Ribotyping of Staphylococcus caprae isolated from goat milk

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Ribotyping of Staphylococcus caprae isolated from goat

milk

Nora Bedidi-Madani, Angeli Kodjo, Laurence Villard, Yves Richard

To cite this version:

Original

article

Ribotyping

of

_{Staphylococcus}

_caprae

isolated from

_goat

milk

Nora

Bedidi-Madani,

_{Angeli Kodjo}

Laurence

Villard Yves Richard*

Unité de

_{microbiologie}

et

_{épidémiologie}

moléculaire,

École

vétérinaire de _Lyon, l, avenue

_Bourgelat,

69280

_{Marcy-l’Étoile,}

France

(Received 3

_September

_1997;

_accepted

I 1 December ₁₉₉₇₎

Abstract - The usefulness of the API-STAPH _system and a method based on ribosomal

fin-gerprinting

was evaluated

_{by studying}

89

_{coagulase-negative staphylococci}

isolated from _goat milk. The bacteria were

_supposed

to

_belong

to the

_{species Staphu}

_l

_ncoccus

_caprae. The results obtained from the API-STAPH _system showed variations in their

_phenotypic

features. DNA

_cleavage

with EcoRl

_yielded

six

_ribotypes

and three distinct _patterns were

_generated

when the DNA of the strains was

_digested

with HindIlI.

_Forty-six

strains were

_correctly

characterized as S. _caprae

_by

the two methods. In addition, 37 isolates

_{having atypical}

biochemical

_profiles

with the API-STAPH system were confirmed as

_being

S. _caprae

_by

the

_ribotyping.

Three strains remained unclassified

_by

both methods.

_Ribotypes

_{generated by} Hintllll were found to be the most infor-mative for

_species

determination, whereas the number of bands

_{generated by}

EcoRI indicated the usefulness of this enzyme in S. caprae

typing.

© Inra/Elsevier, Paris

coagulase-negative-staphylococci

/ _goat / API-STAPH _system /

_ribotype

/

_{Staphylococcus}

caprae

Résumé -

_Ribotypage

de

_{Staphylococcus}

_caprae isolé de lait de chèvre. L’utilisation du sys-tème API-STAPH et l’étude du

_ribotype

ont été mis en oeuvre _{pour confirmer}

_{l’appartenance}

à

l’espèce

S. caprae de 89 souches de

_{staphylocoques}

à

_{coagulase négative}

isolées de lait de chèvre. Les résultats obtenus lors de l’identification

_{par le système}

API-STAPH ont montré une

hétérogénéité

des

_{profils biochimiques.}

L’étude des

_{ribotypes après digestion}

des ADN _{par les} endonucléases EcoRI et Hiniflll a

_permis

d’obtenir

_{respectivement}

six et trois

_ribotypes

différents.

Quarante-six souches ont été ainsi correctement identifiées S. _{caprae par les deux méthodes.}

Trente-sept souches sur 40 _ayant un

_{profil biochimique atypique,}

ont _{pu être identifiées par la}

méthode du

_ribotype.

Les trois autres souches sont restées non identifiées. Les

_profils

obtenus avec

*

Correspondence

and

_reprints

Hin d

I]]

_{permettent la}

détermination de

_l’espèce

S. _{caprae, alors que} ceux obtenus avec _EcoRI

per-mettent une classification

_{plus précise}

des souches au sein de cette

_espèce.

@ Inra/Elsevier, Paris

staphylococci à coagulase negative / chèvre /

_système

API-STAPH /

ribotype

/

Staphylo-coccus _caprae

1. INTRODUCTION

Coagulase-negative

staphylococci

(CNS)

are often _present in _goat milk

sam-ples

collected from

_apparently

normal udder halves and from halves with sub

clinical mastitis

_[22].

_{Staphylococci}

have been associated with clinical and/or sub clinical mastitis and have been much less

widely

studied in farm animals

_{[I I].}

This is

_particularly

true for

CNS,

which are an

important

cause of acute and sub acute

mastitis in cattle

_[10],

in _{goats [22, 23]}

and in ewes

_{[ 16]. However,}

the

signifi-cance of CNS in udder infections in _goats and other animals is unclear.

Staphylococcus

caprae was first

described in 1983 and this

_species

has

been isolated from

_goat’s

milk

_[12].

This CNS

_species

is often isolated from _goat milk

_{[7, 9, 17, 28]}

and seems to be asso-ciated with this host.

_Attempts

to recover

S. caprae from other animals

(i.e.

cows,

ewes)

have _proven to be unsuccessful

_[4,

26]. However,

isolation of this bacterium

from humans affected with

_dermatitis,

uri-nary tract infection

_[19]

and other

infec-tions

_[29]

has been

_reported.

Kloos and Schleifer

_[20]

and Devriese

et al.

