Identification of toxigenic Clostridium difficile strains by using a toxin A gene specific probe

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0095-1137/90/081808-05$02.00/0

Identification of Toxigenic Clostridium

difficile

Strains

by Using

a

Toxin

A

Gene-Specific Probe

BRENDAN W. WREN,* CHRISTOPHERL. CLAYTON, NIGELB. CASTLEDINE, AND SOADTABAQCHALI Department of MedicalMicrobiology, St. Bartholomew's HospitalMedicalCollege, WestSmithfield,

LondonECIA 7BE, United Kingdom Received 8November1989/Accepted 12 April 1990

A4.5-kilobasePstIfragment encoding partof the toxinA gene wasisolated and used as aDNAprobe in

colonyhybridization studies with 58 toxigenic and17nontoxigenic Clostridium

difficile

strains.All58toxigenic strains showedpositivehybridization,in contrasttothe 17nontoxigenicstrains. Southern blotanalysiswiththe toxinAgeneprobeshowed hybridizationtoasingle fragmentofequalintensities forHindIII-digested genomic

DNAs isolated from C.

difficile

strains of wide-ranging toxin production. The positivehybridization signals

wereduetofragments ofheterogeneous lengths (9to 13kilobases)fortoxigenicstrainsof different types but

wereabsent for the nontoxigenic strains.Theseresultssuggestthepresenceofasinglecopyof the toxin Agene

onthegenomeofC.

difficile

strains,and the widevariationoftoxinexpression is notareflection ofgenecopy number. The lack of toxin activity fornontoxigenic strainscanbe explained by theabsence of at least part of

the toxin Agene.Thetoxin Ageneprobewastestedagainst clostridial strains from 18otherspecies, ofwhich

only toxigenic C. sordellii strains showed positive hybridization. Thespecificity ofthetoxin Ageneprobefor toxigenic strainsmay lead toimproved methods forthe specific identification oftoxigenic C.

difficile

strains fromclinical specimens.

Clostridium difficile is well recognized as the etiological

agent ofpseudomembranous colitis and appears also tobe responsible forantibiotic-associated colitis and diarrhea(3, 6, 10). Thepathogenicity of the organism is related to the production ofan enterotoxin, toxin A, and a potent

cyto-toxin, toxin B. The diagnosis of C. difficile-associated

dis-ease depends on the isolation and identification of the

organism or the demonstration of toxin in fecal samples. Toxins A and B appear to be large distinct polypeptides, althoughreportsof molecularmassvalues forthetwotoxins

range from 50 to600 kilodaltons (kDa) (2, 11, 14, 17, 20).

Different strains ofC. difficilevaryinthelevel of toxin A and Bproduction from non-toxin producers to extremely high-level toxinproducers, and furthermoreacorrelationappears to exist between the relative amounts of toxins A and B produced forindividual C. difficile strains (23, 25).

We recently clonedtoxin A from C. difficile into Esche-richia coli K-12 by using the bacteriophage cloning vector

XEMBL3(24). The clone XtA5 containsa 14.3-kilobase (kb)

DNAinsert which encodesa235-kDaprotein. This protein reacts with antisera to the purified toxin A, agglutinates rabbit erythrocytes, and has a cytopathic effect on tissue

culture cells (24). There have been three other reports concerningthecloningof toxin Agenefragments. Muldrow

et al. (13) reported the cloningofa0.3-kb fragment ofthe

toxin A gene which cross-hybridizes with a 4.5-kb PstI fragmentofC. difficile genomic DNA. Avariety of overlap-ping fragments ofthe toxin Agenehavebeen cloned into the vectorpUC12 byvonEichel-Streiberetal. (22). Price etal. (15)cloneda4.7-kb PstI toxin A fragmentinto pBR322 and

have suggestedthat thisregion of the toxinAgeneencodes

the receptor-binding portion of the polypeptide. However, extensivehybridizationstudiesusingatoxinAgene-specific probe have notbeenundertaken. The aim of this studywas to evaluatea4.5-kbPstI fragment from thetoxin-producing

clone AtA5 as aDNAprobe inhybridization studies for the

* Correspondingauthor.

specific identification of a variety of typed toxigenic C.

difficile

strains and to determine whether the probe cross-hybridizes with other clostridialspecies.

