Method for Quantitative Detection and Presumptive Identification of Group B Streptococci on Primary Plating

Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Method for Quantitative Detection and Presumptive Identification of

Group B Streptococci on Primary Plating

Søren Mose Hansen and Uffe B. Skov Sørensen*

Department of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C, Denmark

Received 24 July 2002/Returned for modification 15 October 2002/Accepted 27 November 2002

Maternal prenatal screening for group B streptococci (GBS) followed by offering of intrapartum

chemopro-phylaxis to carriers is one of the strategies used to reduce the incidence of neonatal early-onset GBS infections.

Culturing of vaginal and anorectal swab specimens in selective broth is the screening procedure recommended

by the Centers for Disease Control and Prevention. This technique is sensitive; it does not, however, allow

either evaluation of the degree of colonization or detection of cocolonization with several GBS clones. We have

examined the carriage rate and population dynamics of GBS in a group of Danish women during pregnancy

and 1 year after delivery using a new detection method. In the present paper we describe a mixed blood agar

medium (MB agar) that identifies GBS in the primary cultures by detection of a double hemolysis pattern

consisting of characteristic, large zones of partial hemolysis (“CAMP zones”) and of narrow zones of complete

hemolysis. The MB agar was at least as sensitive as culturing in selective broth for detection of GBS in vaginal

and anorectal swab specimens, and GBS strains could be identified directly on the primary plate due to the

CAMP zones without the need for subculturing. The carriage rate of GBS in a group of Danish women was

found to be more than 30%, a figure considerably higher than the rate that was reported previously.

Streptococcus agalactiae

(group B streptococcus [GBS]) is

the principal cause of neonatal infections in industrialized

countries. A significant decline in the prevalence of neonatal

GBS infections has been observed in the United States as a

result of implementation of preventive measures (7, 28). These

include increased use of chemoprophylaxis during labor in

women at high risk for transmitting the infection to their

new-borns (29). Risk-based or screening-based strategies are

rec-ommended by the Centers for Disease Control and Prevention

to prevent GBS disease (6). Collection of swab specimens at 35

to 37 weeks of gestation is suggested for the screening-based

approach. Rectal and vaginal swab specimens are inoculated

together in a selective broth for 18 to 24 h. The broth is

subcultured on sheep blood agar for another 18 to 24 h, and

suspected colonies are finally examined by conventional

micro-biological techniques. This procedure is not applicable to

semi-quantitative evaluation of colonization intensity, and it is not

feasible for demonstration of colonization with multiple

clones.

For a longitudinal study of the rate of carriage and

popula-tion dynamics of GBS in a cohort of pregnant women, we

needed a medium that compensates for these shortcomings.

We developed a solid medium based on a modification of the

classical CAMP reaction that allows easy detection of

individ-ual colonies of GBS in primary cultures.

GBS generate a unique protein, the so-called CAMP factor,

which interacts with the plasma membrane of red blood cells

(RBCs) and other cell types. CAMP factor causes hemolysis of

sheep RBCs (SRBCs) that have been altered by

Staphylococ-cus aureus

␤

-toxin (sphingomyelinase). This phenomenon is

designated the CAMP reaction, which refers to the authors

Christie, Atkins, and Munch-Petersen (8). CAMP reaction-like

reactions have also been observed after the combined actions

of some other bacterial products on blood, and cohemolysis is

therefore a more adequate expression for a hemolytic reaction

induced by the synergistic actions of two different compounds,

as discussed below. The CAMP factor of GBS has been

char-acterized and has been renamed protein B. It has been

sug-gested that the target of protein B is ceramide (

N

-acyl-sphin-gosine) (4), i.e., a membrane lipid generated from sphingomyelin

by sphingomyelinase cleavage. Binding of protein B causes

disorganization of the lipid bilayer of the cell membrane to an

extent that results in cell lysis (4). The in vivo function of

protein B has not been disclosed yet; however, the hemolytic

activity seems to be an epiphenomenon (19, 27). Binding of

this protein to the Fc region of immunoglobulins has been

demonstrated (12, 19), and the protein seems to be lethal to

rabbits and mice (30). Protein B seems to be essential for

S.

agalactiae

, as the

cfb

gene encoding this protein (24) was found

to be universally present in the genomes of 162 GBS strains

from different geographic areas (20). Furthermore, 96 to 99%

of all human GBS isolates have been found to be positive by

the CAMP reaction (11, 14, 23, 25). Nearly all group A

strep-tococci (

Streptococcus pyogenes

) and

Streptococcus uberis

strains possess equivalent genes named

cfa

and

cfu

,

respec-tively (15, 18). The gene may also be present in some other

streptococcal species (16, 18). The cohemolysis observed with

other species is due to substances different from protein B. The

CAMP reaction-like reaction of

Listeria monocytogenes

is

caused by listeriolysin O (26), and that of

Actinobacillus

pleu-ropneumoniae

is caused by an RTX toxin (17).

