Clinical application of novel sample processing technology for the identification of salmonellae by using DNA probes

0095-1137/90/020237-05$02.00/0

Copyright C 1990, AmericanSocietyforMicrobiology

Clinical

Application of Novel Sample

Processing Technology

for

the

Identification

of Salmonellae

by

Using

DNA

Probes

DAVID R. SCHOLL,l2* CINDY KAUFMANN,3 JOSEPH D. JOLLICK,4 CHARLES K. YORK,1 GAIL R. GOODRUM,' AND PATRICIACHARACHE3

DiagnosticHybrids,

Inc.,'

and Department of Zoology and Biomedical Sciences2 andCollege of Osteopathic Medicine,4 Ohio University, Athens, Ohio 45701, and Department ofLaboratory Medicine, JohnsHopkinsMedicalInstitutions,

Baltimore, Maryland 212053

Received30May1989/Accepted16October1989

Two hundred and fifty clinical fecal specimens collected over a 7-month period were analyzed for the

presenceof salmonella byarapidDNAhybridization procedure. Hybridizationswereperformed by usinga

novelspecimenprocessing protocolcalled wickingandapreviously unreported 1,600-base-pair probecloned fromSalmonella enteritidis DNA. The probewasshowntobe reactive withall 70 Salmonellaserotypestested and notreactivewith 101stock strains of other enteric bacteria. Southern analysisof 30 Salmonella isolates

representing22 serotypes suggested thattheprobesequence washighly conserved, appearingas a 1,600-base-pair band inaBglll digestof isolate DNA in 29 of30 isolates andas a 2,300-base-pair fragmentin 1 of the

isolates.Theprobecorrectlyidentified all salmonella (nine isolates)among47H2S-producingcoloniestested

from among 250 clinical specimenscultured onxylose-lysine-desoxycholate medium. Salmonellae grown on

xylose-lysine-desoxycholate medium gave consistently higher hybridization values than did those grown on eitherMacConkey orHektoen entericagar. Inaddition, ofeight gram-negativebrothenrichments in which

salmonella were identified by conventional means, seven were probe positive. The useof this nucleic acid probeand hybridizationtechnique provides asimpleandrapididentification of Salmonellaspecies.

Rapid and accurate detection of salmonella is a major

concern in food quality testing, environmental water sam-pling, andclinicallaboratories. Althoughprogress has been

made in streamlining the detection and identification of salmonella (5, 6), new methods often do not achieve the

specificity or sensitivity of the standard microbiologie pro-cedures. Typically, culture isperformedon aseriesof solid mediacapable ofselecting forand/ordifferentiatingbetween

salmonella andotherenteric bacteria. MacConkey (MAC) and Hektoen enteric (HE) agars are examples of

gram-negativeselective media which differentiate microorganisms

than can ferment lactose orproduce hydrogen sulfide from

those organismsthatcannot.Themajorityof salmonellaare recognized as non-lactose fermenters (Lac-) and hydrogen

sulfideproducers (H2S+). SuspectedSalmonella isolatesare then identified by biochemical substrate utilization and/or salmonella-specific agglutinationwithpooledantisera. Com-parison ofmetabolic performance with established profiles from known Salmonella species results in a confirmatory identification.

Conventional approaches require follow-up confirmatory testing ofall

H2S'

and/orLac- coloniesandextendthe time

for identification. Since a majority ofthe

H2S'

and Lac-colonies turn out nottobe Salmonella species (e.g., Citro-bacter, Proteus, or another species), a substantial cost is incurred in settingup and reading thebiochemical testson non-Salmonellaisolates.

The use of nucleic acid hybridization and nucleic acid probes formicrobialidentification isanalternativeapproach

thatdifferentiates microorganisms basedon theirgenotypic uniqueness. Elements required for the use of probes in

clinical applications include the development of pathogen-specific probes, functional levels ofassay sensitivity, and development of test protocols that are simple, rapid, cost

*Correspondingauthor.

effective, and adaptable to the work flow of the clinical laboratory.

