AJEBAK 54 (Pt. 1) 91-96 (1976)
STREPTOMYCIN AND TEMPERATURE SENSITIVITY OF
COLICIN-LIKE ACTIVITY IN SALMONELLA CULTURES
by NANCY ATKINSON
(From the Department of Oral Biology,
University of Adelaide, South Australia.)
(Accepted for publication December 15, 1975.)
Summary. Salmonellin production on agar was reduced by adding streptomycin
or raising the incubation temperature to 44°. Production of group 3b salcol was
increased by incubation at 44° and, for one producer only, by adding streptomycin. Production of the diffusible, nontransferable group 4 salcol and also
of diffusible colicin V was reduced at 44°. Group 1 salcol production was
unaffected by raised temperature or streptomycin. Group 2 salcol production also
was unaffected, except in salm-1- transcipients of Arizona Soll6 where it was
reduced along with salmonellin production by incubation at 44° or addition of
streptomycin.
INTRODUCTION.
During work on transfer of colicin-like activity from Salmonella producer
strains to streptomyein-resistant recipients (Atkinson, 1974), some cultures were
seen to produce diUerent sized zones of inhibition according to the streptomycin
content of the agar or the temperature of incubation. Following these observations, cultures producing Salmonella colicins (called 'salcols'; Atkinson, 1973),
salmonellin (Atkinson, 1966) or well known colicins, were compared for inhibition zone size at two different incubation temperatures and on streptomycin
agar. The results of these tests, which are given in the present paper, showed
that the zone size (and thus the apparent activity) of some strains was enhaticed
by streptomycin or incubation at 44°, while other strains showed a reduction.
These effects were mainly related to the type of antibiotic produced rather than
to the producer strain.
MATERIALS AND METHODS.
Sources of producer strains.
Well known colicin producers were kindly supplied by Professor P. Fredericq. Wild type
salcol producers came from 100 strains described and grouped by Atkinson (1973). Their
salcol-t- recipients (subsequently called 'transcipients' after Hedges and Datta, 1973) were
derived from these producers (Atkinson, 1974). Salmonellin producers were represented by
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NANCY ATKINSON
one wild type strain and a number of its salni-f transcipients (Atkinson, 1966). Double
transcipients producing salmonellin and a salcol (Atkinson, unpublished data) were also
included. Transcipients were defined by the name of the recipient followed by the name of
the donor.
Agar activity test for inhibition zones.
Inhibition zones (subsequently called 'zones') were determined by the well known double
agar layer technique (Fredericq, 1963) in which the test strain was incubated overnight at
the required temperature to give a confluent spot of growth on an agar plate, which was
then exposed to chloroform vapour for 15 min and covered with a layer of soft agar (one
part nutrient agar to one part nutrient broth) seeded with the required indicator strain
{Escherichia coli K12-58161, subsequently called 58161, for colicins and salcols, and Arizona
Soll6 subsequently called SoH6, for salmonellin). After incubation at 37° overnight, an
active test culture inhibited the growth of the indicator strain to produce a zone, which was
graded according to the width from the edge of the spot of test growth to the edge
of the zone.
Cellophane test.
This was an adaptation of the agar activity test in which the test culture was grown on
top of cellophane laid over the agar. The cellophane and growth were removed before layering
with the indicator agar. Any diffusible colicin, salcol or salmonellin produced a zone in the
layer after incubation at 37°.
RESULTS.
Agar activity test at 37° and 44°.
To compare their zones at two different incubation temperatures, wild type
producer strains and various transcipients were grown at 37° and 44° in the agar
activity test. The results are given in Table 1. Reduced zones, lacking the typical
double edge seen at 37°, appeared with the group 4 salcol producer and the
colicin V producer, both of which also failed to give a zone in the cellophane
test at 44°, indicating that the diffusible component found at 37° was no longer
detectable at 44°. The zone occurring with the colicin V producer at 44° was
apparently due to a second type of colicin (not V or I), which was also revealed
at 37° by its zone on the indicator strain RV which was resistant to colicins V
and I. Transcipients produced results similar to those of their donors except for
S0II6/SCII/NB8 for which the group 2 salcol zone was reduced along with the
salmonellin zone at 44°.
In summary, the results in Table 1 indicated that raising the incubation
temperature to 44°, while having little effect on the zones of groups 1 and 2
salcols, reduced all salmonellin zones, increased the zones of group 3b and 3c
salcols and prevented the appearance of the diffusible components of the group 4
salcol and colicin V.