Protein B may cause lysis of

␤

-toxin-modified RBCs, as

mentioned above; however, only cells containing more than 45

mol% of sphingomyelin in the plasma membrane are sensitive

to the combined actions of the staphylococcal toxin and

pro-* Corresponding author. Mailing address: Department of Medical

Microbiology and Immunology, Bartholin Building, University of

Aar-hus, DK-8000 Aarhus C, Denmark. Phone: 45 89421740. Fax: 45

86196128. E-mail: [email protected].

1399

on May 15, 2020 by guest

http://jcm.asm.org/

tein B (31). Thus, the CAMP reaction is seen on agar plates

prepared from bovine RBCs and SRBCs, whereas RBCs from

humans, rabbits, and guinea pigs and horse RBCs (HRBCs)

are not lysed (8, 31).

We have taken advantage of the unique feature of protein B

as the indicative principle in a new blood agar medium that can

be used for the easy, sensitive, and specific detection and

enu-meration of GBS in primary cultures of complex specimens

such as anorectal and vaginal swab specimens. The agar plates

described in this article contained a mixture of SRBCs and

HRBCs, which were made sensitive to the action of protein B

by pretreatment with sphingomyelinase. The method was

suc-cessfully applied in a longitudinal study of the GBS carrier

status of a group of Danish women during pregnancy and after

delivery (unpublished data). Compared with five other

tech-niques for GBS detection, the new medium was found to be at

least as sensitive and easier to use, since subculturing was

usually unnecessary. The option for semiquantitative

enumer-ation of GBS and detection of cocolonizenumer-ation with several

clones of GBS is an additional advantage of the mixed blood

agar (MB agar) plates.

MATERIALS AND METHODS

Bacterial strains from collections.Two strains of GBS from our own collection were used for control purposes. Strain RH955 is beta-hemolytic and yields a positive CAMP reaction. Strain RH511A also yields a positive CAMP reaction but is nonhemolytic. Staphylococcal␤-toxin was prepared from cultures of anS. aureusstrain (SSI AB2775) from the Department of Clinical Microbiology, Statens Serum Institut, Copenhagen, Denmark. The toxin was used for treatment of MB agar plates (see below).

Preparation of crude staphylococcal␤-toxin.Partly purified␤-toxin (sphingo-myelinase) was prepared from a Todd-Hewitt broth culture (no. CM189; Oxoid Ltd., Basingstoke, United Kingdom) of theS. aureus strain as follows. The culture was incubated for 24 h at 37°C (optimum, 24 to 28 h), and the bacteria were removed by centrifugation (1,750⫻g, 30 min). The clear supernatant was filter sterilized (pore size, 0.45␮m), and 104 g of (NH4)2SO4per liter was added.

After the mixture was stirred with a magnetic stirrer for 1 h and allowed to settle for 30 min at room temperature, the solution was centrifuged (10,000⫻g) for 20 min. The pellet of the precipitate was discharged, and another 104 g of (NH4)2SO4was added to the total volume of the supernatant. After stirring of

the mixture for 1 h, the mixture was kept at room temperature. On the next day the solution was centrifuged (10,000⫻g) for 20 min. The second supernatant was discharged, and the pellet of crude toxin was dissolved in 20 ml of sterile phosphate-buffered saline (PBS). The toxin concentrate was dialyzed four times against 2 liters of PBS for 24 h at 5°C. Finally, the concentrated toxin stock solution was filter sterilized (pore size, 0.45␮m) and stored at 5°C until it was used. The toxin preparation was stable for several months when stored at 5°C. The sphingomyelinase activity of the toxin stock was determined by titration. The whole surfaces of a number of MB agar plates (see below) were treated with 200 ␮l of twofold serial dilutions of the toxin stock in PBS. A CAMP reaction-positive strain of GBS (strain RH955) was streaked onto each of the toxin-treated plates before they were incubated overnight at 37°C. The plates were inspected for detection of two different hemolysis phenomena around colonies of GBS (double hemolysis). The action of␤-hemolysin caused narrow zones of complete hemolysis, and protein B caused larger zones of partial hemolysis (Fig. 1). The highest toxin dilution that gave optimal double hemolysis around the bacterial colonies was considered the end point. The ready-made␤-toxin solu-tion used for pretreatment of the MB agar plates (see below) was prepared so that it was two times more concentrated than the dilution determined by end-point titration. The use of sphingomyelinase from commercial sources may be a convenient alternative to the in-house preparation of toxin. However, we have not tested such preparations.

