175
FEMS MicrobiologyLetters 9 (1980) 175-177
© Copyright Federation of European MicrobiologicalSocieties
Published by Elsevier/North-HollandBiomedicalPress
A SCHEME FOR THE PHAGE TYPING OF SALMONELLA
HADAR
J.D.H. DE SA, LINDA R. WARD and B. ROWE *
Division of Enteric Pathogens, CentralPublic Health Laboratory, Colindale Avenue, London, NI¢9 5HT, U.K.
Received and accepted 1 August 1980
1. Introduction
Before 1971 Salmonella hadar was rarely isolated
in the British Isles. The increase in the number of human infections, which were generally associated with
food poisoning, was gradual at first but by 1975 the
serotype had become one of the most prevalent seretypes isolated from man in England and Wales. By
1977 S. hadar accounted for 11% of all human strains
received in the Reference Laboratory, being second in
prevalence to S. typhimurium. In 1978 and 1979 the
respective figures were 14% and 16%. In these 3 years
numerous large food-borne outbreaks occurred and
turkeys were the main vehicle of infection. The seretype was found to be established in many turkey
flocks throughout Britain [ 1].
Phage typing is a valuable epidemiological tool and
a phage-typing scheme for S. hadar is described.
on strains ofS. hadar on nutrient agar plates. After
overnight incubation at 38.5°C, single plaques visible
to the naked eye were cut out, together with a small
portion of surrounding culture, according to Callow
[2]. The phages were grown in 3 ml of nutrient broth
with shaking for approx. 4 - 5 h when a clearing of
the broth became noticeable due to lysis of the host
cells by the phage. The culture was spun down, the
supernate drawn off, sterilised with toluene and
titrated against the propagating strain to determine
the Routine Test Dilution (RTD) [3].
150 strains ofS. hadar from human, animal and
em/ironmental sources were screened for lysogeny as
above and 35 phages were obtained. Similarly 32 Salmonella serotypes which possessed either the antigens
O = 6,8;H = Zlo; or H = e, n, x were screened for
lysogeny since S. hadar has the antigenic structure
6,8: zl0: e, n, x. Two phages which reacted with S.
hadar were isolated, one each from S. muenchen and
S. newport.
2. Materials and Methods
2.1. Isolation o f typing phages
The typing phages were from two sources: lysogenic stra~s and sewage strains.
2.1.1. L ysogenic strains o f S. hadar and antigenically
related sere types .
Cultures we.re tested for lysogenicity by growing
them in 3 ml of Difco nutrient broth with shaking at
38.5°C for 5 h. After centrifuging the supernates
were drawn off, sterilised with toluene and spotted
* To whom reprint requests should be addressed.
2.1.2. Sewage
Sewage samples were obtained from areas where
turkey breeding was well established and where S.
hadar was likely to be prevalent in the sewage. Samples were centrifuged at 3500 rev./min and the supernates were separated and sterilised with toluene. The
treated samples were suspended in equal parts of
double-strength nutrient broth, inoculated with broth
cultures of S. hadar strains and incubated statically at
38.5°C overnight or with shaking for 3--4 h~ The
supernates were then separated after centrifugation,
treated with toluene and spotted on a selection of S.
hadar strains, The procedure for selection and purification of the phages was the same as described above.
13 phages were isolated from this source.
176
From
phages
lysogenic
were
a selection
cultures
obtained.
ofS.
to give identical
hadar
All these phages
were
strains and many
reactions.
because
and sewage a total of 50
Others
tested
were
were
of instability
titres and 9 phages
on
scheme.
found
from
discarded
or inability
were selected
Of these, phages
lysogenic
to obtain
adequate
for the final typing
1, 6 a n d 9 w e r e i s o l a t e d
hadar,
strains orS.
phages
2 and 5
TABLE 1
Reactions of
Phage type
S.hadar t y p e
strains with Routine Test Dilutions of the typing phages
S.hadar t y p i n g
p h a g e s at R T D
1
2
3
4
5
6
7
8
9
CL
1
SCL
CL
OL
OL
SCL
CL
OL
OL
2
-
CL
OL
OL
SCL
CL
OL
OL
CL
3
-
-
OL
OL
SCL
CL
OL
OL
CL
4
5
-
CL
+
OL
OL
OL
OL
SCL
-
CL
CL
OL
OL
OL
CL
6
7
-
-
OL
-
OL
OL
SCL
-
CL
-
OL
OL
OL
CL
8
SCL
-
-
SCL
CL
OL
OL
CL
9
.
