AN ABSTRACT OF THE THESIS OF
PATRICK, LUI-SUN KWAN
(Name)
in
Title:
FOOD SCIENCE
(Major)
for the
MASTER OF SCIENCE
(Degree)
presented on
(Date)
INFLUENCE OF COMMENSAL MICROORGANISMS ON
m
CLOSTRIDIUM BOTULIMTJM TYPE E
Abstract approved:
-^
-y.
ong ffj Lee
^,
\ """J"
Achromobacter A43'^produced a compound inhibitory to the outgrowth of Clostridium botulinum type E spores.
The inhibitor could
be produced in various laboratory media and the outgrowth of germinated spores was completely inhibited by 1 /10th dilution of the A43
spent medium.
Germination was not affected.
The spores lost
refractility in the presence of the inhibitor and were darkly stained by
crystal violet.
The germinated spores showed little outgrowth, no
elongation, and no lysis.
The A43 inhibitor was dialysable and could be concentrated by
lyophilizing the dialysate.
The inhibitor was stable at 370C, 250C,
and 50C, but was partly inactivated when heated at 65° C for 10 min.
The inhibitor was not volatile and could not be vacuum distilled at
40° C.
Solutions of acids of pH below 2.0 destroyed the activity.
Molecular weight of the inhibitor was estimated at 800 ~ 1, 000
Daltons by PSAC Millipore Pellican ultrafiltration and by elution time
on a column of Bio-Gel P-2.
The inhibitor could be separated into
fractions containing peptides and lipids on a Bio-Rex 70 X- ion exchange column.
The presence of phosphatidic amino acids was also
suggested by Rhodamine 6G reaction.
The A43 inhibitor was similar, in molecular weight and inhibition characteristics, to tylosin, but appeared to be more heat labile
than tylosin.
Achromobacter species were shown to be selectively inactivated
by the smoking process.
The smoked fish, therefore, may lack the
added safety factor that the inhibitor similar to that of A43 might
provide in other seafoods.
Influence of Commensal Microorganisms on
Clostridium botulinum Type E
by
Patrick, Lui-Sun Kwan
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed June 1973
Commencement June 1974
APPROVED:
Associate P/0fesso»,\M Food Science
in charge of major
Head of Department of Food Science and Technology
Dean of Graduate School
Date thesis is presented
Typed by Clover Redfern for
4/^ /#?3>
Patrick, Lui-Sun Kwan
ACKNOWLEDGMENT
The author wishes to express his appreciations to Dr. J.S. Lee
for his guidance during the course of this investigation and the preparation of this thesis, to Mr. Frank J. Ivey for his valuable suggestions
and technical assistances, and to the members of the Department of
Food Science and Technology for their numerous contributions.
This study was supported by Public Health Service grant
FD-00418 from the Food and Drug Administration.
TABLE OF CONTENTS
Page
INTRODUCTION
1
LITERATURE REVIEW
Sporulation
The Spore
Germination
Outgrowth
Inhibition of Clostridium botulinum
Inhibition of (3. botulinum in Foods
Influence of Microorganisms on Clostridia
4
4
6
6
8
9
10
12
14
14
14
15
MATERIAL AND METHODS
Microorganisms
C^. botulinum Type E Spores
Growth of Achromobacter Species
Inhibition Study
Inhibitor Characterization
Dialysis and Lyophilization
Vacuum Distillation
Ultra Filtration
Stability Testing
Inhibitor Separation
Trichloroacetic Acid Precipitation
Column Separation
Inhibitor Identification
17
17
17
18
18
18
18
19
20
RESULTS
Inhibitor Production
Inhibitor Characterization
21
21
23
DISCUSSION
31
BIBLIOGRAPHY
36
16
LIST OF FIGURES
Figure
1.
2.
3.
4.
Page
Germination and outgrowth of (3. botulinum type E,
Beluga, in Achromobacter A43 spent medijm (SM) of
TPN (TPN-SM), PM (PM-SM) and RCM (RCM-SM).
22
Growth of Achromobacter A43 in perigo medium (PM),
reinforced clostridia medium (RCM) and tryptonepeptone-yeast extract-glucose-NaCl medium (TPN).
24
Inhibitory effects of Achromobacter spent medium
(PM-SM), collected at various growth stages in PM,
on C. botulinum type E, Beluga, spores.
25
Effects of heated Achromobacter A43 spent medium
(SM) on germination and outgrowth of C. botulinum
type E, Beluga, spores.