MB agar plates.Modified CAMP reaction plates containing mixed blood were prepared with a double layer of agar. One liter of agar contained 40 g of blood agar base (no. CM854; Oxoid), 2 g of sodium pyruvate, 0.247 g of MgSO4, and

50 mg ofL-cysteine hydrochloride monohydrate. The ingredients were mixed

during heating to the boiling point and then autoclaved for 15 min at 121°C. The

medium was cooled and kept in a water bath at 50°C. For preparation of the top layer, 2.5% (vol/vol) packed SRBCs and 2.5% (vol/vol) packed HRBCs were added to the 50°C agar base and mixed. The blood cells were washed three times before use in PBS (0.1 M NaCl, 0.05 M phosphate buffer [pH 7.4]) and packed by centrifugation (at 500⫻gfor 5 min). Each plate (diameter, 9 cm) was made of a lower layer of agar base without blood cells (25 ml). After solidification, a top layer of agar base (20 ml) containing 5% mixed blood cells was added. Hemolysis is more easily seen on the relatively thin blood-containing top layer. Untreated plates were stable and could be stored for several weeks in a refrig-erator. Before use, the MB agar plates were pretreated with staphylococcus ␤-toxin, as described below. After treatment with the staphylococcal toxin, the plates were somewhat sensitive to lowering of the temperature. To avoid spon-taneous hemolysis, these plates were kept at room temperature and used on the same day.

g-MB agar plates.MB agar plates containing gentamicin (g-MB agar plates) in both the lower and the top layers were prepared by adding 1 ml of a filter-sterilized (pore size, 0.45␮m) stock solution (4 mg per ml of H2O) of gentamicin

sulfate (658␮g of gentamicin base per mg; no. G 6896; Sigma-Aldrich, St. Louis, Mo.) per liter of agar base at 50°C (see above).

ng-MB agar plates.MB agar plates containing both nalidixic acid and genta-micin (ng-MB agar plates) in both the lower and the top layers were prepared by adding 1 ml of a filter-sterilized (pore size, 0.45␮m) stock solution (8 mg per ml of H2O) of gentamicin sulfate (no. G 6896; Sigma-Aldrich) and 1 ml of a

FIG. 1. Close-up photos of large CAMP zones surrounding

colo-nies of

S. agalactiae

on MB agar pretreated with staphylococcal

␤

-toxin

1 h before they were streaked with bacteria. (A) A typical

beta-hemo-lytic strain of GBS, strain RH955; (B) a nonhemobeta-hemo-lytic strain, strain

RH511A. The plates were incubated overnight at 37°C (see text for

details). Arrows: 1, bacterial colonies; 2, zones of partial hemolysis

around colonies of both bacterial strains; the SRBCs are lysed in these

areas due to the interaction with protein B generated by the bacteria,

whereas the HRBCs are left intact, as these cells are insensitive to

protein B; 3, after removal of the bacterial colony, a narrow spot of

complete hemolysis, in which both kinds of RBCs are lysed due to the

action of

␤

-hemolysin, is seen in the middle of the CAMP zone; 4, as

for arrow 3, except that the bacterial colonies were left in place;

beta-hemolysis is seen as thin clear rims around the bacterial colonies;

5 and 6, no beta-hemolysis appears around the colonies of strain

RH511 or after removal of a colony.

on May 15, 2020 by guest

http://jcm.asm.org/

filter-sterilized (pore size, 0.45␮m) stock solution (15 mg per ml of 0.1 N NaOH) of nalidixic acid (no. N 8878; Sigma-Aldrich) per liter of agar base at 50°C (see above).

Pretreatment of MB agar plates with␤-toxin.A total of 200␮l of sterile ready-made␤-toxin solution (see above) was dispersed evenly with a plastic Drigalski spatula over the surface of the MB, g-MB, and ng-MB agar plates before use. The plates were kept at room temperature until the surfaces had dried (30 to 60 min).