The purpose ofthis study was to characterize the genus specificity ofa new, unique Salmonella probe, SAL6, and demonstrate its potential as auseful diagnostic reagentfor identification when coupled with a simple and rapidsample

preparation(referredtoaswicking)andhybridization meth-ods that had been previously developed for viraldiagnostic applications (8; C. S. Kaufmann, G. Goodrum, C. York, J. Jollick, P. Charache, andD. R. Scholl, Abstr. Annu. Meet. Am. Soc. Microbiol. 1988, C130, p. 353).

MATERIALS AND METHODS

Microbiological evaluation. Rectal swabs from patients suspected ofgastrointestinal infection were inoculated on the fivedifferential and/orselective mediausedroutinelyat

Johns Hopkins Hospital: phenylethylalcohol(PEA),MAC, HE,cefsulodin-Irgasan-novobiocin (CIN),and campylobac-ter (CAMPY) agarplates as well as 5 ml of gram-negative

(GN)broth. Inaddition,xylose-lysine-desoxycholate (XLD)

agarwasaddedforexperimental analysis. PEA,MAC, HE,

and XLD were incubated at 37°C, CIN and GN were

incubated at25°C, and CAMPYwasincubated at42°C. All

culturesexceptCAMPY(72 h)wereincubated for18to20h.

All GN broth cultures were subsequently subcultured to MAC and HEplates. All

H2S'

colonies from HE cultures

and Lac- colonies from MAC cultures were initially

screened withtriplesugariron(TSI)mediumandlysine iron

agar (LIA). Those organisms demonstrating TSI-LIA

bio-chemicalreactions consistent forsalmonella weretestedby using salmonella-specific antisera (Fisher Scientific Co., Pittsburgh, Pa.), and confirmed by using a replicate plate system consistingof 17 biochemical reactions.

Hybriwix probe systems: salmonella DNA probetest. The DNA probe test used in this study was developed by Diagnostic Hybrids, Inc., Athens, Ohio, for research

pur-237

on April 12, 2020 by guest

http://jcm.asm.org/

poses and contained the following reagents: hybridization agent (3.5ml) containing a radioiodinated DNA probe, DNA wicking agent, wash reagent, Hybriwix filters for test spec-imens, and control Hybriwix filters (positive and negative). Hybriwix procedure. A colony from a culture plate was transferred with an inoculating needle to a microcentrifuge tube containing 2 drops of DNA wicking agent. A Hybriwix filter was placed in the tube, and the lysate was allowed to wick and completely wet the filter. Test and control filters (maximum, 14 filters) were placed in hybridization agent containing radioiodinated probe SAL6andincubated at 60°C for either 30 or 120 min. The hybridization agent was decanted, and the filters were rinsed with tap water. Filters were transferred to preheated (73°C) wash reagent and incubated at 73°C for 30 min. They were transferred to absorbent paper to blot dry and were counted for 1 min in a gamma counter (Kemble Instruments, New Haven,Conn.). Individual filter counts greater than 300 cpm were consid-ered positive for salmonella. For testing of GN broth cultures, 1 ml of broth was centrifugedfor 30 s (15,000 x g), andthe supernatant wasremoved. Thepellet wassuspended

in 2 drops of DNA wicking agent, wicked, andhybridized as described above.

Cloning and identification of SAL6. The DNA probe

se-quence (SAL6) used in this study wasobtained bydigesting

Salmonella enteritidis chromosomal DNA with the

restric-tionendonucleaseBglII and ligating this DNA with

BamHI-digested pSP64 DNA (PromegaBiotec Co.,Madison,Wis.).

Theligation mixture wasdigestedwith BamHI before

trans-formation into competent Escherichia coli RR1 cells. Plas-mid DNA was purified from randomly selected clones and

digested with EcoRI tolinearize theplasmid distaltothe SP6 RNA polymerase binding site of vector pSP64 and the

cloned Salmonella fragment. RNAtranscripts

(riboprobes)

weresynthesized byusingSP6 RNApolymerase (Promega)

and [a-32P]rCTP according to the manufacturer's instruc-tions to a specific activity of 2 x 108

cpm/4g.

