Characterization of Staphylococcus aureus isolated from poultry meat in Spain

PROCESSING AND PRODUCTS Characterization of Staphylococcus aureus Isolated from Poultry Meat in Spain R. Capita,1 C. Alonso-Calleja, M. C. Garcı´a-Ferna´ndez, and B. Moreno Department of Food Hygiene and Food Technology, Veterinary Faculty, University of Leo´n, Campus Universitario de Vegazana, s/n. 24071, Leo´n, Spain (RTD) and only 16 (16.7%) at RTD. By using reversed phage typing, we managed to increase the number of phage typeable strains by 46 (47.9%). More than one S. aureus phage type was detected in 14 (35%) carcasses, which emphasizes the convenience of subtyping several S. aureus isolates from the same food sample in epidemiological studies. Two phage patterns (75/84 and 6/1030/ W57) were the most common. The S. aureus isolates were closely related, as 78 strains showed the most common or indistinguishable (<2 phage reaction differences) phage patterns.

(Key words: Staphylococcus aureus, characterization, poultry, typing, Spain) 2002 Poultry Science 81:414–421

carcasses in Leo´n, Spain, by using cultural and biochemical tests and phage typing to trace their origin and their public health significance, to investigate the relationship of the strains, and to determine their diversity within and between samples.

INTRODUCTION Staphylococcus aureus has been tested in meat and poultry products to assess microbiological safety, sanitation conditions during processing, and storage quality of products (Tompkin, 1983). S. aureus is a frequent etiological agent of food poisoning (Halpin-Dohnalek and Marth, 1989; Lo´pez et al., 1993; Jablonski and Bohach, 1997). Its presence in poultry (Waldroup, 1996) emphasizes the need for laboratory surveillance for this bacterial pathogen. However, not all S. aureus strains present in processed poultry carcasses are a cause for public health concern. The animal strains make a very small contribution to human food poisoning (Ha´jek and Marsa´lek, 1971; Shiozawa et al., 1980; Parker, 1983; Isigidi et al., 1990). On the other hand, the S. aureus isolates from human sources may be considered the most dangerous strains of public health significance (Isigidi et al., 1992). In fact, poultry meat has been frequently associated with foodborne illness in which initial contamination is traceable to food handlers (Halpin-Dohnalek and Marth, 1989). The aim of this study was to characterize ninety-six Staphylococcus aureus strains isolated from retail chicken

MATERIALS AND METHODS Bacterial Strains Ninety-six S. aureus strains were isolated in Leo´n, Spain, from forty eviscerated and refrigerated retail chicken carcasses. The samples were aseptically collected, and each carcass was placed in a separate, sterile plastic bag. The samples were brought under refrigeration to the laboratory and analyzed within the following 4 h. Isolation was carried out on Baird-Parker agar containing egg-yolk tellurite emulsion (B-P).2 The bacteria were maintained on tryptic soy agar (TSA)2 slants and stored at 4 C.

Cultural and Biochemical Tests Catalase Test. Isolated colonies on TSA with 1% glucose were tested for evolution of gas bubbles with 30 vol of hydrogen peroxide (Cowan, 1974).

2002 Poultry Science Association, Inc. Received for publication March 28, 2001. Accepted for publication October 18, 2001. 1 To whom correspondence should be addressed: [email protected]. 2 Oxoid Ltd., Hampshire RG24 8 PW England.

Abbreviation Key: BHI = brain heart infusion; B-P = Baird-Parker agar containing egg-yolk tellurite emulsion; NHS = nonhost specific; RTD = routine test dilution; TSA = tryptic soy agar.