Regular blood agar plates.Horse blood (5%) agar plates were purchased from the Statens Serum Institut.

Selective broth.Todd-Hewitt broth (no. 189; Oxoid) was supplemented with 8.0 mg of gentamicin sulfate and 15 mg of nalidixic acid per liter (6). Cotton-stoppered test tubes containing 5 ml of this medium were autoclaved for 15 min at 121°C. After the swabs were streaked on the different solid media they were placed in this selective broth, mixed carefully on a vortex mixer, and incubated overnight at 37°C. A total of 10␮l of broth from each of the test tubes was streaked on regular 5% blood agar plates, and the plates were incubated over-night at 37°C.

Bacterial identification.Suspected isolates of GBS were identified by a com-bination of standard tests (21). The strains were Gram stained, tested for catalase activity, and examined by the traditional CAMP test on CAMP test plates (Statens Serum Institut). The strains were examined for the presence of the group B antigen by latex agglutination (no. ZL52; Streptex; Murex Biotech Ltd., Dartford, United Kingdom).

Clinical samples.A cohort of 77 healthy women was monitored in a longitu-dinal study (unpublished data) from the 19th week of gestation until shortly before delivery at the Department of Obstetrics, Aarhus University Hospital, Skejby, Denmark. All pregnancies were normal, and no complications due to GBS were observed. The project was initiated in January 1999 with approval from the Ethics Committee, County of Aarhus, Denmark, and after informed consent had been provided by all volunteers. The participants were instructed in the technique for obtaining swab specimens at home. Carbon-containing cotton swabs on plastic sticks (Statens Serum Institut) were used for the sampling. Vaginal and anorectal swab specimens were obtained at different times during the pregnancy and again 1 year after delivery (spring 2001). Each swab was immediately placed in a tube containing Stuart’s transport medium (Statens Serum Institut) and shipped to the laboratory by regular mail. All swabs were examined within 36 h after the samples were taken. For the present study, more than 300 paired samples were examined on up to five different media (Table 1). The swabs were inoculated on the solid media as described below for the ng-MB agar plates. Some samples were not examined on all media (exact figures are given elsewhere in the text).

Evaluation of intensity of GBS colonization on ng-MB agar plates. Presump-tive identification of GBS colonies is possible directly on the ng-MB agar plates (see above). These plates were therefore used for semiquantitative evaluation of the degree of colonization, as follows. Upon arrival in the laboratory in Stuart’s transport medium, each swab specimen (vaginal or anorectal) was rolled and rubbed over an area 2 by 3 cm near the edge of an ng-MB agar plate. The inoculum was streaked from this area five to six times with one side of a sterile 10-␮l plastic inoculation loop. To obtain well-separated colonies, spreading was continued by making three sets of streaks perpendicular to the previous streak by using the other side of the same loop. One person performed the inoculation of the agar plates to make it as reproducible as possible. Colonization intensity was evaluated semiquantitatively and blindly for each plate after incubation for 18 h at 37°C in an atmosphere containing 5% CO2. Growth of GBS in streaking area

one or two only was considered light colonization, while growth of GBS in one or more of the following streaking areas was considered moderate to heavy colonization. Samples without detectable growth of GBS were considered negative.

Reagents.All chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich.

Statistics.SigmaStat statistical software (version 1.0; Jandel Corporation, San Rafael, Calif.) was used for analysis of contingency tables by McNemar’s test.

RESULTS

Initial experiments revealed large zones of hemolysis around

colonies of GBS grown on sheep blood agar plates pretreated

with staphylococcal

␤

-toxin. In contrast, only narrow zones of

hemolysis were seen on plates not pretreated with

␤

-toxin. The

hemolysis around colonies of GBS grown on horse blood agar

plates was unaffected by

␤

-toxin pretreatment. On this medium

the bacteria generated only narrow zones of hemolysis. A

dou-ble pattern of partial and complete hemolysis was seen when

the bacteria were cultured on

␤

-toxin-treated agar plates made

with mixed SRBCs and HRBCs (MB agar; Fig. 1). The

com-position of the MB agar plates was optimized in a number of

experiments to promote the most clear-cut reactions for GBS

strains (the recipe is given in the Materials and Methods

sec-tion). On this MB agar, large zones (5 to 8 mm) of partial

hemolysis (“CAMP zones”) and narrow zones (2 to 3 mm) of

complete hemolysis (beta-hemolysis) were generated by the

GBS isolates (Fig. 1A). Nonhemolytic isolates also generated a

large CAMP zone (Fig. 1B), while beta-hemolytic CAMP

re-action-negative isolates did not (data not shown). Experiments

with

S. pyogenes

strains showed that the beta-hemolytic zone

dominates over the CAMP zone for these strains; thus, they

did not look like GBS strains.