28
LIST OF TABLES
Table
1.
2.
3.
4.
Page
Production of anti-C^. botulinum type E factor by
Achromobacter species.
21
Summary of physico-chemical characteristics of
Achromobacter A43 inhibitor.
26
Anti-C. botulinum type E activities of fractions eluted
from Bio-Gel P-2 column.
29
Anti-C. botulinum type E activities of fractions eluted
from the Bio-Rex 70 X column.
30
INFLUENCE OF COMMENSAL MICROORGANISMS ON
CLOSTRIDIUM BOTULINUM TYPE E
INTRODUCTION
Human botulism has always been caused by the consumption of
improperly preserved foods (74).
Sincethe toxic outgrowth of
C^. botulinum appears to require a partial removal of the natural
microbial flora, several possible roles of the commensal microorganisms have been proposedOne of the widely accepted explanations is that the reduction of
competing microbial population would increase the storage life,
thereby, allowing (3. botulinum spores to germinate, grow and produce toxin (74).
The major weakness of this explanation is that the
term "competition" is not defined.
The growth of commensal micro-
organisms in foods was shown to exhaust oxygen that would favor the
anaerobic growth of C^. botulinum (95).
The proteolytic enzymes of
the commensal microorganisms activated type E C^. botulinum proto toxin (100).
Some microbial activity would release growth factors, at
least during the early stages of spoilage (13).
Such activities of
commensal microorganisms would stimulate rather than inhibit C^.
botulinum.
Another possibility is that some of the microorganisms in the
commensal flora that could inhibit (3. botulinum were inadvertently
removed during processing.
Such possibility was investigated in this
study.
In fresh seafoods, gram-negative heterotrophs, such as
Pseudomonas, Achromobacter (Acinetobacter-Moraxella)
Flavobacterium predominate the microbial flora (109).
and
Flavobacter-
ium species rapidly disappear during cold storage, thus leaving
Pseudomonas and Achromobacter the two dominant microorganisms
found in fish (109).
Pseudomonas and Achromobacter also made up
the principle surface flora of meat (59).
Pseudomonas species are considered responsible for the spoilage of fishery products.
Pseudomonas groups III and IV displaced
other microorganisms and became the dominant species (90%) in fish
stored on ice (108).
Pseudomonas species gradually became the pre-
dominant flora on meat stored at 12 C (59).
Achromobacter species are present usually in small numbers in
fresh fish but may increase in proportion after commercial handling
(68).
Achromobacter species are not proteolytic, are not known to
degrade amino acids and are mostly indifferent toward sugars (117).
The taxonomic status of the gram-negative to variable coccobacilli, classified variously as Achromobacter, Acinetobacter, and
Moraxella, has not been settled (12, 117). For the sake of uniformity
this group will be referred as Achromobacter throughout this thesis.
Although some may be lipolytvc (10), the precise role of the
Achromobacter species in food spoilage is not known.
Since Achromobacter species were conspicuously absent in
smoked fish (69), they represented the microbial species which were
removed during processing.
In this investigation, the germination and outgrowth of C^.
botulinum type E spores, in the presence of metabolic products of
Achromobacter species were examined and the substance(s) inhibitory
to C. botulinum type E spores was later isolated and partially characterized.
LITERATURE REVIEW
Clostridium botulinum is an anaerobic, spore forming, gram
positive rod, commonly found in soil.
It is classified by the sero-
logical reactions of the toxins into six groups: A, B, C, D, E, and F.
The physiology of sporulation and outgrowth of C^. botulinum
have not been fully investigated due to difficulties with the anaerobic
system and the extreme toxicity,
Much of our knowledge of (3. botu-
linum has been derived indirectly from non-toxic spore formers such
as Bacillus and C^. sporogenes PA 3 679.
Sporulation
Sporulation of bacteria is usually initiated by nutrient depletion
following the end of logarithmic growth (51).
Exotoxins of clostridia
appear just prior to sporulation and only after an active growth (99).
Autolysis of cells may be involved in the release of the toxins (17).
Except under controlled conditions, sporulation does not occur
simultaneously in all cells in a given population.
In the same medium,
some cells may be actively dividing while others may be sporulating
(33).
It is postulated that sporulation and cell division share a com-
mon mechanism but sporulation, once triggered, is irreversible (55).
Synchronizing sporulation to produce spores at the same physiological
stage is important to later differentiate sporulation, germination and
out-growth.