The DNA template wasremovedbydigestion withDNase Ifor15min

at 37°C. Each labeled riboprobe was hybridized by using

hybridization agent andprobe at 106cpm/ml againstapanel

ofHybriwixcontainingchromosomalDNAfromSalmonella

isolates in addition toseveralnon-Salmonella enteric

bacte-ria. Filters were washed in wash reagent for 30 min and autoradiographed for 4 to 24h at-85°C withanintensifying

screen.

Mapping of SAL6 in S. enteritidis. Restriction

fragments

that contained the SAL6 sequence were determined

by

digesting purified S. enteritidis DNA with the

following

enzymes: BglII, BamHI, BamHI-EcoRI, and BamHI-HindIII. Approximately 1 ,ugofappropriatelydigestedDNA wasloaded per wellandelectrophoresed in a

0.8%

agarose gel in Tris acetate buffer. The gel was blotted to a Gene-Screen Plus filtermembrane(Dupont, NEN Research Prod-ucts, Boston, Mass.) by the method of Southern

(7)

and hybridized to a 32P-labeled SAL6

riboprobe.

Autoradiogra-phywas donefor 16 h at -850C.

Subcloning SAL6 intobacteriophageM13.Purified

pSAL6

DNA and M13mpl9 replicative-form DNA were

digested

withEcoRI and PstI. TheseDNAsampleswere

ligated

with T4 DNA ligase and transformed into E. coli RR1 cells.

Samples of thiscell-DNA mixturewereplatedwith2.0mlof an overnight culture of JM107 on Luria broth agar

plates.

Replicative-form plasmid DNA of recombinants was ex-tracted andpurified by themethod of Birnboim and

Doly (1).

Iodination of M13-SAL6.Single-strandedM13-SAL6 DNA was chemically labeled with 1251 by a modification of the

methodof Commerford

(3)

toanaverage

specific

activity

of

5 x 107

cpm/itg.

Iodinated SAL6DNAwas

purified

freeof iodinatedM13 DNA andwas used for up to2months from thedate of

preparation.

Comparison

of

HE, MAC,

and XLD agar media. Three

Salmonella serotypes

(livingstone, manhattan,

and

mban-daka)

wereinoculatedonto

freshly prepared HE, MAC,

and

XLD agar

plates

and grownat

37°C

for16 h. Five colonies from each

plate

were liftedfrom the agar surface with an

inoculating

needle and

lysed by

using

2

drops

(approximately

35

,ul)

ofDNA

wicking

agent.

All

lysates

werewickedonto

Hybriwix

filters and

hybridized (30

min)

and washed

(30 min)

asdescribedabovefor clinical

testing.

Filterswerecounted

for 1min inagammacounter.

Determination of

1251

probe

SAL6

sensitivity

and

specificity.

To assess both

sensitivity

and

specificity

ofthe 125I

probe

SAL6under assay

conditions,

colonies of

salmonella

and

E. coliwere

picked

from XLD

plates.

Fivecolonies ofeach

organism

were

picked;

themeandiameter ofE. colicolonies

was0.9 mm, and themeandiameterof Salmonella colonies

was 1.1 mm. The colonies were

suspended

inLuria broth, serial 10-fold dilutionswere

made,

and

appropriate

dilutions

were

plated

in

triplicate

onLB agar. After

incubation,

mean

viableCFUonXLDwerecalculatedforeach

colony

picked.

Additionally,

dilutions of both salmonella andE. coli

con-taining

known numbers of viable

CFU

were

hybridized

as

described abovetorelate

hybridization

reactivity

with viable

bacteriapresentin a

colony.

RESULTS

Wicking

technique

and

hybridization

methods. A new

technique

called

wicking

has been

developed

which is a

simple,

clinically

feasible

two-step

method that

effectively

separates DNA from substances that

compromise

DNA

retention and

hybridization

reactivity.

Instep one,

cells

are

rapidly

lysed

and DNA isdenatured

by using

an alkali-surfactantreagent

(DNA

wicking

agent).