[image:3.603.43.284.88.204.2]

More than 300 paired anorectal and vaginal swab samples

were cultured on five different media (Table 1). GBS were

easily detected on the MB agar plates by the presence of the

characteristic double hemolysis around the colonies (Fig. 1A),

and the sensitivity of this medium was found to be considerably

higher than that of regular blood agar plates (Table 1). The

nonselective plates of both media were, however, often

over-grown by the commensal flora in the samples, causing a low

detection rate, especially for the anorectal swab samples.

An-tibiotics were therefore added to the MB agar in order to

improve the isolation of GBS. The best results were obtained

by adding a combination of gentamicin and nalidixic acid to the

agar (Table 1), even though the colonies of GBS appeared

smaller on ng-MB agar medium than on medium containing

gentamicin alone. The sensitivity of the selective MB agar for

detection of GBS in both vaginal and anorectal swab samples

was found to be at least as high as that when a selective broth

was used (Table 1). Full agreement was found between the

tentative diagnosis and the results of the supplementary tests

TABLE 1. Frequency of GBS in swab samples cultured on

different media

Mediumb

Anorectal swab

specimens (n⫽312a₎ Vaginal swab specimens_(n⫽311a₎ No.

positive Sensitivity(%)c _positiveNo. Sensitivity_(%)

Blood agar

27

25.2

68

66.7

MB agar

37

34.6

78

76.5

g-MB agar

88

82.2

91

89.2

ng-MB agar

101

d

_94.4

₉₅

e

_93.1

Selective broth

86

80.4

91

89.2

Total

107

102

a_{Total number of swabs tested on all media.}

b_{Blood agar, 5% blood agar plates; MB agar, agar plates containing SRBCs} and HRBCs and pretreated with staphylococcal␤-toxin; g-MB agar, MB agar with gentamicin sulfate; ng-MB agar, MB agar with nalidixic acid and gentamicin sulfate; selective broth, Todd-Hewitt broth with nalidixic acid and gentamicin sulfate; total, number of swabs yielding growth of GBS on one or more of the media used.

c_{Sensitivity, percentage of swabs that tested positive on the medium of the} total number of swabs that tested positive on one or more of the media used.

d_{The number of anorectal swab specimens positive on ng-MB agar was} sig-nificantly higher (P⬍0.001) than the number of specimens positive in selective broth (see text).

e_{The number of vaginal swab specimens positive on ng-MB agar was not} significantly different (P ⫽0.30) from the number of specimens positive in selective broth (see text).

on May 15, 2020 by guest

http://jcm.asm.org/

when the typical double hemolysis was observed on the MB

agar plates. Neither

S. pyogenes

nor other protein B-generating

streptococci were isolated from the samples.

More than one-third of all pairs of samples were found to be

positive for GBS (36.5%; 131of 358) when MB agar was used.

There was a high concordance (93%) between the results

ob-tained from the analysis of pairs of vaginal and anorectal swab

specimens (Table 2; Fig. 2). No difference in the number of

positive vaginal swab specimens compared to the number of

positive anorectal swab specimens was seen (

P

⫽

0.31), but 5 to

10% more carriers were found when both vaginal and

anorec-tal swab specimens were examined instead of when only one

swab specimen was examined from each person. An increased

rate of detection of GBS was achieved when samples were

obtained from both the vaginal and the anorectal sites, as

opposed to when samples were obtained from either site alone,

but there was no difference in rates of detection among

sam-pling sites. This finding has also been reported by others (22).

The number of anorectal swab specimens positive for GBS was

significantly higher when ng-MB agar was used for cultivation

than with cultivation in selective broth (355 swab specimens

were tested on these two media; of these, 91 were positive on

both media, 240 were negative on both media, 21 were positive

on ng-MB agar and negative in selective broth, and 3 were

negative on ng-MB agar and positive in selective broth [

P

⬍

0.001]). The number of GBS-positive vaginal swab specimens

detected when ng-MB agar was used for cultivation was,

how-ever, not significantly different from the number detected when

selective broth was used (356 swab specimens were tested on

these two media; of these, 96 were positive on both media, 245

were negative on both media, 10 were positive on ng-MB agar

and negative in selective broth, and 5 were negative on ng-MB

agar and positive in selective broth [

P

⫽

0.30]).