MATERIALS ANDMETHODS

Bacterial strains.Seventy-fiveclinical strains of C.difficile isolated from patients at St. Bartholomew's Hospital were

culturedoncefoxitin-cycloserine-fructoseagarselective

me-dium(OxoidLtd., Basingstoke, UnitedKingdom)and iden-tified by Gram stain, smell, colony morphology, and gas-liquid chromatography (25). The C.

difficile

strains were

stored in Robertson cooked-meat medium(SouthernGroup Laboratories, London,UnitedKingdom)untilrequired. All

C.

difficile

strains were typed according to their

[35S]-methionine-labeled sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles (21). C.

difficile

strain typesused in thisstudyareshown in Table1.

Clostridial strains from 18 other species shown in Table 2

were cultured on horse blood Columbia agar plates and

identified by standard procedures (9). The E. coli K-12 derivative 392(lysogenicforphageP2)wastherecipient for transfection cloning manipulations with the bacteriophage

vector XEMBL3, and E. coli K-12 derivative JM105 was

used for transformationexperimentswith theplasmidvector pUC18. E. coli K-12 strains were grown on Luria-Bertani

brothor on Luria-Bertaniagar(12).

Purification of toxin A and antitoxin Apreparation. Toxin Awas purified tohomogeneity from a 5-litergrowth of C. difficile, as previously described (24). Antisera to toxin A

were raised in male Californian rabbits by the method of Redmondetal. (16).

Toxin assays. All C. difficile strains were tested for the

presence of toxins A and B from Robertson cooked-meat

medium. Toxin Awasmeasuredquantitatively bythe direct sandwich enzyme-linked immunosorbent assay, by using antitoxin A as described by Redmond et al. (16). Toxin B titersweredeterminedby usingculturedHEp-2tissuecells,

asdescribed previously (25). Titrations wereperformed by 1808

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TABLE 1. [35S]methionine-labeled PAGEprotein types of

C.difficile analyzed for toxin production and hybridization to thetoxin Agene-specificprobe

No.of strains with Colony PAGE No. of strains toxin A and Bactivity hybridization

type studied Not Lo ih No. No.

detected Low High negative positive

A 5 5 0 0 5 0

B 5 0 1 4 0 5

C 5 2 0 3 2 3

D 3 0 0 3 0 3

E 17 0 0 17 0 17

W 1 0 0 1 0 1

X 28 0 22 6 0 28

Y 10 10 0 0 10 0

z

1 0 1 0 0 1

aLowtoxin producer:Toxin A, <40ng/ml; toxinB titer, <1/128. High toxin producer: ToxinA, >40ng/ml; toxinBtiter,>1/128.

doubling-dilution

series

from unconcentrated

culture

fil-trates, and

titers

were

recorded

at

the

dilution that

gave a 50% cell-rounding endpoint. The strains were divided for purposes

of

analysis into three categories: strains with

nondetectable

toxin,

low-level

toxin

producers, and

high-level-toxin-producing strains. High toxin-producing

strains

were defined as those which produced a titer of 1/128 or greater in the

toxin

Bassay

and

more than 40 ng

of toxin

A per ml

in

the

toxin

A assay. Low

toxin producers

werethose

strains with detectable toxin

production

at levels

below

those stated above.

SDS-PAGE and immunoblot analysis.

SDS-PAGE,

electro-phoretic

transfer

(immunoblotting),

and the

development of

immunoreactive

protein products were

performed

as

de-scribed previously

(8).