_[13]

have

_designed

schemes which

they proposed

for CNS differentiation and identification based on their

_respective

biochemical

_{properties. Although}

these schemes have been able to

_{appropriately}

identify

most

_species,

their

_application

in

routine

_diagnosis

of CNS can be ineffec-tive in some

_instances,

_particularly

for

CNS strains isolated from animals

_[2].

Molecular methods have been

devel-oped

and

_proposed

for bacterial

_typing.

One of

these,

designed

as ribosomal RNA

(rRNA)

gene restriction pattern

analysis

(ribotyping)

has been

_extensively

_applied

in a wide _{range of} bacterial

_species

_[15],

including

staphylococcal species typing

and identification

[3, 7, 14].

We isolated 89 strains of CNS which we

_{thought belonged}

to _{the S. caprae}

species

because of their biochemical fea-tures.

_They

were isolated from _goat milk

sampled

in three different areas of France. The aim of the present

study

was an

exten-sive

_analysis

of these isolates

_by

ribotyp-ing

in order to confirm the

_previous

bio-chemical

_{findings suggesting}

that

_they

were strains of S. _caprae or

_{demonstrating}

that

_they

were other

_{staphylococci species.}

2. MATERIALS AND METHODS 2.1. Bacterial strains

Eighty-nine staphylococcal

isolates were collected

_following

culture of _{goat milk} spec-imens

_{aseptically sampled}

in herds from three different areas in France ₍₁₄ isolates from

Poitou-Charentes, 41 isolates from Ardèche and 34 from

_{Rh6ne-Alpes).}

Clinical mastitis was not observed

_throughout

the

_study.

Two human isolates of S. _caprae

_{(kindly provided}

by

Professor J. Etienne, Centre National de Reference des

_{Staphylocoques, Lyon,}

_France) and

_eight

other _type

_{species (S.}

_caprae ATCC

35538, S.

_xylosu.s

ATCC 35663, S. simulans ATCC 27848, S.

_chromo

_f

_.ienes

ATCC _43764, S. _capitis ATCC _35661, S.

_gallinarum

ATCC

35539, S. cohnii ATCC 35662, S.

epidermidis

ATCC ₁₄₉₉₀₎ were included in the _study for

comparison

purposes.

2.2.

_Species

identification

biochemical identification on standardised API-STAPH

_strip

systems

using

19 substrates (Bio-M6rieux, France).

2.3. DNA

_preparation

Extraction and

_purification

of the total DNA from the

_{staphylococcal}

strains were carried out as

_previously

described [24]. DNA

sam-ples (8 pg)

were cleaved

_separately

with R-oRI and HindIII endonucleases

_(Eurogentec,

Seraing, Belgium), according

to a

_slightly

mod-ified version of the manufacturer’s instruc-tions. DNAs were

_digested

for 4 h with 2U _(in

total) of each endonuclease _per

₁

_N

_g

of DNA. Half of the

_quantity

_{of enzyme (} U) was used for the first 2 h of incubation at 37 °C, then the

_remaining

half was added for a further 2 h of incubation. This

_{procedure, routinely}

used in our

_{laboratory, usually}

ensured

_complete

diges-tion of most of the DNA. Restriction

_fragments

were

_{separated by electrophoresis}

in 0.8 °l°

(w/v) agarose

gel (Appligene, Illkirch,

France)

in TAE buffer (0.04 M Tris Acetate 0.001 M EDTA buffer) at 2.5 V/cm. MM

_digestion

of DNA from Citrohacter diversus

_(kindly

pro-vided

_by

P.A.D. Grimont, Institut Pasteur,

Paris) was used as a molecular size marker.

2.4. Southern

_blotting

and

_{hybridization}

DNA

_fragments

were vacuum transferred

to neutral

_nylon

membrane, (Amersham, Les

Ulis, France), and were UV cross-linked at

0.125

_{joules/cm!’.}

The Southern blots were

hybridized

at 60 °C with I

_pg/mL

of a 16S-23S rRNA

_probe

labelled with acety-laminofluorene _{(AAF), (Eurogentec)} in 10 mL of total

_{hybridization}

fluid. DNA fragments

encoding

rRNA genes

including

those gener-ated _{by digested} Citroba(-ter diver,su.s Mill I were visualized

_{by immunoenzymatic}

detec-tion

_according

to the

_{specifications}

of the man-ufacturer. The DNA

_fragments

of

_digested

Cit-rohacter f7/t!’.BM.! MluI which were used in all blots as size markers included: _{16 751, 12 481, 1,} 7 330, 6 551, 5 75 5 5 097, 4 404, 3 022, 2 777,

1 695, 1 443, 1

_{170 bp.}

2.5.

