Characteristics of Staphylococcus aureus Strains, Isolated from Slaughtered Poultry

Zentralbl. Mikrobiol, 141 (1986) , 163-168 VEB Gu stav Fischer Verlag J en a [Department of Hygiene a nd T echnolo gy of Foodstuffs , Veterinary U n ive rs ity, Brno, State Veterinary Institute , Ostra va , a nd Municipal Institut e of Public H ealth , Ostrava, CSSR]

Characteristics of Staphylococcu8 aureus Strains, Isolated from Slaughtered Poultry J . L UK•.\.SOVA, V. l\IiCKOV•.\. a nd J. PCHALEK With 3 Figures

Summary The ex t en t of contamination in sla ugh t er ed poultry by st ra ins of Staphyloccocus aur eus was investigated, as well as th e or igin of strains and the way of their transfer during operation of the poultry slaughterhouse. 45 st r a ins were isolated from 175 s wa b samples from the surface and the body cavity of poultry a nd 8 st r a in s from 23 workers of th e sla ugh t erh ouse. 22 st ra ins of St. aur eus fr om the poultry were classified as bi otype A (of human origin), according to the bio chemical identifi cation sche me by HAJEK a nd MARSALEK (1971), and 20 st ra ins as b iotype B (of poultry or ig in ). 3 strains wer e not t ypable. 7 strai ns from the workers handling the poultry wer e cla ssified as bi otype A and one strain was not t ypable. Enterotoxins were produced mos tl y by s t ra ins of biotype A (13) , by one st ra in of biotype B , a nd by one s t ra in wh ich could no t b e t ypified. The t ransfer of s t a ph y lococc i from workers into t he poultry wa s verified .

Zusammenfassung In eine m Schlachthof wurde das Au sm af3 der Kontamina t ion des Sch lach t gefluge ls durch v er schied ene Staphylococcus au reu8·S tii m me , der en H erkunft sowie ihre Ubertragung im B etrieb ve r folgt, Aus 175 Ab strich en von der Ob erfliich e und aus der Lei b e shohle des Gef'lugels wurden 45 Stiim m e isoli ert, von den Besch iiftigten des Sch lac htho fes 8 St.amm e. 22 S t. aureus -St iim me des Gefl iigels wurden an Hand de s T ypisierungssch emas von H AJEK und l\IARSALEK (1971) dem B iot op A (ursprungli ch er Wirt : Mensch) und 20 Stiimme dem Biot yp B (urspriinglich er Wirt. : Gefliigel) zugeord net. Drei Stiimme konnten ni cht eingeord net werden, Sieben von den Be sch iiftigten isoli er t e Stii m me gehorten zum Bi ot yp A ; ein Stamm konnte ni cht einge or d net worden. Enterotoxin e er zeugt en vorwiegend di e St iirnm e des Biotyps A (13 ), 1 St amm des Bi ot yps B und einer der n icht. ein zu or d nenden St.amrne. D ie U ber t r agu ng von St. aureU8 von den B eschiiftigten auf das Schl a ch t gefliigel wurde bestiiti gt.

The strains of StaphylocoCC1t8 aureus occur with various animal species and differ in their properties not only from the human strains, but also among each other, in accorda nce with the type of host from whom they were isolated. Among the basic crite ria , determining the origin of isolated st ra ins, HAJEK and MAR SALEK (1971) sugg ested the following t ests: production of fibrinolysin, coagula t ion of human and bovine plasma, growth on agar with crystal violet, production of tX a nd fJ ha emolysin, ph age-t yping, and sensi tivit y to penicillin and tetracy elin. F or the evaluation of st a phy lococci found in raw ma t erial or foodstuffs , t he determination of the origin of st rains is important from t he st andpoint of establishing the human factor, cont ributing to t he cont a mina t ion , det ermination of its source, det ermination of the exte nt of cont a mina t ion of the pr odu cti on equip ment , a nd of

164

J. LuRA.sovA, V. MicKOVA and J. PCHALEK

the possibility of transfer of staphylococci between the animal specIes and man. The hygienic aspect is unquestionable, too. The aim of this work was to determine the extent of contamination of slaughter poultry by strains of Staphylococcus aureus, the origin of those strains, and the way of their transfer to the processing of the poultry.