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ABSTRACT Ninety-six Staphylococcus aureus isolates from retail chicken carcasses in Spain were characterized using cultural and biochemical tests. The strains were phage typed with the international bacteriophage set for typing S. aureus of human origin. Eighty-eight (91.7%) strains were of the poultry ecovar. Strains of human ecovar were not found. These facts are congruent with findings of other authors. Ninety (93.7%) strains were phage typeable. Lysis by phages of Group III was the most frequent with 66 (68.7%) sensitive strains. Twenty-eight (29.2%) strains were sensitive at 100 routine test dilution

CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS FROM POULTRY

415

TABLE 1. Characteristics of Staphylococcus aureus isolated from poultry meat (in parentheses percentage of strains) Staphylococcus aureus group 1

A (72 strains)

B (20 strains)

C (4 strains)

Gram stain Catalase Oxydase Lysostaphin sensitive Facultative growth in thioglycolate Coagulase Thermonuclease Aerobic acid from Mannitol Maltose Acetoin production Staphylokinase β−Hemolysis Bovine plasma coagulation Growth on crystal violet4 Total phage−typeable strains

C + (100) + (100) + (100) + (100) ++ (100) 4 + (100) ≥1 mm (100)3

C + (100) + (100) + (100) + (100) ++ (100) 4 + (100) ≥1 mm (100)

C + (100) + (100) + (100) + (100) ++ (100) 4 + (100) ≥1 mm (100)

+ (100) + (100) + (100) − (100) − (100) − (100) CV:A (100) 72 (100)

+ (100) + (100) + (100) − (100) − (80); + (20) − (100) CV:A (100) 18 (90)

+ (100) + (100) + (100) − (100) − (100) + (100) CV:A (50); CV:C (50) 0

2

1 A = jet-black, shiny, regular, convex colonies, ≥ 2 mm diameter, surrounded by a zone of clearing after 24 h at 37 C and a inner zone of opacity after 48 h on Baird-Parker agar; B = jet−black, shiny, regular, convex colonies, ≥ 2 mm diameter on Baird-Parker agar; C = jet-black, shiny, regular, convex colonies, < 2 mm diameter on Baird-Parker agar. 2 Gram positive cocci. 3 Diameter of pink halo ≥ 1 mm. 4 CV:A = growth spots with a bright or pale yellow color and yellow spots with violet margins; CV:C = blue or violet growth spots with or without an orange tint.

Oxydase Test. Strains were cultivated at 30 C on Columbia agar2 with 5% sheep blood. After 18 h of incubation, one loop of bacteria was smeared onto ordinary filter paper. One drop of 6% tetramethylphenylenediaminehydrochloride3 in dimethyl sulfoxide4 was added to the bacterial material. Oxydase-positive bacteria turned dark blue within 5 to 10 min (Faller and Schleifer, 1981). Anaerobic Growth in a Glucose-Containing Medium. One loopful of a 24-h culture in trypticase soy broth2 was transferred to a tube of Brewer’s fluid thioglycolate medium,5 steamed, and cooled to 50 C. After agitation and solidification at room temperature, the tubes were incubated at 35 C for 72 h (Evans and Kloos, 1972). The tubes were examined by looking through them toward a light source. Symbols of anaerobic growth were ++, dense uniform; +, gradient of growth; ±, individual colonies; −, only aerobic growth. Lysostaphin Test. One-tenth milliliter of a saline (107 cfu/mL) cell suspension was added to a tube containing 3 mL of fluid medium (Schleifer et al., 1981). After agitation, the contents were poured onto the surface of a medium with 1.5% agar. A drop of sterile lysostaphin solution6 (200 µg/mL) was placed on the agar and incubated for 24 h at 35 C. Lysostaphin susceptibility was interpreted according to the following scheme (Schleifer and Kloos, 1975): +, sensitive (complete growth inhibition); ±, slightly resis-

3

Merck Farma y Quı´mica S.A., Barcelona 08110 Spain. Panreac Quı´mica S.A., Barcelona 08110 Spain. 5 Difco Laboratories, Detroit, MI 48232. 6 Sigma-Aldrich Quimica S.A., Madrid 28100 Spain. 4