As mentioned above, a strong correlation was found

be-tween the degree of colonization detected in the two swab

samples (vaginal and anorectal) in a sample pair. For more

than 90% of GBS-negative vaginal swab samples, the

corre-sponding anorectal swab sample was negative as well (Fig. 2;

Table 2). In contrast, among the GBS-positive vaginal swab

samples, only 4 to 5% of the corresponding anorectal swab

samples were negative. A clear tendency toward the detection

of heavier colonization of the vaginal swab samples with

in-creasing colonization of the corresponding anorectal swab

samples was also seen (Fig. 2). Vaginal colonization with GBS

therefore reflects the anorectal colonization density to a high

degree.

DISCUSSION

The CAMP test is a standard procedure for the presumptive

identification of

S. agalactiae

(GBS) (9, 11). In the traditional

assay, a

␤

-toxin-producing

S. aureus

strain is streaked across a

blood agar plate made with SRBCs. A number of suspected

strains of GBS are then streaked on the same plate

perpen-dicular to the streak made with

S. aureus

. For most GBS strains

an arrowhead-shaped hemolysis zone appears near the

inter-section with the

S. aureus

strain after incubation of the plate.

This is due to the cohemolysis caused by the concerted action

of

␤

-toxin from the

S. aureus

strain and protein B from the

strains of GBS. While this CAMP test is useful for the

pre-sumptive identification of pure cultures of suspected strains of

GBS (9), the classical assay is not applicable for direct

detec-tion of GBS in primary cultures of clinical samples. Different

modifications of the CAMP test have been developed for this

purpose (3, 10). However, none of these methods are optimal

for routine examination of a large number of samples, since the

procedures are relatively complicated. Others have used sheep

blood agar plates conditioned with sterile

␤

-toxin-containing

supernatant from an

S. aureus

culture for detection of CAMP

test-positive GBS (1), but such agar plates cannot distinguish

the CAMP reaction and ordinary

␤

-hemolysis.

The Centers for Disease Control and Prevention

recom-mended a procedure for screening for carriage of GBS that

requires culture of anorectal and vaginal swab specimens in a

selective broth which, after incubation, is subcultured on blood

agar plates; the isolates are eventually identified by serological

methods (6). This procedure is specific and sensitive but is

somewhat laborious, costly, and time-consuming and does not

allow quantitative evaluation and proportional isolation of the

individual strains in one sample.

[image:4.603.43.286.90.144.2]

The selective MB agar has several advantages, although

preparation of the MB agar plates is more complicated than

preparation of the traditional plates. Culturing on MB agar

was found to be at least as sensitive as culturing in a selective

FIG. 2. Correlation between growth densities of

S. agalactiae

(GBS) in 310 paired vaginal and rectal swab samples from 77 pregnant

women. Vaginal samples are grouped on the abscissa according to

density of growth of GBS on ng-MB agar plates. The densities of

growth of GBS from the corresponding rectal swab samples were

recorded as negative (open bars), slight (hatched bars), and moderate

to heavy (solid bars) (see text for details).

TABLE 2. Frequency of GBS in 358 pairs of anorectal and vaginal

swab specimens cultured on ng-MB agar

Result for anorectal swab specimens

No. (%) of vaginal swab specimens

Positive Negative

Positive

107 (30)

15 (3)

Negative

9 (4)

227 (63)

on May 15, 2020 by guest

http://jcm.asm.org/

[image:4.603.49.280.458.656.2]

broth, with a sensitivity of over 90%. The primary solid media

can be inspected directly within 18 h, and reculturing is rarely

needed, since typical colonies can be identified as GBS with a

high degree of confidence on the basis of the characteristic

hemolysis phenomenon. The density of GBS colonization can

therefore be estimated semiquantitatively. We found a clear

correlation between the colonization densities of GBS in

paired vaginal and anorectal samples by use of ng-MB agar

plates (Fig. 2). Furthermore, individual bacterial colonies can

be identified on the primary plates and may be selected for

further examination (e.g., for antibiotic resistance testing,

DNA analysis, or serotyping).

Some bacterial species other than GBS may exhibit a

posi-tive CAMP reaction, as mentioned above, but these species do

not constitute a diagnostic problem when MB agar is used for

cultivation of human clinical specimens. On this medium the

hemolytic pattern of group A streptococci differs from that of

GBS (see above).

