Identification of Staphylococcus from French dry sausage

Letters in Applied Microbiology 1992, 15, 73-77

Identification of Staphylococcus from French dry sausage

M A R I E - C H R I S T I N E MONTEL, R E G I N E TALON, M O N I Q U E CANTONNET & J E A N N E FOURNAUD* Station de Recherches sur la Viande, I.N.R.A. Theix, 63122 Saint-Gends-Champanelle, and *Laboratoire de Recherches sur la Viande, I.N.R.A., 78352 Jouy en Josas, France

GWGf157: received 7 April 1992 and accepted 13 April I992

MONTEL, M . - C . , TALON, R . , CANTONNET, M . & F O U R N A U D , J. 1992.Identi- fication of Staphylococcus from French dry sausage. Letters in Applied Microbiology

Fifty-one strains of staphylococci isolated from French dry sausages were mainly identified with Staphylococcus carnosus, S. xylosus, S . warneri and S . saprophyticus. The API Staphylococcus identification system proved to be reliable for S. xylosus and S. carnosus. The identification of S. warneri and S . saprophyticus was performed by DNA-DNA hybridization. These species are better identified by taking into account not only the API Staphylococcus system but also the following characters: novobiocin and lysozyme susceptibilities, production of D-lactate, hydrolysis of triolein.

15,73-77.

In dry sausage, naturally occurring bacteria mainly consist of lactic acid bacteria and micrococcus-type bacteria. These are largely involved in the dry sausage process and are more and more used as starter cultures. Catalase-positive cocci are utilized for their role in the formation and stability of cured meat colour by reducing nitrate to nitrite (Liepe 1983), and for their effects on the flavour and aroma of the products (Liicke 1986; Nychas & Arkoudelos 1990).

Most of Gram-positive, catalase-positive cocci found in dry sausage have until recently been regarded as micrococci. By using the simplified taxonomic schemes proposed by Kloos and Schleifer (1975a,b), Rheinbaben and Hadlok (1979) showed that most of these strains could be identified as being coagulase-negative Staphylococcus.

Nevertheless, Fischer and Schleifer (1980) found 36% Micrococcus varians and 3% Micro- coccus kristinae in dry sausages. Simonetti and Cantoni (1983) noticed that micrococci were dominant in freshly prepared sausages whereas Staphylococcus became important in stored sausages.

The Staphylococcus strains isolated from fermented meat were classified as: S. xylosus, S.

simulans (Fischer & Schleifer 1980), S. carnosus (Schleifer & Fischer 1982), S. saprophyticus, S. warneri (Seager et al. 1986) but it remains that some strains are not identifiable with recognized species (Pirone & Mangaelli 1990; Simonetti & Cantoni 1983).

This study was undertaken because there are no reports concerning the identification of staphylococcus strains at species level in French dry sausage. The identification, on the basis of biochemical and physiological tests, was com- plemented by DNA-DNA homology.

Materials and Methods

B A C T E R I A L S T R A I N S

Fifty-one strains were isolated from traditional French sausage, dried at least 30 days, on Chapman medium (Difco) or P agar medium (Kloos et al. 1974). The organisms were main- tained frozen in APT broth (Difco) with 50% glycerol at -20°C. Reference strains were included in this study. They were obtained from Deutsche Sammlung von Mikroorganismen (DSM), Gottingen Germany : Staphylococcus carnosus DSM 20501T, Staphylococcus xylosus DSM 20266T, Staphylococcus saprophyticus DSM 20229T, Staphylococcus simulans DSM

74 Marie-Christine Monte1 et al. Euclidian distance

N W - I

I

S soprophyficus 869

843 M3 I 750

852 8 4 8 - - - 1 1 1 I - 1

S s

282 ___1 85 I

s xylOs”5

!is 863

S warneri 751 M 2

859 864 849

S epidermidis 855

I

Group A

_-

Group 0

--

Group C

_ -_ Group D

Fig. 1. A simplified dendrogram showing the relationship between strains isolated from sausages and reference species in the ascendant hierarchic classification based on Euclidian distance.

20322T, Staphylococcus warneri DSM 20316’, Staphylococcus cohnii DSM 2O26OT, Staphylo- coccus hyicus DSM 20459T, Staphylococcus scuiri DSM 20345‘, Staphylococcus epidermidis DSM 20044T.