In C^. roseum, a rapid and simultaneous sporulation can
be achieved by using a heavy inoculum of actively growing cells to give
an initial population equal to about 10% of the maximum population
attainable in the sporulation medium (53).
There are some absolute chemical requirements for sporulation.
The Mn
++
ion is essential for sporulation, but not for the vegetative
growth (30).
The Ca
++
ion is needed for the formation of heat
resistant spores (38,80), and a Ca
- specific transport system has
been demonstrated in sporulating B. subtil is (38) and B. megater ium
(21).
Other ions are shown to stimulate sporulation (30).
them NH. , PO.
and SO .
Among
have been shown to enhance the sporu-
lation of C^. botulinum (72).
The list of nutrients reported to be required for the sporulation
of C_. botulinum is long.
Among those are methionine (122); acetate
(31); arginine or its degradation products (32); thiamine (31);
L-alanine, L-proline and L-isolencine (32).
Arginine may be an
important source of exogenous energy required for the maturation of
fore-spores (32).
Some peptides in trypticase (127), acetate and
butyrate (40) and casein derivatives (57^ have stimulated sporulation
of C. botulinum.
The Spore
The fully formed spore is refractile to light (80), resistant to
heat, chemicals, and y radiation (105).
The spore contains numer-
ous spore specific substances, such as diaminopimelic acid, which is
associated with the cortex (83); 3-phospho-D-glycer ic acid, which is a
source of ATP at germination (105); and dipicolinic acid (DPA), which,
together with Ca
, is associated with heat resistance (81).
Germination
Some bacterial spores require activation for germination (73).
Activation is a reversible process which breaks dormancy and conditions the spore for germination under appropriate conditions (29).
It
increases the rate and extent of germination and activates dormant
enzymes in the resting spore (25).
Activation can be achieved by
heating spores in aqueous solutions (29); by reducing agents (47); low
pH (67); Ca
++
-DPA complex (71); radiation (69); water vapor and ethyl
alcohol (73) and by mechanical aberrations (98).
An activated spore retains all of the spore properties except
that it is no longer dormant (73).
If germination is not induced after
activation, the spore can revert to dormancy (29,93).
The germination of spores is an irreversible (71) and degradative process (94) resulting in a cell which has lost the typical
characteristics of the bacterial spore, yet different from the vegetative cell.
During germination, the spore excretes many spore specific
substances such as calcium dipicolinate, diaminopimelic acid and
other spore peptides (94).
The optical density of a germinating spore
suspension decreases as a result of the loss of refractility and the
spore becomes stainable (93).
The usual sequence of events associ-
ated with germination, based on the studies of Bacillus species is as
follows (46): increase in heat sensitivity, increase in sensitivity to
antimicrobial agents, release of K , release of Ca
, release of
DPA, dye stainability, phase darkening, release of hexosamine and
loss of reflectility.
Germination and outgrown are two separate processes that can
be distinguished by different nutritional requirements (78, 126).
Germinated spores may never develop into vegetative cells unless
proper growth conditions and requirements are met (118, 125).
Spores
of anaerobic organisms can germinate under aerobic conditions (1,
119).
Germination can be initiated by addition of a variety of nutrient
initiators or the germinants.
Nutrient initiators are generally strain
specific (46) and may not be required for the subsequent outgrowth
(118).
Among those reported for JC. botulinum are lactate (2),
bicarbonates (2,119,128), glucose (3,129), galactose (1), L-alanine
(2), L-cystine, and glycine (121).
Glucose-6-phosphate,
8
fructose-6-phosphate and fructose-1, 6-diphosphate are also effective
germinants for type E (3. botulinum (2).
Germinants may be supplied
in various combinations (2) and concentrations (115).
Strasdine (1967)
(115) achieved rapid germination of type E C_. botulinum in a
trypticase-peptone-glucose medium under rigidly defined and controlled conditions.
Non nutrient initiators, enzyme and physical
initiators have also been reported for Bacillus species (46), but most
of these have not been fully tested with C^. botulinum.
The optimal conditions for the germination of C^. botulinum type
E spores may vary slightly from strain to strain.
Ando (1971) (1)
found a heat shock (activation) temperature of 65° C for 10 minutes was
optimal for Iwanai strain.