SCL
CL
OL
OL
CL
10
.
.
OL
OL
11
12
13
.
.
.
14
-
++
15
-
-
16
-
17
-
18
.
19
20
-
21
.
22
-
23
-
24
.
25
SCL
CL
.
26
27
-
CL
CL
OL
28
29
30
31
SCL
SCL
SCL
-
CL
CL
-
32
-
33
-
34
35
SCL
CL
36
37
SCL
-
SCL
38
-
SCL
39
SCL
40
.
.
.
.
.
.
-
OL
CL
CL
.
.
OL
.
.
OL
-
CL
OL
OL
OL
CL
OL
OL
-
CL
-
OL
CL
OL
OL
SCL
-
OL
OL
CL
CL
OL
QL
SCL
CL
-
OL
-
CL
OL
OL
SCL
CL
-
OL
CL
OL
-
-
SCL
CL
CL
OL
-
OL
CL
.
.
.
.
.
.
.
OL
-
OL
-
SCL
-
OL
OL
CL
-
OL
OL
-
-
OL
OL
CL
-
OL
OL
-
CL
-
-
CL
SCL
-
OL
-
CL
CL
.
.
.
.
.
-
.
SCL
CL
OL
OL
OL
OL
OL
OL
OL
-
SCL
SCL
CL
-
OL
OL
CL
OL
OL
OL
SCL
SCL
SCL
CL
CL
CL
OL
-
OL
OL
CL
CL
CL
OL
OL
SCL
-
OL
OL
CL
CL
OL
-
OL
-
SCL
CL
-
OL
OL
OL
÷
CL
CL
OL
OL
SCL
CL
-
-
-
CL
_
-
-
OL
-
-
OL
-
OL
-
SCL
-
-
-
SCL
-
OL
CL
CL
SCL
-
-
OL
SCL
SCL
OL
OL
-
-
+
.
.
.
.
CL
.
-, No reaction; +, 1-20
lysis.
.
.
.
SCL
.
plaques; ++, 21-50
.
.
.
p l a q u e s ; S C L , s e m i - c o n f l u e n t lysis; C L , c o n f l u e n t clear lysis; O L , c o n f l u e n t o p a q u e
177
from lysogenic strains of S. muenchen and S. newport
respectively and the remaining phages 3, 4, 7 and 8
were from sewage samples. Each of these phages was
grown in bulk by the standard agar layer technique
[4,5 ] on its specific propagating strain and sterilised
by treatment with- 0.14% toluene or four drops from
a 50 dropper pipette per 20 ml of lysate [6].
2.2. Phage-typing procedure
Fresh cultures, preferably 24-28 h old, were
heavily inoculated into 3 ml of Difco nutrient broth
and incubated at 38.5°C with shaking for 45 min to
1 h. The final density of the culture was approx.
10s-101° cells/ml. Broth cultures were flooded over
the surface of dried nutrient agar plates and then
inoculated with the phages at their RTD. After
adsorption the plates were incubated overnight at
38.5°C and read the following morning.
The 9 phages were used to study approx. 6600
strains ofS. hadar isolated in the British Isles between
1971 and 1979.
3. Results
Forty different phage reaction patterns were
recognised (Table 1).
4. Discussion
The application of the scheme for S. hadar should
prove valuable in epidemiological investigations. The
serotype has a complex antigenic structure and precise serological identification requires highly absorbed
monospecific antisera which are expensive to produce
and not widely available. Phage typing is more rapid
and when provided as a centralised reference facility
is a more economic alternative. The results of the survey showed that the phage-typing scheme provides a
high level of discrimination for strains ofS. hadar.
References
[ 1] Rowe, B., Hall, M.L.M., Ward, L.R. and de Sa, J.D.H.
(1980) Br. Med. J. 280, 1065-1066.
[2] Callow, B.R. (1959) J. Hyg. 37, 346-359.
[3] Anderson, E.S. and Williams, R.E.O. (1956) J. Clin.
Pathol. 9, 94-127.
[4] Adams, M.H. (1950) Bacterial Viruses, Meth. Med. Res.
VoL 2. Chicago Yearbook Publishers, Chicago.
[5] Adams, M.H. (1959) Bacteriophages,Interscience, New
York.
[6] Anderson, E.S. and Felix, A. (1953) L Gen. Microbiol.9,
65.