Material and Methods In the course of the technological processing of poultry 45 strains of Staphylococcus aureus from 175 swabs from the surface and the body cavity of the slaughtered poultry were isolated. Swabs from the neck, nose, and from the hands of 23 workers of the slaughterhouse were taken and 8 strains were isolated. Meat-peptone broth with 6 % NaCl was used for the isolation of strains, followed by the inoculation into the Baird-Parker medium. The following tests were carried out with the isolated strains: - coagulation of rabbit, human and bovine plasma (Subcommittee on Taxonomy of Staphylococci and Micrococci 1965) - production of fibrinolysin, using the dried-off Fibrinogen (SPOFA) and Antilysin (SPOFA) - growth on agar with crystal violet (MEYER 1967) - production of haemolysins (ADAMCZYK and BLAUROCK 1963) - production of enterotoxins (tlOUREK 1976) - anaerobic fermentation of glucose, mannitol, and lactose (Subcommittee on Taxonomy of Staphylococci and Micrococci 1965) - production of thermostable DNAse (LACHICA et al. 1971) - phage-typing with the International Set of Basic Human Phages - proteolytic activity (MARTLEY et al. 1970) - production of phosphatase (HOLDING and COLLEE 1971) - sensitivity to respresentative antibiotics (penicillin, erythromycin, streptomycin, chloramphenicol, tetracyclin) by means of the disk diffusions method (VYMOLA and HEJZLAR 1964).

Results Strains of Staphylococcus aureus were found in 25,7% out of 175 swabs from poultry and in 34,8 % from the workers. The rabbit plasma was coagulated by 98 % of the strains isolated from poultry, the human plasma by 85 %, but the bovine plasma was not coagulated by any strain. With strains from workers the coagulation of rabbit plasma was found in 87 %, the human plasma in 75 % of strains. Again, the bovine plasma was not coagulated by any strain. Fibrinolysin was produced by 50 % of the isolated strains from poultry and 62 % of strains from workers. The crystal violet A typ was shown by 70 % of strains from poultry and 25 % of strains from workers. Phosphatase and thermostable DNAse were produced by all strains. Anaerobic fermentation of sugars was more clean-cut with poultry strains, as well as the proteolytic activity (Fig. 1). Haemolysins were produced by 40 poultry strains, the types IX and IXP prevailed. Type 0 was prevailing with strains from workers (Tab. 1). Twelve strains isolated from poultry and 3 strains from workers produced enterotoxins (Tab. 2). The sensitivity towards the phages used was rather less expressive with most strains. The poultry strains were sensitive mostly to phages of group II and III, the strains from workers to group I and II (Tab. 3). More than 50 % of the poultry strains were resistant to penicillin, erythromycin, and streptomycin, to tetracyclin less than 50 'Yo. The strains from workers were

165

S tap hylococcus au reU8, I s ola t ed fr om Sla ug htered P oult r y

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T abelle 1. H ae mol ysin product ion b y s t ra ins of Staphylococcus aur eus Origin

No.or strains

H aemolysin s
p

0


po

10 5 5 1 3 2 1

13 10 2 1

0 1 0

2 2 0

2 2 0

P oultry biotype A biot y pe B non -t y pable

45 22 20 3

14 6

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2 0 2 0

Worker s b iot y pe A non -t y pabl e

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0 0 0

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T abelle 2. Ent er ot oxin prod ucti on b y strain s of S tap hylococcus aur eus Origin