tant (partial growth inhibition); −, resistant (no visible inhibition). Coagulase Test. A single colony of overnight cultures on TSA was used for a coagulase test following the conventional protocol (Lancette and Tatini, 1992) except that the preincubation times in brain heart infusion (BHI)2 prior to the enzyme assay was 24 h (Chang and Huang, 1996). The BHI broth was used for the coagulase and thermonuclease tests. The formation of a clot was examined at 2, 4, 6, and 24 h (Kloos and Lambe, 1991; Martin and Myers, 1994). The types of coagulase reaction were classified from 0 to 4 following the recommendations of the American Public Health Association (Lancette and Tatini, 1992). Any degree of clot formation (1 + or above) was considered a positive reaction. Thermostable Nuclease. BHI broth (0.1 mL) was heated in a boiling water bath for 20 min and placed in wells made in toluidine-DNA agar (Pascual-Anderson, 1992) plates. Plates were incubated at 37 C for 4 h. The development of a pink halo extending at least 1 mm from the periphery of the wells was considered a positive reaction. Carbohydrate Dissimilation. The production of acid from maltose and mannitol under aerobic conditions was tested for as described by Baird-Parker (1980). Purple agar base5 with 1% (wt/vol) carbohydrate was used. Incubation was carried out at 30 C for 5 d, with daily readings. Acetoin Production. BHI with 2% glucose was inoculated and incubated for 15 d at 30 C. After incubation, Barrit reagent (0.6 mL of 5% a-naphthol and 0.2 mL of 40% KOH) was added, and the mixture was shaken vigorously. A definite reddening of the culture supernatant indicates production of acetoin (Baird-Parker, 1980).

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Tests

416

CAPITA ET AL. TABLE 2. Biotypes and phage groups of Staphylococcus aureus strains isolated from poultry meat in Spain

Biotype1 Poultry NHS

K K K K

− − − −

β β β β

− + − −

BC BC BC BC

− − + +

CV:A CV:A CV:A CV:C

Phage group

Biotyped strains (96)

Phage-typeable strains (90)

III

Miscellaneous

Mixed

Additional phages2

NT3

88 4 2 2

86 4 − −

62 4 − −

4 − − −

2 − − −

18 − − −

2 − 2 2

1 K = staphylokinase; β = β-hemolysin production; BC = bovine plasma coagulation; CV = growth on crystal violet agar type (A or C); NHS = nonhost specific. 2 1030, W57, 2009, and 18042. 3 Not typeable with the international set for typing Staphylococcus aureus strains of human origin.

TABLE 3. Results of phage typing of 96 Staphylococcus aureus isolates from poultry

III Miscellaneous Mixed Additional Phages4 Total

RTD1

100 RTD 2

16 (16.7) − − − 16 (16.7)

24 (25) 4 (4.2) − − 28 (29.2)

Reversed phage typing 3

26 (27.1) − 2 (2.1)3 18 (18.7) 46 (47.9)

TOTAL 66 (68.7) 4 (4.2) 2 (2.1) 18 (18.7) 90 (93.7)

1

Routine test dilution. Number (percentage) of phage typed isolates. 3 All strains were also lysed by the additional phages. 4 Phages 1030, W57, 2009, and 18042. 2

Biotyping. Each of 96 S. aureus strains was biotyped following the simplified system proposed by Devriese (1984). Characteristics examined were staphylokinase production, β-haemolysin production, bovine plasma coagulation, and growth on crystal violet agar. The strains were alloted to host-adapted ecovars and nonhost-specific (NHS) biotypes. Phage Typing. All phages were typed according to standard methods (Blair and Williams, 1961). Twentythree phages of the international bacteriophage set for typing S. aureus of human origin were used: 29, 52, 52A, 79, 80 (Group I); 3A, 3C, 55, 71 (Group II); 6, 42E, 47, 53, 54, 75, 77, 83A, 84, 85 (Group III); and 81, 94, 95, 96 (miscellaneous). For reversed phage typing, the phages 1030, W57, 2009, and 18042 were also used. Initially strains were typed at routine test dilution (RTD; the highest dilution producing confluent lysis). The nontypeable strains were tested at 100 RTD. Reversed phage typing was used for strains that could not be typed at RTD or 100 RTD. The lytic reactions were read as follows: ++ = more than 50 plaques (strong lysis); + = 20 to 50 plaques (moderate lysis); ± = less than 20 plaques (weak lysis); − = no plaques (no lysis). Two cultures were considered to be different when one culture was lysed strongly by at least two phages that did not lyse the other culture to any degree (≥2 phage reaction differences). Phage typing was carried out in the Centro Nacional de Microbiologı´a del Instituto de Salud Carlos III.7

7

Majadahonda, Madrid, Spain.

