.
RtBUTtON AND tTS PUBLtC HEALTH StGNtFtCANCE.
Hanıza Keskintepe*
Hayvan Staphylococ'Iarmm özellikleri, Dağılınıı ve Halk Sağlığı yönünde Önenıi
SunılUary: 133 staphyloeoeei isolated from various specimcm of different speeies of domestic animals wc re studied from the stand point of their properties and ditribution. İn addition, a brief diseussion of the publie health signifieanee of staphyloeoeeie infeetion whieh has deınonstrated zoonotie assoeİation wa~ presented.
Majority of the isolates weee recovered from pus abeesses from horse, eattle, dog and eat. Limited ııumber of staphyloeoeei were obtanied from samples of vaginal diseharge, syno\'ial f1l1id and milk. All of these speeimens were reeeived from the clinies of Veterinary Faeulty. '27 strains out of 133 staphyloeoeei were isolated from livers. heart blood, kidney aııd affeeted joints of ekieken's.
More strains of Staphyloeoeeus aureus were recovered than of Staphyloeoeus albus from these speeimens. İt was found that Si isolates produeed golden-eoloured, Alpha or Bet,ı hamol)'tie eolonies on sheef blood agar and eoagulated rabbiı plasma. 27 strains also produeed golden-eoloııred eolonies and were eoagulase pasiıive, bul did nol produee hae-molysis. 20 strains pradueed white and Beta-haemolytie eolonies, bul they were eoagulasc-positive. Caagıılase negatif 35 strains produeed white and nonhaemolytie eolanies.
Although fermentatian af mannitol and jelatin ligııefaetion is reeognised as a eharae-ter of pathogenie strains, results of the present study did not serve to distinguish pathogenie strains fram non-pathogenie varieries.
İ t is clear from ıhe results of differenI workers including the author that the eoagulase test is the most reliable eriterion of pathogenieity.
Özet: Çeşitli eveil hayvan türlerine ait marazi maddelerden izole edilen 133 staphy-loeoe suşunun dağılım ve özeııikleri üzerinde çalışıldı. Buna ilaveten zoonotik ilişki gösteren ye halk sağlığı yönünden önemli staphyloeoc enfeksiyonu hususunda kısa bir lartışma su-sunuldu.
Suşların büyük bir çoğunluğu at, sığır, köpek, kedi apse içeriğinden ve belirli adette staphyloeoe suşu ise vagina akıntısı, sinovya sıvısı ve süt numunelerinden izole edildi. Bu
Staphylococd in Animal •. Clıaraeteristies. Distribution And İt •.
marazi maddeler bakteriyolojik muayene için Fakültemiz kliniklerinden 133 staphyloeoe suşundan2Tsi muayene için laboratu"arımıza gönderilen raeiğer, kalp kanı, böbrck veya arızalı ekle",lerindeıı izole edildi.
91
göııderilnıiştir. eivei"lerin
ka-Bu marazi maddeleı-den Staphyloeoeeus albus'tnn daha çok Staphyloeoeeus aureus suşu elde edildi. Si suş koyun kanlı agan üzerinde altın sarısı renginde Alpha-veya Beta hemolitik koloniler oluşturdu ve tavşan plasmasını koagule ettiler. 27 suş ise altın sarısı ko-loni oluşturmalarına ve koagulaz-pozitif olmalarına rağmeıı heınoliz meydana getirmemiş-lerdir. Beyaz ve hemolitik koloni oluşturan 20 suş koagulaz testi ilc pozitif sonuç vermiştir. Koagulaz-negatif olan 3') suşta beyaz ve heInolitik olmayan koloniler halinde üremİşlerdir. :\fannit fermentasyonu ve gelatini eritme patogen suşların bir karakteri olarak tanınırsada, bizim yapmış olduğumuz çalışmalar bu sonuç:ları teyit etmemiştir.
Fakat bizimki dahil bir çok araştırıcılarııı çalışma sonuçları, koagulaz te.>tinİn cn gü-venilir birpatojenite testi olduğunu göstermekH'clir.
İntroduction
Staphylococci are wide spread İn many places in nature and most of them live a purely saprophitic existancc. Some are pathogenic and capable of causing disease in man and animale (6, 23).