Isolation of DNA andrestriction endonuclease analysis. C.

difficile

genomic

DNA

for Southern blot

analysis

was

pre-TABLE 2. Colonyhybridization of clostridial strains other than

C.

difficile

withthe toxinAgene-specific probe

Colony hybridi-Straina zation with 4.5-kb

PstIprobe

C.

beijerinckii

NCTC 11920. C. butyricum NCTC 7423.

C. carnis NCTC 10913.

-C. chauvoei NCTC8070.

C. histolyticum NCTC 503.

-C.novyi typeANCTC538.

C.

paraputrificum

NCTC11823.

C.perfringensNCTC 1265.

C.putrefaciensNCTC9836.

C. septicum NCTC547.

C. sordelliiNCTC 6800. +

C. sordelliiNCTC 8780. +

C. sphenoidesNCTC 507.

C. sporogenesNCTC532.

-C. tertiumNCTC541.

C. tetani NCTC540.

C. tetanomorphum NCTC 540.

-C. innocuum R657.

C.fallaxR2720.

aAll strainsusedfor this studyexcept C.innocuum R657andC.fallax

R2720wereobtained fromtheNational CollectionofTypeCultures, Public HealthLaboratory Service, Colindale, United Kingdom. C. innocuum R657

and C.fallaxR2720 wereobtained fromM.Phillips,Public HealthLaboratory

Service,Luton,UnitedKingdom.

pared as previously described (27). Attempts to prepare high-molecular-weight plasmid DNA with lysates of repre-sentatives of each of the nine

[35S]methionine-labeled

PAGE-type protein groups A to E and W to Z were made by the method of Anderson and McKay (1) for isolating high-molecular-weightplasmid DNA from streptococcal species and thatof Strom et al. (19) for isolating plasmid DNA from clostridial species. The restriction endonucleases BamHI, CfoI, DdeI, EcoRI, EcoRV, HindIII, PstI, SalI,

SmaI,

TaqI, XbaI, and XhoI were used to digest C. difficile genomic DNA, as recommended

by the suppliers (Amersham

Inter-national, Amersham,

United Kingdom).

OnlyHindIII

con-sistently

resulted in

complete digestion of

C.

difficile

DNA

and was the most suitable restriction enzyme for the pur-poses of thisstudy.

Isolation of recombinant DNA and subcloning. The

plate

lysate method

(12) was

used for isolation of

DNA

from

the

clone XtA5.

Plasmid

DNAs

from

the

recombinant

subclones were isolated in bulk by the alkaline lysis method (12). Restriction endonuclease

digestion

of XtA5 was

performed

by using the enzymes described above and mapped by

standard

procedures (12). The

vector

pUC18

was used for

subcloning,

and recombinants were transformed into E. coli

K-12 strainJM105

by

the calcium chloride

procedure

(12). All recombinant E. coli clones were grown on Luria-Bertani broth or agar

supplemented

with

ampicillin (100 ,Lg/ml).

Colony hybridization and Southern blot tests.

Samples

for

colony hybridization

were grown

directly

on

gridded nylon

filters (Hybond-N; Amersham International) overlaid on

cefoxitin-cycloserine-fructose

agar

plates

for 24 h at

37°C

under anaerobic conditions.

When

colony growth

was

visi-ble,

the

nylon filters

were

placed

on

filter

paper saturated

with

1%

SDS

solution

and denatured

by

the method of

Grunstein

and

Hogness

(7).

Anextra

denaturing

and

neutral-ization

step was

included

to ensure

complete

lysis of all

bacterial

cells.

C.

difficile

DNA

for Southern blot

hybridiza-tion analysis was digested to completion with

HindIII,

and

the

fragments

were

separated

by

electrophoresis

ina

hori-zontal

gel containing

0.5%

agarose at 60 V

for

20

h

and

transferred

to a

nylon membrane

by the method of Southern

(18).