In a systematic study, Strasdine (114)
demonstrated that C^. botulinum spores germinated best at 30° C and
pH 7.0 in a trypticase-glucose-peptone medium. Although an anaerobic
condition was not required for germination, thioglycolate increased
the extent of germination for type E (3. botulinum (1).
Outgrowth
The fate of a germinating spore is dependent on the availability
of the exogenous nutrients and the growth conditions, which may be
different from those of germination (27).
Synthesis of macromolecules
and structures during outgrowth would result in a vegetative cell.
A comparative evaluation of media used for C^. botulinum by
Kauter jst al. (1970) (66) has shown that most of those employed invariably contained glucose, peptone, and trypticase (61, 102, 115).
Glu-
cose is important for toxin production (52) but it may suppress
sporulation (40).
C^. botulinum type E grows between pH 4. 8-8. 5 (8)
but the optimal pH is 7.0 (116).
30° C and at an A
w
of 0. 995 (84).
Type E C^. botulinum grows best at
The growth of this organism at
temperature as low as 3. 3° C and at an A
w
of 0. 94, however , have
been reported (8).
Inhibition of Clostridium ^botulinum
Foster and Wynne (1948) (42) demonstrated that low concentrations of some unsaturated C
0
Io
fatty acids , e.g., oleic, linoleic and
linolenic acids inhibited the germination of C^. botulinum spores.
A
specific inhibitor for C^. botulinum has been discovered in a medium
heated with sodium nitrite (91).
This so called "Perigo" factor is a
very potent inhibitor of C^. botulinum and, unlike NaNO?, its activity
was not pH dependent (91).
The inhibitor, yet to be characterized,
appears to be an inorganic coordination complex of sulphur, iron,
nitrogen, and oxygen (B.Jarvis, personal communication).
10
Inhibition of C. botulinum in Foods
Clostridia are primarily soil organisms (62).
C_. botulinum type
E, however, has a wide geographic distribution in marine and esturian
environments and can be found in the fish products throughout the
world (106).
Following the outbreaks in 1963 (5), type E C^. botulinum
was found to be indigenous in many species of fish of the Great Lakes
(20).
The ubiquitous presence of (3. botulinum type E has to be kept
in mind for processing smoked or other semi-preserved fisheries
products.
In semi-preserved foods, heat treatment is probably the most
important safety factor (36).
The heat treatment prescribed in the
Good Manufacturing Practice (GMP) (44) for smoked fish (hot smoking
at 82° C (180oF) for 30 minutes if the water phase salt (WPS) of the
finished product is 3. 5 or above; or 660C (150oF) for 30 minutes if
the WPS is at least 5.0) is probably sufficient in eliminating most of
the original aerobic microbial flora (69) and C^. botulinum type E (86).
The curing salts added to smoked fish augment the effect of heat (36).
The 2. 5-5.8% of NaCl would inhibit the outgrowth and/or toxin production of C^. botulinum type E (9,41, 103) and sodium nitrite, if added,
would further inhibit C^. botulinum type E (112), especially in combination with heat (36).
C. botulinum type E spores, however would sur-
vive the smoking process (24,87,88,89,90), stressing the significance
11
of inhibiting growth and toxin production in smoked fish.
Inactivation of microorganisms by smoking depends on the heat
and moisture levels.
A relative humidity of 70% in the smoke cham-
ber in addition to the hot smoking temperature of at least 176. 60F for
30 minutes is needed to assure a safe product (86).
Any variation of
the smoking conditions from those cited in the GMP or above may
invite C^. botulinum type E hazard.
heat (4,82).
Spores are more resistant to dry
Dry areas on the surface of the fish may contribute to the
survival of type E spores in hot smoked fish (24).
The heat inactivation and NaCl inhibition of spores are proportional to the number of spores initially present (97).
Higher tempera-
tures and greater concentrations of NaCl are needed to inactivate a
heavy concentration of spores in food.
The commercially accepted
level of curing salts may not be sufficient to inhibit the surviving C^.
botulinum type E spores (92, 110), especially at pH above 7.0 (112).
Salt concentration which has no effect on spore outgrowth at optimum
temperature can not prevent the outgrowth at lower temperature (41,
104).
(3. botulinum type E can germinate, grow and produce toxin at
3.3-50C (102), although the growth at the low temperatures maybe
extremely slow (85).
Vacuum packaged smoked fish, with an extended
shelf life, may permit C^. botulinum type E spores to germinate and
grow.