Analysis

of

_ribotyping

The

_fragment

size of the

_ribotype

_patterns were calculated from

_migration

distances and

analysed

using

the TAXOTRON software

(Institut Pasteur, Paris, France).

3. RESULTS

3.1.

_Biotyping

Forty-nine

of the 89 isolates were

iden-tified

_according

to biochemical

charac-terisation,

recorded in a five

_{digit profile}

number,

as S. _caprae.

_Forty

strains were

given

an

_{atypical profile according}

to the

API-STAPH database, which could lead to

false identification

_{(table I).}

In this last

group, six different clusters were found

on the basis of their

_phenotypic

proper-ties.

3.2.

_Ribotyping

3.2.1. EcoRl

_{hybridization}

_pattern

analysis

Six

_ribotype

_patterns

_designated

as

ribo-type

I to

_6,

were found when the DNA

from the 89 isolates was cleaved with

EcoRI endonuclease. The EcoRl rDNA

banding

patterns varied between 10 and

18 bands _per

lane,

with

_fragment

sizes between 0.9-9.3 kb.

Comparison

of S. _caprae

_(type

strain

and

_wild-type

_isolates)

_{hybridization}

pat-terns with other different

_{staphylococci}

type

species

analysed through

the

_study

demonstrated the _presence of

species-spe-cific

_{ribotypes (figure}

_I).

_Ribotype

I

which

_regrouped

14 strains

(8

from

Ardeche,

5 from

_Rh6ne-Alpes

and 1 from

Poitou-Charentes),

showed 12

bands,

sim-ilar to the

_{hybridization}

pattern of the S. co

p

rae type strain

(0.9; I .0; 1.2; 1.3; 1.3;

1.7; 2.2; 3. I ; 3.3; 3.6; 4.3 and 4.8

_kb).

The

respectively

40 isolates

₍₁₉

from

Ardeche,

18 from

_Rh6ne-Alpes

and 3 from

Poitou-Charentes),

20 strains

(14

from Ardeche and 6 from

_Rh6ne-Alpes)

and three

iso-lates

(2

from Poitou-Charentes and I from

Rhone-Alpes),

showed the same or almost

the same _pattern as

_ribotype

I but lacked

the 1.73 kb

band,

or the 1.73 and 2.22 kb

bands for

_ribotype

2 and

_ribotype

_3,

respectively.

An additional band of 1.49 kb

was also found within

_ribotype

3.

Ribo-type 4, which

_regrouped

six strains

_(five

from Poitou-Charentes and one from

Rh6ne-Alpes),

showed the same 12 bands

as

_ribotype

_{I plus}

a further six bands of

size 1.4; 2.4; 2.5; 5.8; 7.5 and 9.3 kb.

Ribo-type 5 which included three strains from

Poitou-Charentes,

_displayed

all the bands

of the

_ribotype

I with three additional

bands

_{(2.4; 2.8;}

and 6.0

kb)

(figure

1

).

3.2.2. HindIII

_{hybridization}

_pattern

analysis

Three

_{ribotypes, designated}

I to

3,

were

found after

_digestion

with HindIII. DNAs

from the

_samples

cleaved with HindIII

gave an _{average of} ten bands _per

frag-ments, and this observation made it

pos-sible to

_identify

86 of the isolates as S.

caprae.

Cleavage

with HindIII

_generated

nine

common bands

_representing

the

_ribotype

of the _{S. ccrprae}

_type

strain

(0.7; 0.8;

1.7;

2.4; 4.1; 4.4; 4.9;

7.9 and 8.8

_kb).

The

comparison

between the

_ribotype

of the S. _caprae

_type

strain and the

_ribotypes

of

S. caprae isolates

_analysed

in this work

regrouped

72 of the field strain S. _caprae

(4

strains from

Ardeche,

28 from

Rh6ne-Alpes

and three from

_{Poitou-Charentes).}

When

_compared

to

_ribotype

1,

_ribotype

2,

including

8 S. caprae strains

_(two

strains

from

_Rh6ne-Alpes

and six from

Poitou-Charentes),

lacked the 3.0 kb band. It also

had an additional band of 15.5 kb

_(figure

2).

Ribotype

3, which

_regrouped

six S. caprae strains

(one

strain from

Rh6ne-Alpes

and five from

_{Poitou-Charentes),}

was _very similar to

_ribotype

2 _except that

an additional band of 0.8 kb was observed.

Three strains from the

_Rh6ne-Alpes

pro-duced three distinct

_{hybridization}

_patterns

when their DNA was cleaved with Hindlll and two distinct

_{hybridization}

patterns,

two of these three strains

_yielded

identical

hybridization

patterns. The

_{hybridization}

patterns

of these three strains were _very

different from that of S. _caprae ATCC

type strain

_(figures

I and

2).

Human

strains _{also gave different patterns} with

the two restriction _enzymes

_(figures

I

and

_2).