RESULTS AND DISCUSSION Table 1 shows characteristics of 96 S. aureus strains isolated from chicken carcasses. On B-P, S. aureus reduces tellurite to form gray-black shiny colonies. Colonies are generally surrounded by an opaque halo (lecithinase activity) and show an iridiscent film in and immediately surrounding colonies (lipase activity). However, there are strains of S. aureus that are lecithinase negative (frequent in meat) and that do not produce clear zones around the colonies (Isigidi et al., 1985, 1989; Purdy et al., 1988; Feuersenger et al., 1994; Matos et al., 1995; Oxoid, 1998; Adesiyun et al., 1999). In our study, 24 (25%) strains formed on B-P jet-black colonies without a halo of clearing after 48 h. Twenty-one (21.9%) strains showed clot formation (coagulase positive) within 2 h, and 100% strains formed firm clots after 6 h at 37 C. Kloos and Lambe (1991) and Martin and Myers (1994) also increased the percentage of coagulase-positive strains by increasing incubation time. The percentage of coagulase-positive strains in our study is higher than those reported by Chang and Huang (1996) who found that the sensitivity of the coagulase test was 98.1% after 6 h of incubation. Table 2 presents the biotyping results combined with the phage-typing results with regard to the 96 S. aureus strains. It appears from this table that a high proportion (91.7%) of the strains belonged to the poultry ecovar. This ecovar is widely found on fresh and frozen poultry carcasses (Isigidi et al., 1992) and does not play an important role as far as public health is concerned (Shiozawa

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Group

417

CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS FROM POULTRY TABLE 4. Grouping of the phage patterns and strains

Phage patterns Strains

Total

Common phage lysis patterns

One phage reaction difference with the common phage lysis pattern

16 (100%) 90 (100%)

2 (12.5%) 20 (22.2%)

10 (62.5%) 58 (64.4%)

TABLE 5. Occurrence of two most common phage lytic patterns in the 90 typeable Staphylococcus aureus strains

4 16

28 30

75/84 6/1030/W57

et al., 1980; Harvey et al., 1982; Parker, 1983; Mead and Dodd, 1990; Isigidi et al., 1992). Because the majority of these strains are partially (mixed types) or solely sensitive to phages of Group III of the international set for typing human S. aureus strains, these strains probably come from the birds themselves (Ha´jek and Marsa´lek, 1971; Devriese

4 (25%) 12 (13.3%)

et al., 1985; Isigidi et al., 1990; Mathieu et al., 1991) and not from other origins. Four strains showed a NHS biotype and were lysed by phages of Group III, as happens in the majority of the NHS strains (Mathieu et al., 1991). Strains that are NHS, together with strains of human ecovar, are frequently toxigenic and may be considered as the most dangerous to public health. Four of the nonclassified strains could belong to poultry ecovar because the bovine plasma coagulation reaction was not strong (only 3 +). According to Devriese (1984), important differences exist among the interpretations of the bovine plasma coagulation test. The absence of strains belonging to human ecovar agrees with the findings of other authors. The presence of these strains in poultry meat is generally considered as an indicator of postprocessing recontamination and is not frequent in developed countries (Devriese et al., 1985; Isigidi et al., 1990; Mathieu et al., 1991).