No.of s trains

E n t erotoxins A

B

C

D

E

4 4 0 0

2 2 0 0

0 0 0 0

1 1 0

1 0 1

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Poultry biot y pe A biotype B non-t y pa ble

45 22 20 3

0 0 0 0

6 5

Wo rke rs bi ot y pe A non -t ypable

8

0 0 0

1 1

7

1 0

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Tabe lle 3. P hage-groupi ng of staphylococci, iso lated from pou lt ry an d worker s Origin

Po ult ry Worke rs

Phage groups I

II

III

M

- /3 2/3

- /18

- /3

2/ 11 1/8

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166

J. LUKASOVA, V. l\IiCKoVA and J . PCHALEK

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Fig. 2. Bi ochemical proper ti es of staphyl ococci, isolated fr om poultry (% positive reactions).

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poultry- biotype A poultry- biotype B workers' biotype A

80 60

40 20

PNC

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STR

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Fig. 3. Antibiotic resi stance pat,terns of S tap hylococcus aur eus stra ins . A; PNC = penicillin, ER = ery tr omyc in, STR = streptomycin, CH LP TTC = t etracyclin

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chloramphen icol.,

res istant to penicillin and tetra cyclin. Most of t he st rains were sensitive to chloramphe nicol, 46.7% of strains were resistant to 3 or more ant ibiot ics . 11 % of the strains were sensitive to all t he antibiot ics used. Considering t he determ ined properties, t he strains were divided in t o biotypes acco rding to HAJEK a nd MARS.A.LEK (1971). 22 st rains of staphylo cocci isolated from po ultry were classified as biotype A and 20 strains as biotype B, 3 strains were not typable. 7 strains isolated from workers were classified as biotyp e A ; 1 strain was non-t ypa ble. The bio chemical propert ies are given in Fi g. 2 and 3 and Tables 1 an d 2.

Discus sion The degree of contamination of t he slaughtered pou ltry, due to strains of Slaphy aureus , depends on the way of processing, st oring, and on the hygienic conditions du ring product ion. The human factor is very import ant as well. There are different opinions with regard to the origin of staphylococci, fou nd in t he slaughtered poultry. Some a uthors consider the strains to be mostly of human lOGOC'OU8