S. ubris

is not associated with humans, and in

contrast to most GBS strains, strains of this species are not

beta-hemolytic (21).

A. pleuropneumoniae

is found only in pigs.

Occasionally, stool samples may contain

L. monocytogenes

(2),

and colonies of this species resemble colonies of

beta-hemo-lytic streptococci on blood agar. However, L

. monocytogenes

is

sensitive to gentamicin and will, therefore, not appear on the

selective MB agar plates. Furthermore, differentiation from

GBS is easily accomplished by demonstrating the positive

cata-lase reaction of

L. monocytogenes.

Among a group of 77 pregnant women, we found 21% to be

persistent carriers of GBS and an additional 27% to be

tran-sient carriers of GBS. These rates are considerably higher than

the rates previously reported for a large group of Danish

preg-nant women (13), in which only 8 to 15% carriers of GBS were

found. Other recent rates of carriage are not available from

Denmark. Our prevalence findings based on the use of

selec-tive MB agar are similar to those recently reported for younger

women from the United States. In a prevalence study in which

the selective broth technique was used, 34% of female

univer-sity students were found to be colonized with GBS (5), and

among a group of pregnant women in a prospective cohort

study, 28% were found to be colonized with GBS (22).

ACKNOWLEDGMENT

This work was supported by grant 9900316 kg/mp from the Danish

Medical Research Agency.

REFERENCES

1. Anthony, B. C., D. M. Okada, and C. J. Hobel.1978. Epidemiology of group B streptococci: longitudinal observations during pregnancy. J. Infect. Dis.

137:524–530.

2. Armstrong, D.1995.Listeisteria monocytogenes, p. 1880–1885.InG. L. Man-dell, J. E. Bennett, and R. Dolin (ed.), ManMan-dell, Douglas and Bennett’s principles and practice of infectious diseases. Churchill Livingstone, New York, N.Y.

3. Bae, B. H., and E. J. Bottone.1980. Modified Christie-Atkins-Munch-Pe-tersen (CAMP) test for direct identification of hemolytic and non-hemolytic group B streptococci on primary plating. Can. J. Microbiol.26:539–542. 4. Bernheimer, A. W., R. Linder, and L. S. Avigad.1979. Nature and

mecha-nism of action of the CAMP protein of group B streptococci. Infect. Immun.

23:838–844.

5. Bliss, S. J., S. D. Manning, P. Tallman, C. J. Baker, M. D. Pearlman, C. F. Marrs, and B. Foxmann.2002. Group BStreptococcuscolonization in male and non-pregnant female university students: a cross-sectional prevalence study. Clin. Infect. Dis.34:184–190.

6. Centers for Disease Control and Prevention.1996. Prevention of perinatal group B streptococcal disease: a public health perspective. Morb. Mortal. Wkly. Rep.45:1–24.

7. Centers for Disease Control and Prevention.2000. Early-onset group B streptococcal disease—United States, 1998–1999. Morb. Mortal. Wkly. Rep.

49:793–796.

8. Christie, R., E. Atkins, and E. Munch-Petersen.1944. A note on a lytic phenomenon shown by group B streptococci. Aust. J. Exp. Biol. Med. Sci.

22:197–200.

9. Darling, C. L.1975. Standardization and evaluation of the CAMP reaction for the prompt, presumptive identification of Streptococcus agalactiae (Lancefield group B) in clinical material. J. Clin. Microbiol.1:171–174. 10. DiPersio, J. R., J. E. Barrett, and R. L. Kaplan.1985. Evaluation of the

Spot-CAMP test for the rapid presumptive identification of group B strep-tococci. Am. J. Clin. Pathol.84:216–219.

11. Facklam, R. R., J. F. Padula, E. C. Worthham, R. C. Cooksey, and H. A. Rountree.1979. Presumptive identification of group A, B, and D streptococci on agar plate media. J. Clin. Microbiol.9:665–673.

12. Fehrenbach, F. J., D. Jurgens, J. Ruhlmann, B. Sterzik, and M. O¨ zel.1988. Role of CAMP-factor (protein B) for virulence. Zentbl. Bakteriol. Para-sitenkd. Infektkrankh. Hyg. Abt. 1 Orig. Suppl.17:351–357.