The

toxin

Agene

probe

was

radiolabeled in vitro with

[-y-32P]dCTP

(Amersham

International) by

the random

primer

hexamer

method of

Feinberg

and

Vogelstein

(5).

Southern blot

hybridizations

were

performed

with dextran

sulphate

enhancer and 50%

formamide,

whereasfor

colony

hybridizations

the

best

results were

obtained

by

using

25%

formamide

(26). All

filters were washed in 0.3 M sodium

chloride-0.06

M

Tris

hydrochloride (pH

8.0)-0.002

MEDTA

for 5 minand the same solution

including

1.0% SDS for 30 min at

68°C. All blots

were

dried and exposed

to

Fuji-RX

X-ray

film

at

-70°C for

16 h.

RESULTS

Figure

1 shows a

partial restriction

map

of

the

bacterio-phage

clone

XtA5,

which

contains

a 14.3-kb DNA insert

originally

isolated from a clinical isolate of a

high-level-toxin-producing

C.

difficile

strain

(Wl).

A4.5-kb PstI

frag-ment was

ligated

into the

plasmid

vector

pUC18

to

produce

subcione

pBWW47.

Immunoblot

analysis

with antitoxin A

against

a

lysate of clone pBWW47

revealed a

protein band of

an

approximate molecular

mass

of

140

kDa

(Fig. 2,

lane

1),

in contrast to

AtA5, which

showed a

protein

band

of

an

approximate

molecular mass of 235 kDa

(lane

3).

No

cross-reactions,

apart from

background

bands due to

nonspecific

reactions with the host strain E. coli

K-12,

were observed

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B H X P X EvXh E P H B

- Il'

I

'

I'

I

1_

0kb 14kb

DNA PROBE

FIG. 1. Partial restriction cleavage map of the 14.3-kb insert

from thebacteriophage clone XtA5. The DNA probe used for this

study is indicated. B,BamHI;E,EcoRI;Ev, EcoRV;H,HindIII;P,

PstI; X,XbaI;Xh,XhoI.

with pUC18

alone (lane

2). On the basis of these results and

nucleotide

sequence data (unpublished

results),

it is likely

that the

4.5-kb DNA fragment represents an internal region

of

the whole toxin Agene, and thus it was chosen as a toxin Agene-specific probe for hybridization studies.

Seventy-five

typed C.

difficile

strains were tested for toxin

A and B

production,

and 58 were positive by both assays

(Table

1). Thelevel of toxin A activity appeared to correlate

with

toxin Bactivity for

individual

strains; for example, all

strains defined

aslow-level toxin A producers were all low

toxin

B

producers.

The

remaining

17 strains were

negative

for toxin production in both

of the

toxin

assays.

Colony hybridization experiments with

the

radiolabeled

4.5-kb DNAprobe revealed positive hybridization with all 58

toxigenic strains

ofvarious degrees of toxicity (mainly types

Eand X) and nohybridization with types A, C, and Y, which

consistently

have shown no

toxin

production (Table 1).

Of

theotherclostridial strains from 18 other species, 2 toxigenic C.sordellii strains (which were

positive

in the

toxin

Aand B assays)

cross-hybridized

with thetoxin A gene probe.

Genomic

DNA

from nine

strains of differing

types (A to E

and

Wto Z) were

digested

with

HindIlI,

andequal

concen-trations of

DNA were loaded onto an agarose gel and

electrophoresed. After Southern transfer,

the samples were

hybridized

with the 4.5-kb

PstI

probe (Fig. 3). The probe

hybridized

to a

single

HindIlI

fragment

of

equal

autoradio-graphic intensities for

the six

toxigenic strains

(B,

D,

E, W,

X, and Z) tested. The sizes of the fragments to which the

probe hybridized varied between

9 and 13 kb

for strains from

different

typing

groups. No

plasmid

DNA larger than 9 kb

wasisolated from the toxigenic strains used in Southern blot

studies

using

the

methods of Anderson

and McKay (1) and

Strom

etal. (19).