In steptwo, the

resulting lysate

is

applied

tothe base ofa

Hybriwix,

wherein

absorption

ofthe

lysate

mixture takes

place by

vertical

capillary

action. This

wicking

method

results in the

migration

ofDNA

present

in the

lysate

to a

specific

linearlocationonthe

Hybriwix

filter in an immobi-lized form that can be

readily

hybridized,

i.e.,

a

single-stranded form. The wicks have a

large (but

undetermined)

capacity

for

binding

DNA. Inthecaseof

high

concentrations

oftarget

DNA,

a

wide,

irregular

band

extending

backfrom thefront isobserved.This DNA is

hybridizable

and

contrib-utes to the

positive

signal.

The smear-back effect is

espe-cially

noticeablein the

positive

controls shownin

Fig.

1.The

position

of thenucleic acid frontonthe

Hybriwix

filtercan

belocated

by

adding

anindicator suchas

phenol

red tothe

lysate.

The

phenol

red

comigrates

with the DNA under

wicking

conditions.

Figure

1isan

autoradiograph

that illustrates

wicking.

The

target DNA on wicks

1,

3, 5,

7,

and 29wasfrom

randomly

chosen Salmonella serotypes, whereas wicks

9,

11, 13, 15,

and 17 contain target DNA from non-Salmonella enteric bacilli. The

positive

controls contained

pSAL6

plasmid

DNA as the target, and the

negative

controls contained

wicking

reagent

only.

The

probe

was

32P-labeled

pSAL6

riboprobe.

The target DNA was

placed

in 2

drops

ofDNA

wicking

agent and absorbed onto the

Hybriwix

filter

by

vertical

capillary action,

i.e.,

wicking,

before

hybridization.

Mixing

of a small cell

pellet

or

colony

of the various

Salmonella isolates with the DNA

wicking

agent

resulted in

on April 12, 2020 by guest

http://jcm.asm.org/

I

FIG. 1. Wicking of DNA from salmonella and non-Salmonella entericbacteria. After autoradiography, the developed X-ray film

was positioned overthe actual wicks and photographed for this

figure. The bands ofreactivityseenheredonotappear onthe wicks; rathertheyare onthe overlaidX-rayfilm.Thefigureisdescribed

furtherinthetext.

rapid lysis of the cells and denaturation of the double-stranded DNA. When thelysate was applied tothe base of theHybriwix filter, the DNAmigratedas abandtoaspecific location approximately halfwayup thefilter (Fig. 1).

Probe selection and characterization. The DNA probe SAL6 was one ofnumerous random clones derived from

BglII fragments of S. enteritidis chromosomal DNA. Ribo-probestranscribed from the cloneswereusedtoevaluate the reactivespecificity of variouscloned Salmonellasequences. Hybridization of 32P-labeled riboprobes from different cloned Salmonella sequences (including SAL6) to panels of chro-mosomal DNAs from Salmonella isolates and non-Salmo-nella isolates indicated that clone SAL6 could detect dif-ferent serovars of salmonella. DNA from 70 distinct Salmonellaserotypes waswickedontoHybriwix filters and hybridized with 32P-labeled pSAL6 riboprobe. Positive hy-bridization signals essentially identical to the positive con-trols were seenfor each Salmonella isolate tested(datanot shown). This indicates that the SAL6sequencewaspresent ineach of the70serotypes tested.

Determination of SAL6 sequence conservation among

Salmonella isolates was then further assessed.

Chromo-somal DNA from 30 Salmonella isolates representing 22

serotypes weredigested with BglII and analyzed by South-ern hybridization. Twenty-nine of the isolates showed a bandhybridizingatapproximately 1,600 base pairs (bp), the sizeofthe cloned DNAsequenceinpSAL6. Oneisolate (no. 27) demonstratedaband atafragment length of 2,300 bp.

The Salmonella sequence present in clonepSAL6 was a unique, previously uncharacterized sequence as shown by Southern analysis of S. enteritidis chromosomal DNA. The data(notshown) demonstrated that pSAL6 contains aBglII fragment approximately 1,600 bp in length. Additionally, the

TABLE 1. Hybridizationreactivity of three Salmonella serotypes grown on threedifferent selective media

Salmonella Hybridizationvalue(cpm) on the following media:

serotype XLD MAC HE

Livingston 556 ± 142 510± 99 179± 27

Manhattan 468± 137 370 ± 72 223± 88

Mbandaka 358 ± 68 244± 55 166± 30

a Values are expressed as the averages of five colonies on five separate wicks.