Most of the lesions produced by organisms of these species are superficial infiamation with pus formation in all domestic animals. Once pathogenie strains gaİn a foothold in the depper tİssues of the exposed anİma!, theİr multİplication cause neerosİs and abeess for-~atİon. İn some instances, the organİsms may İnvade the lymhatİcs, blood stream and matastatie foeİ usually develop (6, 9).
Staphylococci are as well the causative agcnt of some serİous infec-tİons such as mastitis İn most species of domestic animals; pyaemia in lambs; septicamia and arthrİtis in poultry; aene, furuneulosis and batryomiyeosis in horse and cows; dermatitİs and septieaemia in dog and eat (2, 4,
ı
O,ı
2,ı
4,ı
5, 22, 28).İn man staphylococci are usually found İn pyogenic lesions and infections such as osteomyelitis, renal carbuncle, peri-renal abcess, bronchopneumonia. Some cases pyacmia, septicaemia and malig-nant endocarditİs may result from spread from a primary localised abcesses (9). Cases of food poisoning are frequently due to the ente-rotoxin produced by certain staphylococci growing in cook cd meats, milk and milk-products, fish and gravİes (ı 9, 2ı).
l\.1any strains show a marked degrec of variation to antibiotic and chemotherapentic agents. These antibiotic resistance strains has beconie of a great epidemiolojical and therapeutic impertance (3,
ı
7,92 Hamza Keskintepe
Some zoonotic infections of staphylococci may be maintained in nature either by animals or man and transmission may occur in either or both directions (8, 20, 2
ı,
24).Staphylococci are now classified in separate two species according to several laboratory tests which are based on their biological features. Organisms of this genus are the only bacteria that produce coagulase which causes citrated or oxalatcd plasma to coagulate. Due to this fact, the coagulase test has bcen accepted as an inportant criterion of pathogenicity. Coagulase-positivc pathogenic strains most of which produce Alpha-Beta-or Delta-ha( molysins and golden coloured colo-nies are called Staph. aureus. Coagulase-negative less virulent strains which produce white and non-haemolytic colonics are designated as Staph. epidermidis (I, 6, 13, 16).
The purpose of the present sudy was to deseribe some of the pro-perties and the distribution of the strains isolated from various speci-es ofanimals. İn addition ve discussed the public health significance of staphylococcic infection which has dcmonstrated zoonotic associ-ations and opportunities for rcciprocal transmissions between domes-tic animals and man.
Materials and Methods
A total of 150 specimens receivcd from the clinies were examincd during the period of 4 years. 60 per cent of these samples were pus from abcesses of various specics of domestic animals such as horse (19), Cattlc (21), dog (27), cat (17) and sheep (6). Of the 150 speci-mens 40 percent wc re individual milk samplcs from known cases of mastitis (36), vaginal dischargc (14), synovialf1uid (10).106 strains of staphylococci were isolated from the specimens mentioned above. İn addition, in the course of carryiııg out diagnostic examination of polutry, 27 strains wcre recovered trom livers, heart blood, kidneys and affected .ioints.
İsolation
The media use d for isolation and su bculture of staphylococci was sheep blood agar plates. This medium contained 2percent trypto-se (Difco),
o.
i percent glucose, 0.5 percent sodium chloride and 2 percent agar (pH 7.0); sheep blood was added to 5 percent. All spe-cimens were streaked upon this medium which was then inculated at 37 oC. for overnight and Ieft at room temperature for an additional 24 hours to observe any further develepment of pigment. Coloniesresembling those of staphylococci were studied microscopicaHy for the characteristic Gram reaction and grapclike formation of cocci. Gram staining was conducted according to standard procedure. The-se typical isolates regardlcss of pigmentation were inoeulated into nutrient broth for carring oııt of their biological features.
Coagulasc prduction test
Citrated 0,5 ml of rabbit plasma, prcviously dilutee! ı:4 with saline was uscd for the tube test. Five drops of an overnight broth cu 1-ture were added to this plasma. The tubes were incubated in a wa-terbath at 37 oC. for one hour and at intervals up to 4 hours. İf the strains did not show coagulation at the end of this period, the tu bes were kept in incubator for overnight and final reading was then made.