12
Influence of Microorganisms on Clostridia
The effects of commensal microorganisms on C^. botulinum have
been variously described as stimulatory or inhibitory.
Microbial
growth can affect C^. botulinum by changing the pH and/or the redox
potential of the medium (3), or by production of certain metabolites (7).
Salmonella typhimiurium and C^. perfringens (79) and some lactic
acid bacteria (13) enhanced the growth of C_. botulinum by lowering the
oxidation reduction potential.
some growth factors (13).
Lactic acid bacteria may also supply
Yeast (77) and P. aeruginosa (95) also sup-
ported the growth of C^. botulinum in food by increasing the pH.
C^. botulinum type E toxin was shown to be potentiated by some
proteolytic clostridia (28, 100).
Some acid producing organisms from
the soil, however, destroyed or prevented the toxin formation of C^.
botulinum (53).
The growth of C^. botulinum in fermented foods such as vegetables or milk can be inhibited by naturally occurring lactic acid
bacteria; and the addition of lactic acid starters to some foods was
shown to inhibit C^. botulinum type A (101).
Free fatty acids and anti-
biotics produced by Brevibacterium linens in surface ripened cheeses
inhibited the growth of C^. botulinum (50).
Nicin produced by Strepto-
coccus lactis in milk products also was inhibitory to C^. botulinum
(49).
Bioticin E produced by some non-toxigenic variants of C^.
13
botulinum type E was bacteriostatic for spores of type E and to the
non-proteolytic types of B and F (39, 65).
Culture filtrates of C.
sporogenes, _S. faecalis and Escherichia coli, as well as the proteolytic enzymes produced by other microorganisms also prevented
the germination of C^. botulinum (28).
14
MATERIAL AND METHODS
Microorganisms
C^. botulinum type E, Beluga strain was selected as the test
microorganism for its predictable high spore yield.
The culture was
obtained from Mr. Keith Ito of the National Canners Association,
Berkeley, California.
The spores were germinated by heat-shock at
65° C for 10 min and grown at 30° C.
Achromobacter A43 was initially isolated from seafoods and has
been maintained as a reference strain in this laboratory.
organism is a non-motile gram negative coccobacillus.
The
It possesses a
cytochrome oxidase and is resistant to 3.0 I. U. penicillin G, thus may
belong to either Moraxella or Acinetobacter species.
classification scheme of Bauman et al. (12),
According to the
Moxaxella species should
be cytochrome oxidase positive and sensitive to 3. 0 I. U. of penicillin G.
Acinetobacter, on the other hand, should be cytochrome oxidase negative and resistant to 3. 0 I. U. penicillin G.
Achromobacter strain A44
was similarly isolated from seafoods and Achromobacter NCMB 131
and NCMB 132 were obtained from Dr. J. M. Shewan of Torrey
Research Station, Aberdeen, Scotland.
_C. botulinum Type E Spores
The biphasic culture system of Bruch et al. (1968) (22) was used
15
for the vegetative growth and sporulation of C_. botulinum type E.
It
consisted of a layer of TPG agar of Schmidt ejt al. (1962) (102), overlayed with distilled water containing 1% suspension of a 48 hr C.
botulinum cells.
The maximum sporulation of approximately 90% of
the vegetative cells was accomplished after 48 hr at 30° C.
The spores were collected by a combination of the methods of
Hitzman et al. (1957) (57) and Burch et al. (1968) (22).
The overlying
liquid was centrifuged and the sediment-containing spores, sporangia
and vegetative cells were washed 5 times with sterile distilled water.
After the last wash, the sediment was resuspended in distilled water
and refrigerated for 3 days to allow autolysis of the vegetative cells.
After this period, the cell debris collected at the bottom while the
spores remained in suspension.
The supernatant was then siphoned
off and the spores collected by centrifugation at 44 G, and washed 10
times with sterile distilled water.
Viable spore counts were made by
plating the heat shocked spores on TPG agar. The spore concentration
g
was adjusted to 2.3 x 10 spores/ml which when 0.15 ml was added to
9.85 ml of Per igo's medium in a 16x125 mm (Kimax 71350-4)
screwed capped tube, would give an O. D. of approximately 0. 35 at
630 nm..
Growth of Achromobacter Species
Achromobacter species were cultured in TPN (27), Reinforced
16
Clostridia Medium (RCM, less agar and sodium thioglycolate) and
Perigo Medium (PM) (91), minus sodium thioglycolate and bromocresolpurple.