Activity tests on streptomycin agar.
Streptomycin-resistant derivatives of wild type salcol and salmonellin producer strains were grown on streptomycin (500 /ng/ml) agar and nutrient agar
at 37°, except that group 3b and 3c salcol producers also were grown at 44°.
Zones were compared on the two agars. The results are given in Table 2 which
also includes results for tests on various types of transcipients.
PRODUCTION OF SALMONELLA COLICINS
93
Double producers of salmonellin and group 2 salcol on streptomycin agar
provided two different reactions, in which transcipients of So 116 showed reduced
zones for both salmonellin and group 2 salcol, hut transcipients of SCll or
SW2, while showing reduced zones for salmonellin, had no change in zone for
group 2 salcol. Here the type of recipient appeared to affect the group 2 salcol
response to streptomycin.
TABLE 1.
Effect of increased incubation temperature on satcol and salmonellin producers.
Type of test strain
Zone size at increased temperature
on 58161
wild type salcol producers
group i, group 2, group 3a
group 3b, group 3c
group 4
wild type salmonellin producer
colicin producers
B, C, F, I, K
S4
V
salcoH- transcipients
group 1, group 4
group 3b
salm+ transcipients
salm+ group 1 salcol + transcipients
salm+ group 2 salcol + transcipients
SC11/NB8
S0II6/SCII/NB8
salm+ group 3b salcol + transcipient TM85/SC11
salm+ group 4 salcol + transcipient
on Sol 16
unchanged
increased
reduced
reduced
unchanged
increased
reduced
unchanged
increased
unchanged
reduced
reduced
unchanged
reduced
increased
increased
reduced
reduced
reduced
reduced
- = no zone.
Strains tested:
wild type producers of salcol or salmonellin:
salcol group 1. Ad37 Ad59 A20 BL24 BM151 BM168 BM200 BM340 BM368 D9 D22 D131
D143 E28 G3 SC68 Lxl L3 L8 M4 M7 M41 M51 Nel3 Pc3 SC127 SCI32
TM522S139.
salcol group 2. NB8 BM102 BM516.
salcol group 3a. D33 SC83 M5.
salcol group 3b. TM43 TM62 TM85 TM122.
salcol group 4. S358.
salmonellin.
SCll.
colicin producers:
CA18 colicin B CA57 colicin C CA42 colicin F CA53 colicin I K235 colicin K
CA7 colicin V S385 Shigella dispar colicin S4.
transcipient producers of salcol or salmonellin:
salcol group 1. 58161/A20 58161/Nel3 Soll6/A20 Soll6/Ad37.
salcol group 3b. 58161/TM85.
salcol group 4. 58I61/S358 (nondiffusible) Sol 16/S358 (nondiffusible).
salmonellin.
SW2/SCI1 (previously SW2B) S0II6/SCII (previously S0II6B)
17K/SC11 (previously 17KA) 58161/SCll (previously 58161A).
transcipient double producers of salcol and salmonellin:
salcol group 1 and salmonellin. Soll6/SCll/BL24 Soll6/SCll/Lxl SW2/SCll/NeI3.
salcol group 2 and salmonellin. SCI 1/NB8 Sol 16/SCl 1/NB8.
salcol group 3b and salmonellin. TM85/SC11.
salcol group 4 and salmonellin. SCI 1/S358 58161/SCl 1/S358*.
* The salmonellin production of this strain was transferred from Sol 16/SCl 1.
NANCY ATKINSON
94
Reduction in zone size on streptomycin agar could be due to reduction of
growth of the test strain as compared to nutrient agar. However, all the
salmonellin producers grew well on streptomycin agar, suggesting that their
reduced zone size was due to the streptomycin inhibiting salmonellin production.
TABLE 2.
Effect of streptomycin agar on salcol and salmonellin producers.
Type of test strain
Zone size on streptomycin agar
on 58161
wild type salcol producers group 1, group 2,
group 3b, group 4
wild type salmonellin producer
salcol + transcipients
group 1, group 4
group 3b
salm+ transcipients
salm+ group 1 salcol + transcipients
salm+ group 2 salcol + transcipients
SC11/NB8, SW2/SC11/NB8
Sol 16/SCl 1/NB8
salm+ group 3b salcol+ transcipients
salm+ group 4 salcol + transcipients
on Sol 16
unchanged
reduced
unchanged
increased
unchanged
reduced
reduced
unchanged
reduced
unchanged
unchanged
reduced
reduced
reduced
reduced
- = no zone.