B I O C H E M I C A L C H A R A C T E R I S T I C S

Acid production from carbohydrates, urease, arginine dihydrolase, phosphatase, nitrate reduction and acetoin production were studied using the API STAPH system according to the manufacturer’s instructions (API System, Biomerieux).

The configurations of lactic acid produced from glucose in the supernatant fluids of 48 h cultures at 30°C in APT broth were enzymati- cally determined with L- and D-lactate dehydro- genases (Boehringer, Mannheim).

Novobiocin (5 pg/ml), lysostaphin (200 pg/ml) and lysozyme (400 pg/ml) susceptibilities were

tested by the agar plate method described by Schleifer and Kloos (1975a).

Lipase activity was revealed according to the procedures of Kouker and Jaeger (1986) with 3% triolein as substrate.

Haemolytic activity was detected on P agar supplemented with sheep erythrocytes (5% v/v). Free coagulase was tested on rabbit plasma and deoxyribonuclease (DNase) production on DNase test agar (Difco).

C O M P U T E R A N A L Y S I S

The API 19 tests were coded in a numerical profile according to the manufacturer’s instructions (API system, Biomerieux). The interpreta- tion was performed by using the APILAB plus software. Twenty characters were coded in the following states: 0, negative; 1, positive; 2, strongly positive. The data were examined with STAT.1.T.C.F. software by Hierarchical Ascendant Classification by measuring Euclid- ian distance and using ponderal distance means as aggregation criteria.

D N A - D N A H Y B R I D I Z A T I O N

DNA was extracted and purified by the method of Brenner et al. (1982). Native DNA was labelled in uitro by nick translation with deoxy (3H) cytidine 5’ phosphate nucleotide as detailed in the commercial kit (Amersham). DNA-DNA hybridizations were performed at 60°C by following the S1 nuclease trichloroacetic acid pro- cedure as described elsewhere (Grimont et al. 1980).

BIOCHEMICAL I D E N T I F I C A T I O N

All the isolates were identified as Staphylococcus species as they shared the typical characteristics of this genus. They were Gram-positive, catalase-positive, non-motile and were suscep- tible to lysostaphin. In addition all strains were coagulase-negative, DNase-negative and haemolysin-negative on sheep blood agar. They could be regarded as harmless.

The APILAB software provided an identification for the staphylococcal strains. The quality of the identification was judged according to the relative relationship between the profile observed and those of the different taxons of the data base.

IdentiJkation of Staphylococcus dry sausage Table 1. DNA-DNA hybridization among strains from groups A

and C, and reference strains of Staphylococcus species

% Hybridization with labelled DNA from

75

M3 1 863 Source of Group Group

unlabelled DNA A C

Isolates from sausages - Group of Fig. I

A A A A A A A A A A A A A A A A A A

C C C C C C C C C C

DSM 20229T DSM 20316T DSM 20266T DSM 20501T DSM 20322T DSM 20260T DSM 20459T DSM 2034Y DSM 2 W T

Number M31 866 283 865 843 750 869 848 852 860 847 822 850 86 1 282 284 844 85 1

863 853 854 752 751 M2 859 864 849 855

S . saprophyticus S . warnmi s. xylosus S . carnosus S . simulans S . cohnii S . hyicus S . scuiri S . epidermidis

100 85 75 65 84 67 78 89 83 99 60 22 18 65 93 74 16 8

10

6 -

- -

16 9

10

25

75 9

23 9 5

11 15 20

5

-

5 14 11 - -

18 - - -

-

- 11 14

6 15 13 11

100 75 91 67 65 73 80

100 8

13

17 78 17 24 13 10

5 7

11

-

A very good identification was obtained for 19 strains: 10 strains were identified as S. xylosus, two as S. carnosus, three as S. saprophyticus, two as S. warneri and two were assigned to S. epidermidis. Of 51 isolates, 32 could not be clearly identified; their profiles did not fit totally with any of the reference strains in the data base.