TABLE 6. Activity, frequency of lysis, specificity index, percentage of strong reactions of the phages used for routine test dilution (RTD), and relationship of each one to the rest of the phages Lytic action of the 23 phages on the 16 typed strains Frequency of lysis Phage number 29 52 52A 79 80 3A 3C 55 71 6 42E 47 53 54 75 77 83A 84 85 81 94 95 96 1

(Number (Number 3 (Number 4 (Number 100. 2

of of of of

Sensitive strains

Total reactions1

Total strains2

Index of specificity3

Percentage of strong reactions (++)4

6 11 14 10 8 2 2

11.3 20.7 26.4 18.9 15.1 3.8 3.8

37.5 68.7 87.5 62.5 50 12.5 12.5

20 23.9 30.4 22.7 20 20 50

66.6 36.4 85.7 60 75 0 0

reactions produced by this phage/number total of reactions at RTD) × 100. strains lysed by this phage/number of total strains studied typed at RTD) × 100. strains sensitive to this phage/number of total reactions on these strains) × 100. strong reactions produced by this phage/number of total reactions produced by this phage) ×

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Most common lytic pattern

Number of strains showing common phage lysis pattern

Number of strains showing one lytic reaction different (i.e. indistinguisable) from common phage pattern

Two or more than two phage reaction differences with the common phage lysis pattern

418

CAPITA ET AL. TABLE 7. Activity, frecuency of lysis, specificity index, percentage of strong reactions of the phages used for 100 routine test dilution (100 RTD), and relationship of each one to the rest of the phages Lytic action of the 23 phages on the 28 typed strains Frequency of lysis Phage number

1

(Number (Number 3 (Number 4 (Number 100. 2

of of of of

Total reactions1

Total strains2

Index of specificity3

6

18.7

21.4

60

22

68.7

78.6

84.6

4

12.5

14.3

100

Percentage of strong reactions (++)4

100 22.7

0

reactions produced by this phage/number total of reactions at RTD) × 100. strains lysed by this phage/number of total strains studied typed at RTD) × 100. strains sensitive to this phage/number of total reactions on these strains) × 100. strong reactions produced by this phage/number of total reactions produced by this phage) ×

The proportions of strains phage typed at RTD, 100 RTD, or by reversed phage typing are shown in Table 3. Results indicate that the greatest percentage of strains (93.7 and 97.7% of total and poultry ecovar strains, respectively) were phage typeable. These percentages are greater than the majority of those reported by other authors. Shimizu (1979) indicates that the strains of the poultry ecovar are poorly typeable unless special phages are

used. The phage-typeable S. aureus strains isolated from foods with the international bacteriophage set for typing S. aureus of human origin ranges from 0% (strains of poultry origin; Kuramasu et al., 1967) to 79.9% (isolates from milk samples in Denmark; Aarestrup et al., 1995). Other percentages of phage-typeable strains are as follows: 4.0% (isolates from milk in Yugoslavia; Milojevic, 1990); 12.1% (isolates from milk in India; Mallikarujuna-

FIGURE 1. Number of sensitive Staphylococcus aureus strains to each one of the human phages. RTD = routine test dilution.

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29 52 52A 79 80 3A 3C 55 71 6 42E 47 53 54 75 77 83A 84 85 81 94 95 96

Sensitive strains

419

CHARACTERIZATION OF STAPHYLOCOCCUS AUREUS FROM POULTRY TABLE 8. Activity, frecuency of lysis, specificity index, percentage of strong reactions of the phages used for reversed phage typing, and relationship of each one to the rest of the phages Lytic action of the 27 phages on the 46 typed strains Frequency of lysis Phage number

1

(Number (Number 3 (Number 4 (Number 100. 2

of of of of

Percentage of strong reactions (++)4

4.3

8.3

0

1.3 1.3 18.5 1.3

4.3 4.3 60.9 4.3

12 6 10

7.9 4.0 6.6

26.1 13.0 21.7

8.3 8.3 22.2 8.3 15.4 16.7 16.7

0 0 75 0 0 0 0

2

1.3

4.3

8.3

0

2 2 35 46

1.3 1.3 23.2 30.5

4.3 4.3 76.1 100

8.3 8.3 25 30.5

0 0 68.6 95.6

Total reactions1

2

1.3

2 2 28 2

Total strains2

reactions produced by this phage/number total of reactions at RTD) × 100. strains lysed by this phage/number of total strains studied typed at RTD) × 100. strains sensitive to this phage/number of total reactions on these strains) × 100. strong reactions produced by this phage/number of total reactions produced by this phage) ×