Staphylococcu8 aureU8, Isolated from Slaughtered Poultry

167

origin (KUSCH 1972), while others admitted that up to 50 % of the strains were of poultry origin (GIBBS 1978). Our results give evidence that staphylococci, either of poultry or of human origin, were found in the slaughtered poultry; 49 % were strains of biotype A and 44 % strains of biotype B. The maximum differences between the strains of biotype A and B concerned the production of fibrinolysin. Enterotoxins were mostly produced by strains of biotype A (11) and only 1 strain of biotype B. These data correspond to the results by other authors, e. g. SHIOZAWA (1976), HAJEK and MARSA,LEK (1973). It is interesting to note that enterotoxins were produced mostly by strains resistant to the used phages. The crystal violet A typ exhibited 90 % of strains of biotype B, with the biotype A we found it in 50 % of occasions, with other strains there was the type C and E. More marked differences in resistance to antibiotics were found with penicillin and chloroamphenicol. Small differences or none were noted with the other biochemical properties. In the course of processing most staphylococci in poultry are found after plucking and drawing (TUREK et al. 1983), the source of poultry staphylococci being the live poultry (feather, infection processes). The possibility of transfer of human staphylococci arises everywhere, where persons come in direct contact with the poultry. This may happen practically during the whole course of processing. We succeded, by means of the phage-typing, to prove a direct transfer of staphylococci from workers to poultry in 3 cases. The occurence of staphylococci in slaughtered poultry does not represent any serious health risk if the poultry is quickly chilled and further on heat-treated. Staphylococci may, however, become a source of contamination of other foodstuffs. A dangerous situation may arise on processing of semi-products and products from poultry meat and of mechanically boned, separated meat, or when not maintaining the hygienic principles for the preparation of meals in cookeries collective boarding. References ADAMCZYK, B., und BLAUROCK, G.: Zum Nachweis spezifischer Staphylokokken.Hamolysine. Z. Ges. Hyg. 9 (1963), 456-471. Gums, P. A., and PATTERSON, J. T.: Biochemical characteristics and enterotoxigenicity of Staphylococcus aureus strains isolated from poultry. J. App!. Bact. 44 (1973), 57-74. HAJEK, V., and MARSALEK, E.: The differentiation of pathogenic staphylococci and a suggestion for their taxonomic classification. Zb!. Bakt. Hyp. Abt. Orig. A 217 (1971),176-182. HAJEK, V., and MARSALEK, E.: The occurrence of enterotoxigenic Staphylococcus aureus strains in hosts of different animal species. Zb!. Bakt. Hyg. 1. Abt. Orig. A 223 (1973), 63-68. HOLDING, A. J., and COLLEE, J. G.: Routine Biochemical Tests. In: Methods in Microbiology 6A. London 1971, 25. KUSCH, D.: Biochemical characteristics and phage-typing of staphylococci isolated from poultry. Zb!. Bakt. Hyg. 1. Abt. Orig. B 164 (1977), 360-367. LACHICA, R. V. F., GENIGEORGIS, C., and HOEPRICH, P. D.: Metachromatic agar diffusion methods for detecting staphylococcal nuclease activity. App!. Microbiol 21 (1971), 585-587. MARTLEY, F. G., .TAYASHANKAN, S. E., and LAWRENCE, R. C.: An improved agar medium for the detection of proteolytic organisms in total bacterial counts. J. Appl, Bact. 33 (1970),363-370. MEYER, W.: Uber die Brauchbarkeit des Kristallviolettestes zur Differenzierung von Staphylococcus aureus-St.ammen. Z. med. Mikrobio!. Imunol. 153 (1967), 158-168. SHIOZA WA, K.: Enterotoxigenicity and some properties of Staphylococcus aureus strains isolated form chickens. Jap. J. Vet. Res. 24 (1976), 114. SOUREK, J.: Prfspevek ke stanoveni typ u st.afylokokoveho enterotoxinu typovymi sery vyrobenyrni v Institutu mikrobiologie a epidemiologie. Cs. epid. mikr. imun. 25 (1976), 159, 166.

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J. L UKA§oVA, V. l\HcKovA and J . P CHALEK, Staphylococw 8 aureu8, I solat ed fr om . . •

T UREK, B., GRIEGER, C., B URDOVA, 0. , a nd LENGYEL, G. : Povr ch hy di n y v pri ebehu jatocn eho opracovani a z hl ad iska vysky t u niekt or ych hygien ick y vyznarnnyc h mi kroor gan izm ov . H y d . priern . XXV (1983), 286 -291. VyMOLA, F., a nd H EJZLAH, M.: Di fu sni d isko va m et oda p ro ureovanf cit livost i mi kr ob u na antib iotika . Zpra.vy t echnicko-poradcnske alu zby , Sp olana 6 (1964), 15-1 8. Subcomm itee of Taxonom y of Staphylococci an d Micro cocci. Int. Bull. Ba ct. 15 (1965), 109-110. Ac k nowledge me nt

' Va th a n k MU D r V. H ,I.JEK, Sta p hylococca l R eferen ce La borat ory , Olo mo uc, for phagetyp ing of Stap hsjlococcus aur eus stra ins. Authors' addresses : MVD r J INDRA L UKAs o vA, ka t edra hy gien y a tech nologi c potravin , Vyso ka sko la veterin arni, P alack eh o 1- 3, 61 242 Brno, ~IVDr VLASTA ~Ii CKOVA , Statni veter inarni ust a v , 723 08 Ostrava Martinov , a nd MUDr J AN P CHALEK, Mestsky ustav narodnfho zdravi 72300 Ostra va, CSS R .