To attempt to clarify this identification a dis- criminant analysis was carried out with 20 char-

acters: 16 tests of the API Staph strip were considered. Fermentations of glucose, fructose, melibiose were omitted because they were similar for all the strains. Four complementary tests were taken into account : susceptibility to lysozyme and novobiocin, production of D-lactic acid and hydrolysis of triolein.

Strains were divided into five main groups (A to E) at the Euclidian distance level of 3.5 (Fig. 1).

76 Marie-Christine Monte1 et al, Table 2. Useful discriminating characteristics for identifying Staphylococcus species from sausages

S. S. S. S. S. S . S . carnosus warneri epidermidis saprophyticus cohnii scuiri xylosus

Susceptibility : -Novobiocin S S S R R R R -Lysozyme R R R (R) S S S

Triolein hydrolysis - + + + + + + + ADH + + +

+ Urease -

+ - V + -GIcNAc - - -

-Lactose + V + - + + -Mannose + V + V + + + -Trihalose + + + + + +

+ + + + + -Saccharose - -Xvlose - - - - - -

Lactate isomer D, L D, L L L > D L > D L L - -

- - - -

- - + + + Acid from:

-

-

- +

ADH, arginine dihydrolase; GlcNAc, N-acetyl glucosamine; S, sensitive; R, resistant; v, variable.

The clustering obtained for S. xylosus and S . carnosus was in agreement with those obtained with APILAB software. Ten strains previously identified as S. xylosus were grouped in cluster B with the reference strain S. xylosus DSM 20266T. Furthermore two strains, identified as S . carnosus, were classified in cluster D with the reference strain S. carnosus DSM 20501'.

Cluster A contained 32 strains including the reference strains of S. saprophyticus DSM 20229T, S. scuiri DSM 20345T, S. cohnii DSM 20260T and 29 isolates from sausages. Four of these strains were identified as S. saprophyticus by the APILAB sofware.

Cluster C contained 12 strains characterized as follows: reference strains S. epidermidis DSM 20044T and S. warneri DSM 20316T; 10 strains from sausages, four of them identified as S. warneri and S . epidermidis by APILAB software.

Clusters A and C seemed heterogenous and the assignation of some strains to a species was not obvious. So further characterizations by DNA-DNA hybridizations were carried out with representative isolates from each of the two groups A and C.

D N A - D N A H Y B R I D I Z A T I O N S

Results of the hybridization experiments are summarized in Table 1.

With labelled DNA from M31 belonging to cluster A, the degree of DNa reassociation was 65% to 99% for 13 strains of cluster A. The homology was below 19% for four strains of group A and six strains of cluster C. M31 exhibited a high degree of homology (75%) with

the reference strain S. saprophyticus DSM 20229' but not with the other reference species tested.

This DNA relatedness has demonstrated that 14 strains of this cluster A obviously belong to the species S. saprophyticus. Ten strains of cluster A could be assigned to S. saprophyticus on the basis of their Euclidian distance with strains identified to that species by DNA-DNA hybridization. But this cluster is heterogenous. Four strains at least could be regarded as intruders in the S . saprophyticus group. Their identification needs further work.

The levels of DNA homology of eight strains from cluster C with strain 863 from this cluster ranged from 65% to 100% except for two strains, 849 and 855, which gave values of 8% and 13%. Furthermore strain 863 showed a level of homology of 78% with S. warneri DSM 20316T, and was not related to other reference strains.

From these results it appeared that cluster C could be divided into two groups. Eight strains could be assigned to S. warneri. By their Euclid- ian distance, two strains 849 and 855 are close to S. epidermidis DSM 2 W T and, as suggested by APILAB software, they may be regarded as that species.

Discussion

The API Staphylococcus system seems to be suf- ficiently diagnostic to identify S . xylosus and S . carnosus. By referring to the biochemical and genomical identifications, it was possible to select some features for a better differentiation

Identification of Staphylococcus dry sausage 77 of S. saprophyticus and S. warneri from other staphylococci (Table 2). Novobiocin susceptibility is the most useful character. Staphylo- coccus warneri is easily separated from S. epidermidis by two tests: fermentation of trihalose and production of D-lactic acid. Accord- ing to Schleifer and Kloos (1975a), S. warneri and S . saprophyticus do not ferment mannose, but in this study some strains identified as these species fermented this carbohydrate.