Haemolysin production
The basic medium used for this purpose was the same as they emp-loyed for prcliminary isolation. An overnight broth culture of a colony from a strain was spread over a sheep erythrocyte agar plate and in-eubated at 37 oC. for 24 hours. Eaclı colon)' was inspected for a sur-raunding haemolysin effect.
Biochemical reactions
Glucose, laetose, maltose, manntitol, saliein, xylosc and arabi-nose were used. Readings were made af ter incubation for 3 days at 37 oC.
Gelatin liguefaction
Tubes of nutrient gelatin were stab inoculated, and incubated at 37 cC. for 6 days. The tubes were kcpt for 20 minutes at 4 oC. before reading eaeh day.
Results and mscussion
İn the eourse of about more than 4 year, studies were condueted on
ı
33 strains of staphylocoeei which were isolated from various speci-mens of domestic animals. 5ı
isolates produced golden-coloured, Alpha or Beta-haemolytic colonies on 5 per een t sheep blood agar and were coagulase-positive in tube test using rabbit plasma. 27 strains also produced golden-eoloured colonies and were eoagulase- positive, but did not produce haemlysis. 20 coagulasc-positive strains ofstaphy-loeocci formed white and Beta haemolytic colonies on primary iso-lation. 35 strains produced white, non-haemolytic colonies and were eoagulase-negative. The amount of haemolysin produced was judged9ı Hamza Keskintepe
by the diameter of the zone around the eolony. All of the strains iso-lated from milk samples produced Beta-haemolysin. Strains from po-uItry speeimens produeed Alfa-and Beta-haemolysin. Isolates from pus of abeesses, vaginal discharge and syno"Vial fluid showed wide Alpha haemolysis, and little Beta-haemolysis.
All coagulase-positive strains were the same in that they fermen-ted glucose and mannitol. i8 of the eoagulase-ncgative strains exa-mined were active biochemically. These strains formed acid from glu-eose, laetose, salicin and xylose. Mannitol was also fermented by 5 of these strains.
İsolates obtanicd from speeimens of dogs and eats differed from ether animal strains in that they did not form a golden pigment.
Strains of animal origin have been investigated by a number of investigators employeng tests such as eoagulase produetion, forma-tion of toxin, pathogenieity for laboratory animals and fermantation reaetions (I, 1I, 12, i3, 16, 30). The sensitivity of staphyloeoeei to Iysis by various baeteriophages has been aeeepted as a method of studying the epidemiology of staphylococeal infeetions in man and animals (5, 8, 27, 29). Most of the workers (6, 3, 18, 19) has eriticised the aeeeptanee of host pathogenieity as a eriterion for Staph. aureus. İn the peresent study, therefore, eertain laboratory tests were employed to classify staphyloeoeei as pathogenie or not. The limited number of fermentable substanees employed, as well as the property of gelatin liqucfaetion and haemolysis produetion did not serve to distinguish the pathogenie strains from non-potho'genic ones. Likewise, produetion of golden yellow pigmented eolonies wc re not an adequate indicator of pathogenieity. Because eoagulase-po-sitivestrains devoid of golden pigment wc re frequently eneountered, even on primary isolation. The property that corrolates best with pat-hogenicity was the claboration of eoagulase. All eoagulase pozitive strains from clinical cases, therefore were designated a as Staph. aureus.
Koenig et. aL. (I 6) have reported some inportant faetors that influenee the virulcnee of Staph. aureus as follows: antiphagoetic surface components of the strains; the produetion alpha-toxin whieh may promote neerosis, interfere with infiamation and injure leucocy-tes; the produetion of delayed hypersensitivity which enehances tis-suc necrosis and increases the susceptibility to infection. Despite the combined effects of these several factors mentioned above, the over all virulence of Staph. aureus for animal and laboratory animals is
Staphylococci in Animals. Charactcristics, Distribution And İts... 95
comparatively low. ScriOliSstaphylococcal disease occurs only when the local or the general antibacterial defences of the host have been depressed (26).
A great deal of ressearch has resulted, some of which has demonst-rated zoonotic associations and opportunities for reciprocal trans-mission bctween domestic animals and man. Smith and Crabh (25) reported, for example, that antibiotic-resistant staphylococci were much more prevalent among human attendants of swinc which were fed antibiotic supplements than among attendants of swine not fed antibiotics.