Swamy and Krishnamurthy, 1998); 26.0% (strains from restaurant workers in Kuwait; Al-Bustan et al., 1996); 50.2% (strains from milk samples in Trinidad; Adesiyun et al., 1995); 54.4 to 54.8% (isolates from poultry; Kusch, 1977); 59.6, 66.4, and 72.5% (isolates from human handlers, bulk milk and composite milk, respectively, in Trinidad; Adesiyun et al., 1997); 63.8% (strains from food samples, including poultry meat, in India; Gill et al., 1994); 67.7 or 77.5% (strains from foods or raw meat, respectively, in Trinidad; Adesiyun et al., 1992); 68.0% (strains of various origins in Atlanta; Bannerman et al., 1995); 69.2% (isolates from meat and fish in India; Das and Khanna, 1995); 76.2% (strains from raw milk in Denmark; Aarestrup et al., 1995); and 88.1% (strains from poultry by-products in Spain; Capita et al., 2001). Lysis by phages of Group III was most frequent with 66 (68.7%) sensitive strains (Table 3). Eighteen, four, and two strains were lysed by additional phages, group miscellaneous, and phages in the different groups (mixed), respectively. According to Ha´jek and Hora´k (1971), the majority of the strains of poultry origin are sensitive to phages of Group III. Also other authors (Genigeorgis and Sadler, 1966; Harry, 1967; Devriese et al., 1972; Gibbs et al., 1978; Mathieu et al., 1991; Adesiyun et al., 1992; Isigidi et al., 1992; Rahman et al., 1992; Desai and Kamat, 1998)

found that phages from Group III lyse, with great frequency, S. aureus strains isolated from food (including poultry). Only sixteen (16.7%) strains could be phage typed by RTD. By using phages at 100 RTD, the number of phagetypeable strains was 28 (29.2%). Similarly, Kusch (1977) and Adesiyun et al. (1992) managed to type a large number of S. aureus isolates at 100 RTD using the human bacteriophage set. By using reversed phage typing, we managed to increase the typeable strains by 46 (47.9%). In the 90 phage-typeable strains, 16 different phage patterns were observed. Two patterns were the most common and were exhibited by 20 strains (Table 4). A further 58 strains (10 patterns) gave one phage strong reactionfrom the two common patterns and would thus be considered indistinguishable from at least one of these patterns. The remaining 12 strains, which showed two or more than two phage strong reactions different from at least one of the two common patterns, formed four more phage patterns. These data indicate a close relationship among the S. aureus isolates studied. Table 5 shows the number of strains included in each pattern and the number of indistinguishable strains of each pattern. For these tables, all lytic reactions (+ +, +, ±) were considered.

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29 52 52A 79 80 3A 3C 55 71 6 42E 47 53 54 75 77 83A 84 85 81 94 95 96 1030 W57 2009 18042

Index of specificity3

Sensitive strains

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CAPITA ET AL.

More than one phage type of S. aureus was detected in 14 (35%) carcasses. This fact emphasizes the convenience of subtyping several S. aureus isolates from the same food sample in epidemiological studies. Figure 1 shows the number of sensitive S. aureus strains to each phage. Tables 6 to 8 show the results of the behavior evaluation of the phages at RTD, 100 RTD, or when using reversed phage typing. At RTD, Phage 75 had the greatest number of sensitive strains and percentage of strong reactions and high specificity index (“best behavior”). Phages with greater activity at 100 RTD or using reversed phage typing were 84 and W57, respectively. Phages 52, 52A, 79, 80, 3A, 3C, 47, 81, 94, 2009, and 18042 did not lyse any of the S. aureus strains tested.

The authors thank the Comision Interministerial de Ciencia y Tecnologı´a (CICYT, project number ALI910294) for its financial support and the Servicio de Bacteriologı´a del Centro Nacional de Microbiologı´a (Madrid, Spain) for the phage typing of the Staphylococcus aureus strains studied.

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ACKNOWLEDGMENTS

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