The present study indicates that the staphylococcus species in French dried sausages are essentially composed of S. carnosus, S. xylosus, S . warneri and S . saprophyticus. Fischer and Schleifer (1980) reported the occurrence of S. simulans in dry sausages made in Germany. This species was not found in this study. The occurrence of S. saprophyticus and S. warneri in dry sausage is not often mentioned. Only Seager et al. (1986) found that S. saprophyticus, S. xylosus and S. warneri represented 76% of their isolates from fermented meats. Staphylococcus saprophyticus and S. warneri probably come from the skin of pork where they were found living.

We are grateful to Dr Jean-Louis Berdague for his helpful contribution with computer analysis.

References BRENNER, D.J., MCWHORTER, A.C., LEITE KNUTSON,

A.C. & STEIGERWALT, J.K. 1982 Escherichia uul- neris: a new species of Enterobacteriaceae associ- ated with human wounds. Journal of Clinical Microbiology 15, 1133-1 140.

FISCHER, U. & SCHLEIFER, K. H. 1980 Vorkommen von Staphylokokken und Mikrokokken in Roh- wurst. Fleischwirtschaf 60(5), 1046-1049.

GRIMONT, P.A.D., POPOFF, M.Y., GRIMONT, F., COY- NAULT, c. & LEMELI, M. 1980 Reproducibility and correlation study of three deoxyribonucleic acid hybridization procedures. Current Microbiology

K m s , W.E. & SCHLEIFER, K.H. 1975a Isolation and characterization of staphylococci from human skin.

325-330.

I1 Description of four new specimens: Staphylo- coccus warneri, Staphylococcus capitis, Staphylo- coccus hominis and Staphylococcus simulans. International Journal of Systematic Bacteriology 25,

KLOOS, W. E. & SCHLEIFER, K. H. 1975b Simplified scheme for routine identification of human Staphy- lococcus species. Journal of Clinical Microbiology 1,

KLOOS, W.E., TORNABENE, T.G. & SCHLEIFER, K.H. 1974 Isolation and characterisation of micrococci isolated from human skin, including two new species: Micrococcus lylae and Micrococcus kristinae. International Journal of Systematic Bacte- riology 24, 79-101.

KOUKER, G. & JAEGFX, K.E. 1986 Specific and sensitive plate assay for bacterial lipases. Applied and Environmental Microbiology 53,211-213.

LIEPE, H.U. 1983 Starter cultures in meat production. Biotechnology 5,400-424.

Lucm, F.K. 1986 Microbiological processes in the manufacture of dry sausage and raw ham. Fleisch- wirtschaf, 65(10), 1505-1509.

NYCHAS, G.J.E. & ARKOUDELOS, J.S. 1990 Staphylo- cocci: their role in fermented sausages. Journal of Applied Bacteriology (Symposium series No. 19),

RRONE, G. & E. MANGAELLI, J.S. 1990 Caratteriza- zione di Micrococcaceae isolate da salami tipo Napoli. Industria Conserue 65,220-223.

ferenzierung von Mikroorganismen der Familie Micrococcaceae aus Rohwiirsten. Fleischwirtschajl

SCHLEIFER, K.H. & K m s , W.E. 1975a A simple test system for the separation of staphylococci and micrococci. Journal of Clinical Microbiology I, 337- 338.

SCHLEIFER, K.H. & K m s , W.E. 1975b Staphylococci from human skin. International Journal of System- atic Bacteriology 25, 50-61.

SCHLEIFER, K.H. & FISCHER, U. 1982 Description of a new species of the genus Staphylococcus: Staphylo- coccus carnosus. International Journal of Systematic Bacteriology 32, 153-156.

SEAGER, M.S., BANKS, J.G., BLACKBURN, C. & BOARD, R.G. 1986 A taxonomic study of staphylococcus spp. isolated from fermented sausages. Journal of Food Protection 51,295-297.

SIMONET~I, P. & CANTON], C. 1983 Coagulase negative staphylococci for dry sausage ripening. Industrial Alimentaria 22 (204), 262-264.

62-79.

82-88.

167s-188s.

RHEINBABEN, K.V. & HADLOCK, R. 1979 Gattungsdif-

59, 1321-1324.

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Identification of Staphylococcus from French dry sausage