DISCUSSION

This

study showed

that C.

difficile strains which

lack toxin

activitv

aopear

to have at

least

4.5 kb

of

the

toxin

Agene

I1 2 3

__ 235

140

FIG. 2. Immunoblot analysis of recombinant clones by using antitoxinA. Lanes: 1, 2

pug

ofE.coliJM105transformedwithclone

pBWW47containinga4.5-kbDNAinsert;2, 2 ,ugofE.coliJM105 transformedwithpUC18 (negative control);3, 2 ,ugofE.coli P2 392

transfected with clone AtA5 containing a 14.3-kb DNA insert. Numbers to the left and right of the gel show molecular masses

(kilodaltons) of proteins.

FIG. 3. Hybridization analysis after Southerntransferwiththe

32P-radiolabeled

4.5-kb PstI fragment against 3 ,ug of

HindIlI-digestedC.difficilegenomicDNAsfrom standardtypestrainsAto

EandWto Z.

absent. Immunoblot analysis revealed that the 4.5-kb PstI

fragment encodes

a

protein

of 140 kDa, which is

approxi-mately

half the

size

of

the

molecular

mass

of the

purified

toxin

A (11, 24) and

approximately

half the size of the

protein encoded by the original clone XtA5. Nucleotide data from

this

laboratory (unpublished data) suggest that the 4.5-kb

PstI

fragment is completely within the toxin A gene. Theapparent

translation of

an

internal

region of

a

clostridial

gene in E. coli is

surprising.

It

is

likely

that

transcription of

the 4.5-kb Pst

fragment

in

pBWW47

starts

from

anexternal promoter

(lac)

of the

pUC

vector. Similar

observations

have been made by von

Eichel-Streiber

et al.

for

their set of

overlapping fragments

of the toxin A gene cloned into

pUC12 (22).

Use

of

the 14.3-kb BamHI insert

from

XtA5 as

a

toxin

probe failed

to

descriminate

between

toxigenic and

nontoxigenic strains (unpublished data), indicating

thatthe

strains

probably

sharehomology with DNA

flanking

atleast

oneend

of

the

toxin

Agene. Further

hybridization studies

arenecessary to

determine

ifall of the

toxin

A

and/or

toxin B genes are absent in

nontoxigenic strains.

Southern

blot

analysis of toxigenic

C.

difficile

strains of

various

toxigenic activities

revealed a

single

hybridization

band

of

equal intensity for

thesix strains

(B,

D,

E, W, X,

and

Z)

tested.

Since

equal

amounts

of

DNA were

probed

with

the4.5-kb

PstI

fragment,

these resultssuggest that a

single

copy

of

the

toxin

Agene is present on the C.

difficile

genome

and that the

variation

in

toxin

expression is

not a

reflection

of

gene copy number. In the absence of

detectable

plasmid

DNA

fragments larger

than 9kb

from

the

toxigenic strains

tested,

the toxin A gene is assumed to be

chromosomally

located.

Price et al.

(15)

have

reported

the use of a 2.1-kb PstI

fragment

as atoxin A

probe

in Southernblot

hybridization

studies.

In

their

report, five

toxigenic

strains were

positive

by

Southern

blot

hybridization

and three

nontoxigenic

strains were

negative.

However, no

colony

hybridization

experiments

against

C.

difficile

and other

clostridial

strains

were

performed.

The 2.1-kb PstIfragment used by Priceet

al.

(15)

waspart of a 4.7-kb

PstI

DNAinsert froma

pBR322

clone

originally isolated

from the

highly

toxigenic

C.

difficile

strain VPI 10643. von Eichel-Streiber et al.

(22)

have also

reported

the presence of a 4.7-kb PstI

fragment,

which

encodesa

protein product

of

approximately

140

kDa,

aspart ofa set

of

overlapping

toxin A clones

isolated

from thesame

C.

difficile strain,

VPI10643.