13. Feikin, D. R., P. Thorsen, S. Zywicki, M. Arpi, J. G. Westergaard, and A. Schuchat.2001. Association between colonization with group B streptococci during pregnancy and preterm delivery among Danish women. Am. J. Ob-stet. Gynecol.184:427–433.

14. Fuchs, P. C., C. Christy, and R. N. Jones.1978. Multiple-inocula (replicator) CAMP test for presumptive identification of group B streptococci. J. Clin. Microbiol.7:232–233.

15. Gase, K., J. J. Ferretti, C. Primeaux, and W. M. McShan.1999. Identifica-tion, cloning, and expression of the CAMP factor gene (cfa) of group A streptococci. Infect. Immun.67:4725–4731.

16. Hassan, A. A., A. Abdulmawjood, A. O. Yildirim, K. Fink, C. Lammler, and R. Schlenstedt.2000. Identification of streptococci isolated from various sources by determination ofcfbgene and other CAMP-factor genes. Can. J. Microbiol.46:946–951.

17. Jansen, R., J. Briaire, E. M. Kamp, A.L. Gielkens, and M. A. Smits.1995. The CAMP effect ofActinobacillus pleuropneumoniae is caused by Apx toxins. FEMS Microbiol. Lett.126:139–143.

18. Jiang, M., L. A. Babiuk, and A. A. Potter.1996. Cloning, sequencing and expression of the CAMP factor gene ofStreptococcus uberis. Microb. Pathog.

20:297–307.

19. Jurgens, D., B. Sterzik, and F. J. Fehrenbach.1987. Unspecific binding of group B streptococcal cocytolysin (CAMP factor) to immunoglobulins and its possible role in pathogenicity. J. Exp. Med.165:720–732.

20. Ke, D., C. Menard, F. J. Picard, M. Boissinot, M. Ouellette, P. H. Roy, and M. G. Bergeron.2000. Development of conventional and real-time PCR assays for the rapid detection of group B streptococci. Clin. Chem.46:324– 331.

21. Kilian, M.1998. Streptococcus and Lactobacillus, p. 633–667.InA. Balows and B. I. Duerden (ed.), Topley and Wilson’s microbiology and microbial infections, 9th ed. Arnold, London, United Kingdom.

22. Orafu, C., P. Gill, C. Nelson, B. Hecht, and M. Hopkins.2002. Perianal versus anorectal specimens: is there a difference in group B streptococcal detection? Obstet. Gynecol.99:1036–1039.

23. Phillips, E. A., J. W. Tapsall, and D. D. Smith.1980. Rapid tube CAMP test for identification ofStreptococcus agalactiae(Lancefield group B). J. Clin. Microbiol.12:135–137.

24. Podbielski, A., O. Blankenstein, and R. Lutticken.1994. Molecular charac-terization of the cfb gene encoding group B streptococcal CAMP-factor. Med. Microbiol. Immunol.183:239–256.

25. Ratner, H. B., S. W. Lyndell, and C. W. Stratton.1986. Evaluation of spot CAMP test for identification of group B streptococci. J. Clin. Microbiol.

24:296–297.

26. Ripio, M. T., C. Geoffroy, G. Dominguez, J. E. Alouf, and J. A. Vazquez-Boland.1995. The sulphydryl-activated cytolysin and a sphingomyelinase C are the major membrane-damaging factors involved in cooperative (CAMP-like) haemolysis ofListeriaspp. Res. Microbiol.146:303–313.

27. Ruhlmann, J., B. Wittmann-Liebold, D. Jurgens, and F. J. Fehrenbach.

1988. Complete amino acid sequence of protein B. FEBS Lett.235:262–266. 28. Scharg, S. J., S. Zywicki, and M. M. Farley.2000. Group B streptococcal disease in the era of intrapartum antibiotic prophylaxis. N. Engl. J. Med.

342:15–20.

29. Schuchat, A.2000. Neonatal group B streptococcal disease—screening and prevention. N. Engl. J. Med.343:209–210.

30. Skalka, B., and J. Smola.1981. Lethal effect of CAMP-factor and uberis-factor: a new finding about diffusible exosubstances ofStreptococcus agalac-tiaeandStreptococcus uberis. Zentbl. Bakteriol. Parasitenkd. Infektkrankh. Hyg. Abt. 1 Orig. Reihe A249:190–194.

31. Sterzik, B., and F. J. Fehrenbach.1985. Reaction components influencing CAMP factor induced lysis. J. Gen. Microbiol.131:817–820.

on May 15, 2020 by guest

http://jcm.asm.org/