Strains tested:
wild type producers of salcol or salmonellin (streptomycin-resistant isolates):
salcol group 1. A20Sr S139Sr LxlSr Ad37Sr Pc3Sr M27Sr.
salcol group 2. NB8Sr BM102Sr.
salcol group 3b. TM85Sr.
salcol group 4. S358Sr.
salmonellin.
SCllSr.
colicin producers:
S385 Shigella dispar colicin S4 Sr (grew better on streptomycin agar).
transcipient producers of salcol or salmonellin:
salcol group 1. 58161/A2O 5816I/Nel3 17K/Nel3 Soll6/BM15I S0II6/BMI68
S0II6/BM2OO S0II6/M5 S0II6/SC68 S0II6/BL24 S0II6/AD37
So 116/A20 So 116/Ad32 So 116/Pc3 So 116/He3 So 116/L8.
salmonellin.
SW2/SCI1 Sol 16/SCl 1 17K/SC11 58161/SCll AdLiz4/SCIl
bunnikLiz23/SCll minnesotallll/SCll ruirull24/SCll Adl4/SCll
AdCey4/SCll.
transcipient double producers of salcol and salmonellin:
salcol group 1 and salmonellin. Pc3/SCll Lxl/SCll M27/SC11 Sol 16/SCl 1/A20
Sol 16/SCl 1/Lxl 17K/SCIl/Lxl Sol 16/SCl 1/BL24.
salcol group 2 and salmonellin. SC11/NB8 SW2/SC11/NB8 Sol 16/SCl I/NB8.
salcol group 3b and salmonellin. TM85/SC11.
salcol group 4 and salmonellin. SW2/SC11/S358 58161/SCl 1/S358*.
* The salmonellin production of this strain was transferred from S0II6/SCII.
Increase in zone size on streptomycin agar occurred only with one test
strain, the transcipient of group 3b salcol, 58161/TM85, and could be due to the
streptomycin stimulating group 3b salcol production. However, the donor, TM85,
showed no increase in zone size on streptomycin agar. Furthermore, 58161/TM85
PRODUGTION OF SALMONELLA GOLIGINS
95
on streptomycin agar had similar zones at 37° and 44°, whereas TM85 had
increased zones at 44°. Thus, in the transcipient, the group 3b salcol production
appeared to be activated by streptomycin in the agar, and was no longer
susceptible to change of incubation temperature. The loss of temperature
sensitivity and the enhancing effect of streptomycin could be due to the effect
of the new host, 58161, on the transferred group 3b salcol factor.
In summary of Table 2, growth on streptomycin agar reduced the salmonellin zones of all producers, increased the zones produced by the transcipient of
group 3b salcol, 58161/TM85, and had no effect on the zones of any other strain.
DISGUSSION.
Accepting that reduced zone size indicated reduced production of the
active material, the present work showed sensitivity of salmonellin production to
temperature of incubation and to the presence of streptomycin in the agar.
Transfer of the salmonellin factor to a number of different recipients, some
producing salcols, had no effect on the temperature and streptomycin sensitivity
which were therefore considered to be typical characteristics of salmonellin
production and could be used to define it more accurately.
In contrast, wild type salcol group 3b production was also temperaturesensitive, but was increased at higher incubation temperatures, thus allowing
better investigation of the group 3b salcol producers, hitherto recorded as weak
producers, which were therefore difficult to work with (Atkinson, 1973).
Increased production on streptomycin agar occurred with the transcipient of
group 3b salcol, 58161/TM85 but not with the donor TM85. This effect could
be due to the infiuence of the recipient strain, 58161, on the group 3b salcol
factor (a host-mediated effect).
Production of group 1 and the non-diffusible, transferable group 4 salcol was
unaffected or increased by temperature or streptomycin even in transcipients also
producing the sensitive salmonellin. However, group 2 salcol production, while
unaffected in the wild type strains, was reduced by higher incubation temperatures and by streptomycin in transcipients of Soll6 which also produced
salmonellin. This suggested that an interaction had occurred between the two
factors in this host. However, transfer of group 2 salcol factor to other salmonellin producers (SW2/SG11, SGll) resulted in no reduction of group 2 salcol
production. Here the type of recipient markedly affected the ability to produce
group 2 salcol. Further work on these transcipients supported this view
(Atkinson, unpublished data).
Acknowledgements.
this work.
Mrs. Felicity Underwood provided excellent technical assistance in
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NANCY ATKINSON
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(1973):
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