SAL6 sequence mappedto a very large

(>24-kbp)

BamHI fragment.

Testingwasalso conductedtodetermine thespecificityof the pSAL6 probe sequence. No detectable hybridization occurred between the pSAL6 riboprobe and purified chro-mosomal DNA from 101 stock strains (representing 22

genera) of bacteria other thansalmonella (datanotshown). These determinations show that SAL6 is clearly different from previously reported, less specific, morecross-reacting

Salmonellaprobes(4).

Hybridization reactivity of colonies from HE, MAC, and

XLD. The effect of the growth medium on hybridization reactivity of Salmonella colonies was evaluated. Table 1 representstheaveragehybridizationvaluesforthree Salmo-nellaserotypestested aftergrowthonHE,MAC, andXLD. The results indicate that the hybridization reactivity of Salmonella coloniesgrown on XLDand MACwas superior

to that of colonies grown on HE with all three serotypes tested, and that Salmonella colonies grown on XLD

pro-videdthehighestreactivity. Althoughnoneoftheserotypes

exceededan average hybridization valueof300 cpmwhen

taken from growth on HE and hybridized for 30 min, all

average countsfrom growthonXLDexceeded300 cpm. Clinical detection of salmonella: colony assay. Two

hun-dred andfifty fecal specimens over a7-month period were

processed in the clinicalphase ofthe study. Hybridization

assays were performed on any bacterial colony that was

capable of producing H2S on either HE or XLD, as

evi-denced by the appearance ofablack colony. The test was

performed on 47

H2S'

colonies, which were

subsequently

biochemicallyidentifiedasCitrobacter(20isolates),Proteus

(17 isolates),

Morganella

(1

isolate),

and Salmonella (9 isolates) species.Theonly isolatestodemonstrate

reactivity

greaterthan300cpm werethe nine Salmonella isolates. The

enhanced reactivity seen in isolates grown on XLD with control isolates was also documented when fresh clinical isolateswere tested (Table 2).

Clinical detection of salmonellae: GN brothassay. Each of the 250 fecal specimens was inoculated into GNbroth and enriched foran averageof18 h; GN brothsweretested for

the presence of salmonella byusingthe

`251-labeled

SAL6

probe. Table 2 shows the 120 min hybridization values obtained fromeight GN brothculturesin whichthe presence

ofsalmonella wasconfirmedby culture. Salmonellae were

detectable from 1 ml of GN broth in seven of

eight

salmo-nella culture positive broths tested. Of note is that one

plate-positive specimen (no. 2) failed to produce a pellet

upon

centrifugation

of the GN

broth,

suggesting

that

mini-mal, if any, Salmonella growth had occurred. As

impor-tantly,the DNAextractedfrom240

salmonella-negative

GN

brothcultures showednofalse-positive cross-reactivity with

probe

SAL6.

Figure

2shows the distribution ofcountsfrom

salmonella-negative GN broth cultures after

hybridization

on April 12, 2020 by guest

http://jcm.asm.org/

TABLE 2. Hybridization values of clinical isolatecolonies picked from HE, XLD, and GN media and

hybridized for 30 and 120 min Hybridization value (cpm)

Clinical isolate 30-min 120-min

serotypeb hybridization hybridization

HE XLD HE XLD GN

Typhimurium 197 1,251 404 2,352 5,358

Heidelburg 123 272 160 760 82C

Newport 342 528 601 774 948

Enteritidis 630 1,020 237 1,729 6,538

Newport 561 1,655 1,201 1,838 NTd

Enteritidis 701 977 1,791 3,241 856

SaintPaul 765 720 1,015 2,060 876

Enteritidis 436 853 1,279 2,616 6,494

Enteritidis 454 398 792 1,934 734

a Recovered from 250consecutive stool specimens.

b One to fivecolonies of each isolate were individually picked, wicked, and hybridized.