Also, Muldrow

etal.

(13)

have

reported

a 0.3-kb

fragment

of the toxin A gene which

cross-hybridizes

with a 4.5-kb PstI

fragment

of C.

difficile

genomic

DNA from the same strain.

Judging from

the

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relative positions of the restriction enzyme sites for EcoRI, EcoRV, and XbaI on the restriction mapsfor

AtA5

andfor toxin A clones reported by Price et

al.

(15) and von Eichel-Streiber et

al.

(22) and the observation that plasmid clones with two

different

sizes of fragments produce proteins of approximately 140 kDa which cross-react with antitoxin A,it is likely that the two

PstI

fragments encode the same portions of the toxin A gene. However, apart from the

different

sizes of the

PstI

fragments, there are other differ-ences,

notably

the lack of an

internal PstI

site in the 4.5-kb

PstI

clone. Theseanomalies are probably due to the

different

sourcesof C. difficile DNA, from which the toxin A clones were obtained (strain Wl [this

study]

and strain VPI 10643

[15,

22]).

These observations are not surprising, particularly

inview of the heterogeneity of

HindIII

restriction sites noted among

different

toxigenic C.

difficile

strains in this study and by Price et

al.

(15).

Of

the 18 other clostridial species tested with the toxin A gene probe,

only

2 C.

sordellii

strains showed positive hybridization. These

results

are inkeeping with the fact that the two C.

sordellii

strains were positive on the toxin A and B assays and that C.

sordellii

antitoxin is known to cross-react with the C. difficile toxins, which are routinely used on tissue culture

cells

as a test to neutralize C. difficile toxin activity from patient fecal specimens. However, despite the fact that C. sordellii and C. difficile have closely related toxins, as confirmed at the DNA level by this study and by Priceet

al.

(15), C. sordellii has notbeen

found

tobea cause of pseudomembranous colitis or antibiotic-associated dis-ease in humans. This suggests that factors in addition to toxin A are probably

responsible

for C.

dîfficile-related

disease.

A striking observation made with

all

75 C. difficile strains used in this study was the wide range of toxin A and B production among strains of different types. For example, type E strains were highly toxigenic and type X strains showed variable toxin production, whereas type Y strains are nontoxigenic. Furthermore, a correlation between the amounts of toxins A and B produced forindividual strains appears to exist. This

1:1

ratio in the production of toxins A and B by C. difficile strains hasalso been noted by Wilkins et

al.

(23). An explanation for the apparent coregulation of toxins A and B is

offered

by the recent studies of Dove etal. (4), who have shown that the two toxins are separated by a 1.4-kb DNA fragment and are therefore likely to be part of the same operon.

This study demonstrates that a 4.5-kbPstI fragment of C.

difficile

toxin A is an efficient probe for the identification of toxigenic C.

difficile

and C. sordellii strains. However, we

have

found

that using the toxin A probe directly on fecal

specimens to identify toxigenic C. difficile strains generally fails due to nonspecific binding offecal debris to the nylon membrane support (unpublished data). The toxin A gene-specific probe may prove a useful alternative to tissue culture

cells

for the identification of toxigenic C.

difficile

strains, particularly if the problems ofnonspecific binding can be overcome and the development of more rapid hybrid-ization

procedures

can be attained. Alternatively, the poly-merase chain reaction amplification of a suitable region of toxin A, for example,within the 4.5-kbPstI fragment which is absent in nontoxigenic strains should overcomethe neces-sity for using nylon membranes and provide a more sensitive

detection

method. Evaluation by the polymerase chain

re-action method of toxigenic C. difficile colonies and

clinical

specimens is in

progress.

ACKNOWLEDGMENTS

We thank Sandy Gale for typingthemanuscript.

This work was supportedby theMedical Research Counciland

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