3.2.3.

_{Comparison of biotypes,}

ribotypes

and origin of isolates

Data

_{regarding biotypes}

and

_ribotypes

versus the

_origin

of the isolates are

recorded in table II.

_Analysis

of these data indicated that there was no evidence of a

relationship

between one

_biotype

or

ribo-type with a

_given

_origin.

In other

_words,

according

to the methods

used,

the same

strains were found in the three herds

inves-tigated,

whatever their localization.

Nev-ertheless,

biotypes

D and E

_(API-Staph

scoring

6506103 and

_6736103)

_appeared

to be the main

_biotypes

of S. _caprae iso-lated from _goats, both

_representing

48 %

of total isolates.

4. DISCUSSION

Ribotyping

has been

_proposed

as a

method for

_identifying

_{staphylococcal}

species

and

_subspecies

_{[6-8, 14].}

Other

identification methods have been

_applied,

including plasmid profile

analysis

[

I

_]

and

antibiotic

_{susceptibility}

_[25

_{J. However,}

these methods are not

_always

able to dis-criminate between

_species.

_Analysis

of

the

_complex

_patterns

obtained after

diges-tion with

_only

total DNA endonucleases is

too difficult to

_interpret

and makes this

method insufficient for

_{discriminating}

iso-lates [21

].

In this

_study

40 of the 89 CNS isolates

were not

_correctly

identified

_by

the

phe-notypic

methods.

_Ribotyping

was used as an additional tool for accurate

identifica-tion and 37 of these

_atypical

isolates were

finally correctly

identified

_using

this method. Three of these 40 isolates remained unidentified whatever the

method used. Because

_they

did not fit the

phenotypic

features of S. c’aprae, these

isolates should be included in another

species.

Similar results were also

_reported

previously

when a radio labelled

_probe

was used

_[5

_1.

Digestion

with HindIII

_produced

fewer

DNA

_fragments

than the EcoRI

_cleavage.

These results confirmed those of

Thomp-son and Carter et al.

[27].

Six

_ribotypes

were found with EcoRl and three

ribo-types were found with

Hindlll,

the same

results were recorded

_by

Izart et al.

_[18].

_J.

The biochemical

_profiles

did not make it

possible

to

_distinguish

between the dif-ferent taxa. However, we found that

iso-lates included in the same _group

_through

biochemical characterization were differ-entiated into six different clusters with

ribotyping

when the DNA was cleaved

with EcoRI and in three different clusters

when the DNA was cleaved with/!tf/in. 1.

De

_Buyser

et al.

_[8]

used the same

endonucleases and concluded that Hificflll

yielded

a better discrimination of most of the

_{staphylococcal}

taxa; however results

from these workers indicated that the two

intermedius carnivora and S. intermedius

pigeon

were

_{only separated}

_by

EcoRI.

Two human isolates included in this

study

were

_previously

characterized

_by

ribotyping by

Vandenesch et al.

[29].

Our

results,

obtained when

_performing

ribo-typing,

showed additional

bands,

partic-ularly

within the EcoRI

_digested

frag-ments, when

_compared

to those

_previously

found. This

_discrepancy

could result from

a

_{partial digestion}

of DNA with this

enzyme due to a

_high

amount of DNA to

be

_{digested (8 pg)}

or a

_high

_background

from our non-radio labelled

_probe.

Initial

settings

of reaction _parameters with

regards

to amount of DNA to be used were

satisfactory

when

_using

_{8 pg for}

_digestion

and vacuum transfer

_prior

to

hybridiza-tion with our non-radio labelled

_probe.

Less DNA

_(i.e.

4 to 5

_pg)

could be used in

in time and

_quantity

of transfer buffer. These remarks

_point

out the need for

stan-dardizing

between

_ribotyping

methods when the

_resulting

databases are to be

compared

between laboratories. Be that as it may, the human strains still

_appeared

significantly

different from the

_caprine

strains as had been found

_previously.

The

_ribotyping

method _may be of value as an additional tool for the identification

of

_{staphylococci}

when

_phenotypical

char-acterization is not efficient. Therefore this

method can be used before

_progressing

to

a DNA/DNA

_{hybridization study}

or other more

_{specialized investigations.}

In

con-trast, its value as

_{epidemiological}

marker

for differentiation of strains of S. _caprae

should be re-evaluated with _respect to

other molecular tools

_{(i.e. pulse-field gel}

electrophoresis, randomly amplified

poly-morphic

DNA)

since it failed to

_provide

evidence on the

_{relationships}

between

strains and

_geographic

_origin

in this

_study.

ACKNOWLEDGMENTS

The authors are indebted to

_{Evelyne Borges,}

Fran!oise

Maurin and Pascale

_Exbrayat

for their technical assistance.

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