C Growth was not observed in this culture.

dNT, Not tested.

for30 and 120 min. Ofnote is the fact that the majority of

counts werewell belowthe 300cpmpositive cutoffvalue. Relationship of hybridization reactivity to bacterial

num-bers with positive and negative test organisms from XLD

plates. Since itwouldbe usefulthat the test beperformedon suspected Salmonella colonies from primary isolation

plates, we determined thehybridization reactivity relativeto

the number of viable CFU of positive salmonellaa) and

negative(E. coli)bacteria fromXLDplates. Wicks contain-ing lysate from6 x

108,

4 x

108,

2 x 108,1 x 108,0.6x 108,

and 0.3 x 108 viable salmonella showed hybridization reactivities of2,817, 2,676,2,102, 1,702, 1,136,and 647 cpm,

respectively. Wickscontaining1 x

109,

2.5 X 108,and 1.5 x

108E. colicellsshowed reactivities of171, 51, and 21cpm,

respectively.

Additionally,thenumber of viableE.coli andsalmonella fromtypicalXLDcolonies was determined

by plate

counts

to range between 2.16 x 108 and4.95 x 108CFU.

DISCUSSION

Theavailability ofdiagnostic methodsandreagentsforthe

identification ofsalmonella has been

historically

limitedto

40

35

Co

en

Q 'a. E

o

.0

E

z

30 25

20

151

10

5.

100 200 300

CPM

FIG. 2. Distribution of reactivity of probe SAL6 among 240 salmonella-negative GN broth cultures. Bars; 1 30-min hybrid-ization; O, 120-minhybridization.

serological

andbiochemical approaches. Recently, Fitts et

al.

(4)

describedthe

cloning

of

salmonella-specific

sequences

that demonstrated reactive

specificity

for 80 to 95% of

Salmonella isolatestested.Our

study

has beensuccessfulin

identifying

a

probe

specific

forsalmonella.

Comparison

of

restriction endonuclease

mapping

of SAL6 with maps of other

published sequences (4)

demonstrated that SAL6 is indeeda

newly

characterized

probe.

Hybridization

studies of

probe

SAL6 to 153 Salmonella

serotypes

representing

90.1% of all Salmonella serotypes

recorded in 1985

by

the Centers for Disease Control

(2)

indicatedthat the

homologous

sequencewaspresentin allof

the isolates.

Further,

molecularcharacterization

by

South-ern

analysis

of30Salmonella isolatesdemonstrated that 29

isolates contained a reactive

1,600-bp fragment

similar to

thatused for

cloning

SAL6.

Additionally,

an

H2S-

Salmo-nella isolatewasfoundto containthe

homologous

1,600-bp

fragment.

Specificity

of SAL6 for

only

theSalmonellagenus

wasconfirmed

through

thelack of

hybridization

between the

probe

and 101 different non-Salmonella bacteria

represent-ing

22distinctgenera.

Theclinical

phase

of this

study

succeededin

coupling

this

highly specific probe

with aninnovativeand

simple

sample

processing

technology

termed

wicking.

This

technique

sep-arates

specimen

target DNA from substances that compro-mise both targetretention and

hybridization

reactivity

when

moretraditional immobilizationmethodsare

employed.

Ad-ditionally,

the

simplicity

of this

technique

avoids more

cumbersome and

time-consuming

methodsof cell

lysis

and DNA extraction which are

only

appropriate

for use in research laboratories.

In addition to the

simplicity

and

speed

of the

wicking

technique,

there are two other

potential advantages

over

conventional DNA

processing approaches.

First,

the

con-centration and easyidentification ofthetarget DNAon the

Hybriwix

filter

permits sectioning

ofthe filtertoseparatethe

target DNA from other material in

specimens

that may

contribute to

nonspecific binding

of the labeled

probe.

Although

much fecal material was wicked onto the filter

fromthe GN broth concentrates inour

study,

sectioning

of

the

Hybriwix

filterwas not necessary inthis system, since

the counts from non-Salmonella cultures remained well below the

positive

threshold of 300 cpm.

Second,

the

wick-ing

of target nucleic acid resulted in a

relatively

equal

distribution oftarget DNAacrossthewidth of the

Hybriwix

filter.

Thus,

ribboning

the

Hybriwix

filter

equally

into

strips

ina

longitudinal

fashionwouldresult in

multiple

filters with

similar amounts oftarget that could be

hybridized

simulta-neously

against

a

panel

ofdifferent

probes.