Meat-proceccing plants provide particularly favorahle oppor-tunities for interspecific transmission to occur. Rabenholt et. aL. (21) found evidence that an ou tbreak of type 80/81 staphylococcal pyo-derma in a poultry processing plant was related to a period in whieh chlortetracycline was used in the plant for ice-water bath immersion of eviscerated poulrty.
Domestic animals mayaıso acguire their staphylocoecal infee-tions from man. Smith et. aL. (26) and Moeller et. aL. (20) studied 295 human patients with coagulase-positive Staphyloeoeeus au.reus infeetions from rural areas and have obser vedthat a mumber of their patients had transmitted infeetions to their eattle. Similarly, Wallace and his eolleagues (27) have reported the association of type 80/81 infeetion in man ""ith mastitis in cattle.
Veterinary clinics may constitute heavily contaminated envi-ronments in whieh reciprocal infeetions are possible, Live and Niehols (17) reported that 50 per cent of 4 th year clinical veterinary stu-dients but only 3 per cent of preclinieal veterinary studients at the university of Penusylvania carried antibiotic resistant staphyloeoeci of phage type 80/81. Silberg et aL. (24) studied the human and ani-mal populations in another veterinary hospital environment but found little or no evidenee for interspecies transmission.
İn summary, the results of many investigators indieate that inter-host transmission does occur and heavliy contiminated environments may be inportant sources of outbreaks Gl' infeetion in both animals and man.
References
1- Anderson, J.C. (1976): Mechanisms of staphylococcal virulence ın relatian to bovine mastitis. Br. Vet. Jour., 132, 229-245.
96 Hamza Keskintepe
2- Bdjljosov, D., Sachariev, z., and Georgiev. Z. (1974):
Be-sonde~heiten on Staphylokokken, isoliert om sclz.ıehtgeflügel. Monats-hcfte für Veterinarmcdizin, 29, 292-294.
3- Balı,!
R.J.,
and Schmidt, J.P. (1965): Phage type and antibiotic sensitiviry of stap!ıylococci isolated from monkeys and their human hand-lers.4- Blakburn, P.S.. (i 959): Botryomycosis of the udder. Br. V ct.
J.,
115, SI1-313!5- Blau~e, L., Husted, P., Mckee, A., and Gonzalez, J. (1964): Epizôrftiology oj staph)'lococci in dogs. Am.
J.
V ct. Res., 25,1195-1200\,\\ ..,ı
6- Braner, D.W., and Gillespie, J.H. (1973): Hagan's infection diseases of domestic animals. pp. 274, Corneli Uni\"ersity Press, Itho~~ and London.
7- Carriaghan, R.B.A. (i 966): Spinal cord Compression in fowls due to spondylitis caused by Staphylococcus f;yogmes.
J.
Comp. path., 76,9- 14: 1 •
8- Coles, E.H.;' and Eisenstark, A. (1959): Staphylococcic phages. I. The use of human ryping phages in ryping of Staph)'lococcus aureııs of animal origin. II. Typing of S. aureus cl/ltures isolated from the bovine udder.~Am .
.J.
Vet. Res., 20, 832-840.9- Cruiekshank, R., Duguid, J.P., Swain, R.H.A. (i 975): Medi-cal Microbiolog)'. pp. 139, E. and S. Lıbıngstone limited, Edinburg and London.
10- Derbyshiıre; J.B. (i958) :The experimental Production of Staphylo-coccat mastilis in the goaı .
.J.
Comp. path. and therap., 68, 232-241. 11- Derbyshire, J.B., and Berrnan, D.T. (1969): Themultiplica-tion of staphylococci oj boıine or humen origin in ccus milk. Res. V ct.
Sei.,'10, 161-167.
12- Devriese, L-A. Oeding, P. (i975): Coagulaçe and heat-reçistant nucleare producing Staph)-lococcus epidtrmidis strainsfrem animals ..Jour. Appf~ Baet.,' 39, 197-207.
,\\\
13- Duthie E.S. (1954): Evidence of staphylococcal coagulase.
J.
Gen. Mie:~bioı., 10, 427-429.14- Hearst B. R. (i967): Low inciden ce of staphylococc dermatitides animals with high incidence of Staphylococcus aureus. Part 1:Preliminary study of cats. Vet. Med., 62, 475-477.
Staphylocoeei ;n Aaimals. Chararteri,tie,. Distribution And tt,... 97
15- Hearts, B.R. (1967): Low incidence of staphylococcal deırıatitides in
animal with high incidence of Staph)'lococcus aureus. Part 2:I?reliminary study of dogs. Yet. Med., 62, 541-544.
16- Koenig, M.G., Melly, M.A., and Rogers,.D.E. (1962):
Fac-tors relating to the vindence of staphylococci.
J.
E~p. Med."ı 116, 601-607.17- Live, L., and Nichols, A.C. (1961): The animal h{!~fpital as a source of antibioticresistant staph.-ılococci.
J.
ınfeet. Dis., 108, 195-204. 18~ Mann, P.H. (1959): Bacteriophage lJping staph]lococci #ıund in thenostrils of dogs and ca/s.
J.
Am. Yct. :\1ed. Ass., 134, 4Q~-470. 19- Markham, N.P., and Markham, j.G. (1966): Staphylococci inman and animals. Distribution and characteristics of strain~cJ. Comp.
Path., 76, 49-56. ,l .y,
20- Moeller, R.W., Smith, j.M., Shoemaker, A.C. and Tjalm, B.A. (1963): Transfer of hospital staph)10cocci from man~~ofarm ani
mals.
J.
ArTı. Yet. Med. Ass., 142, 613-617. . ~\21- Ravenholt, R. T., Elkema, R.C., Mulhern, M., and Watkins, R.B. (1961) : Staphylococcal infection in meat animals and 17,1eatworkers.
Pub. Health Rep., 76, 879-88~.
22- Reid, W.B., and Wilson, j.B. (1959): A study of the staphylncocci
associated with the bovine udder. Am.
J.
Yet. Res., 20, 825-831. 23- Rountree, P.M., Freeman, B.M., andjohnston, K.•G.
(1956):Nasal caniage of Staplzjlococcus a//reus by various domestic'land labora-tmy animals.
J.
path. and Bac!., 72, 319-321. .124- Silberg, S.L., Blenden, D.C., and Novick, A. (196}J):Risk of
staplzylococcic infeetion among human beings ünd animals in- lı veterinary lıospital erıvironment. Am.
J.
Yet. Res., 28, 267-273.• '<1
25- Smith, H. W., And Crabb, W.E. (1960): The effect
qf
diets con-taining tetracylines and penicillin on the Staph.J10ceccus aur~~s flera of the nose and skin of pigs and Chiekens and their human attenda[is..J.
path.and Bact., 79. 243-249. . - l'l
.~,
26- Smith, I. M., Moeller, R.W., jones, M., and Hazard, E.C.
(1963): Staph]'iococei in rural patients diseharged from lıospıtals. Am.
J.
Med. Sc., 245, 12-22. id27- Wallace, G. D., Q.uisenberry, W.B., Tani~oto, It.H., and Lynd, F.T. (1962): Bacteriophage type 80/81 staphylococcal infection
in human beings associated with mastitis in caule. A.m.
.J.
Pub. Health., 52, 1309-1317.98 Hamza Ke,kintepe
28- Watson, W.A. (1964): Studies on tlZe distribution of the slzeep tiek
(lxodes rieinus L.) and the oecuneliee of en<.ootiestaphyloeceeal infeetion Qf lambs in north-west Yorksbire arıd nortlz-east Laneııshire. Vet. Rec.,
76, 743-746.
29.,- Wentworth, B.B. (1963): Baettriophage t}ping of staphyloeoeei.
Bact. Rev., 27, 253-261.
.i
30- Yoshida, K., and Eckstedt, R.D. (1968): Relation of mueoid growtlz of Staplzyloeoecııs allreus to clumping faetor reaetion, morpholog)'
in Serum-so lt agar, and virulence. Jour. BacterioL., 96, 902-908. 31- Zin!!: R.D., hderson, G.R., and Skaggs, J.W. (1961): The
p~blit.healt/ı 'Signijicanee of stap/ı)'loeoeeic infiretivn in eaule.
J.
Am. \ et. Med. Ass., 138, 382 -386.Yazı "Dergi Yazı J(urlllu"ııa 21.2.1977 günü gelmiştir.
f.