Our results indicated that media used for recovery of

salmonella affectedthe

reactivity

ofthetest.

Inspection

of Salmonella colonies grown on

XLD, HE,

andMAC

gener-ally

indicatedthatcoloniesonXLDwerethe

largest

and that those on HE were the smallest.

Additionally,

Salmonella

colonies grownonXLDwerecollectedmore

efficiently

from the agar surface with an

inoculating

needle;

i.e., a

greater

percentageofthe

colony

could be

processed

for

hybridiza-tion. It is

probable

that the increased

reactivity

seen from

colonies from XLD is due to more target available from these

larger,

more

easily

picked

colonies.

The

adaptability

of the

wicking

technique

to the clinical detection ofsalmonellawastested inkit form. The

wicking

technique

and

hybridization

methodswere

simple

and

rapid

and

adapted

wellto the work flow within a clinical

labora-tory.

This

study

hasdemonstrated thataSalmonella

probe

can

on April 12, 2020 by guest

http://jcm.asm.org/

beusedinthe clinicallaboratory toidentify H2S-producing

colonies recovered from fecal specimens. Use of the test could provide same-day, accurate identification of salmonel-lae and earlier notification to the physician, avoiding an additional 24 h required to screen out non-Salmonella iso-lates before agglutination with salmonella-specific antisera becomes feasible.

The sensitivity and specificity of this culture-amplified

hybridization test oncolonies from primary isolation plates

isclearly adequate, sincehybridization reactivity to lysates of as few as 3.2x 107CFU exceeded by at least threefold the

reactivityseen with 1 x 109negative bacteria. Also the data indicate that 2 x 108 to 6 x 108 CFU is expected from a typical XLD colony with positive reactivity between approx-imately 1,000 and 3,000 cpm, whereas negative reactivities in this cell range do notexceed 171 cpm.

Although media used to recover salmonella affected the reactivity of the test,modifying the hybridizationprocedure (increasinghybridization time or pooling colonies)permitted accuratetesting ofcoloniesfrom either HE or XLD agars. Screening for salmonella in GN broth cultures with the

probewas less sensitive, mostlikely as a result of inhibited

growth of salmonella in mixed culture. Modifications of

enrichment mediamay improve detectability.

Simple technical approaches to rapid detection and/or identification ofenteric pathogens are sought by both clinical andindustriallaboratories. The accuracy and ease of

perfor-manceoftheSalmonella probe concurrent with the wicking technique suggest that an enteric probe panel with this approach would be extremely useful forboth applications.

LITERATURECITED

1. Birnboim, H. D., and J. Doly. 1979. Arapid alkalineextraction procedure for screening recombinant plasmid DNA. Nucleic AcidsRes. 7:1513-1523.

2. Centers for Disease Control. 1985.Salmonella surveillance. 1985 annualsummary. Centersfor DiseaseControl,Atlanta. 3. Commerford, S. L. 1971. iodination of nucleic acids in vitro.

Biochemistry10:1993-1999.

4. Fitts, R., M. Diamond, C. Hamilton, and M. Nevi. 1983. DNA-DNA hybridization assay for detection of Salmonella spp. in foods. Apple. Environ. Microbiol. 46:1146-1151.

5. Greene, L. C., P. C. Applebaum, and J. A. Kellogg. 1984. Evaluation of a two-hour method for screening pathogens from stoolspecimens. JClin. Microbiol.20:285-287.

6. Metzler, J., and I. Nachamkin. 1988. Evaluation of a latex agglutinationtest for thedetection of Salmonella and Shigella spp.byusing broth enrichment.J.Clin. Microbiol. 26:2501-2504. 7. Southern, E. M. 1975. Detection ofspecific sequences among DNAfragments separated by gelelectrophoresis. J. Mol. Biol. 98:503-517.

8. Swierkosz, E. M., D. R.Scholl, J. L. Brown, J. D. Jollick, and C. A. Gleaves. 1987. Improved DNA hybridization methodfor detection of acyclovir-resistant herpes simplex virus. Antimi-crob. AgentsChemother. 31:1465-1469.

on April 12, 2020 by guest

http://jcm.asm.org/