The PM contained 2% tryptone , 1% peptone, 1% beef
extract, 0. 5% yeast extract, 0.5%NaCl, 0. 25% K-HPO^ 0.2%
glucose and 0. 1% soluble starch and the reaction of pH 7. 0.
Since
PM supported the maximum production of inhibitory substance by
Achromobacter A43 (Figure 2), and was equally suitable for (3.
botulinum type E (with 0. 1% sodium thioglycolate), most inhibition
studies were conducted with this medium.
Achromobacter A43 was grown in PM for 48 hr at 25° C in a New
Brunswick G 24 Environmental Incubator-Shaker, the spent medium
(SM) was collected by centr ifugation.
The pH of the SM was adjusted
to 7. 0 and sterilized by filtration through Millipore HAWP 04700 filter.
Growth studies of A43 in different media was conducted in
Bellco side arm flasks and the optical density of the cultures was followed spectrophotometrically at 600 nm.
Inhibition Study
Experiments for studying inhibition were carried out in PM
minus bromocresolpurple indicator.
Unless otherwise stated, all
inhibition studies were conducted with 1/10 dilution of SM, in order to
compensate for any depletion of nutrient by Achromobacter species.
Accordingly, control study was conducted with 9/10 strength of PM.
17
The pH of SM was adjusted to 7. 0 and 1. 0 ml of this was pipetted
into the screw capped tube containing 8. 85 ml of fresh PM.
The
0. 15 ml distilled water suspension of C_. botulinum type E spores was
inoculated into this medium immediately after heat shock and the
germination and outgrowth of spores followed spectrophotometrically
at 630 nm during 30° C incubation.
Some cultures in which inhibition
was apparent were examined microscopically after staining with 1%
crystal violet for 30 sec (57).
Inhibitor Characterization
Dialysis and Lyophilization
The A43-SM was dialyzed overnight against running tap water at
2° C and the inhibitory activity of the non-dializable fraction measured.
Also the dialysate collected in a given volume of water after 7 days at
2°C was concentrated by lyophilization and tested for inhibitory
activity.
Vacuum Distillation
The lyophilized dialysate, which retained the inhibitory activity,
was dissolved in distilled water and vacuum distilled at 40° C.
The
distillate and the residue were then examined for inhibitory activities
against C^. botulinum type E spores.
18
Ultra Filtration
The A43-SM was passed through millipore Pellican ultra filtration membranes, PSED and PSAC, with the respective molecular
weight cut-off of 25, 000 and 1,000.
The filtrates and the residue were
tested for the inhibitory activity.
Stability Testing
The heat stability of the inhibitory compound produced by
Achromobacter A43 was tested after autoclaving at 1210Cfor 15 min,
heating at 65° C and 850C for 10 min, and storing at 370C for 12 days.
The effect of pH was measured by treating SM with 0. 5 M HC1
to give the final pH's of 1. 0, 2. 0, 3. 0, 4. 0, 5. 0, 6. 0 and 7. 0 for
approximately 1 hr.
The pH of the treated SM, however, was read-
justed to 7. 0, with 0. 5 M NaOH, prior to testing for inhibitory activity.
Inhibitor Separation
Trichloroacetic Acid Precipitation
The lyophilized dialysate was dissolved in distilled water and
treated with an equal volume of 20% trichloroacetic acid (TCA).
visible precipition was observed.
No
After centrifugation, the super-
natant was assayed for the inhibitory activity.
19
Column Separation
The dialysate concentrated by lyophilization was fractionated
with two different columns, Bio-Gel, P-2 and Bio-Rex 70 X.
Bio-Gel
P-2 column is essentially a molecular sieve, and Bio Rex 70 X column
separates peptides or compounds with free amino groups according to
the ionic strength.
Bio-Gel P-2 resin (Bio-Rad Laboratories, 200-400 mesh) was
packed in a 2. 5 x 80 cm column to a height of 50 cm.
The sample was
eluted by gravity with distilled water at room temperature at a rate of
approximately 30 ml/hr and the eluate collected in 10 ml fractions.
The collected fractions were pooled according to the reactions with a
0. 5 M AgNO
solution and the pooled fractions tested for the inhibitory
activity.
The Bio-Rex 70 X ion exchange resin (Bio-Rad Laboratories,
200 mesh) was prepared according to the methods of Stevens and
Bergstrom (114).
It was packed to a height of 80 cm in a
2. 5 x 120 cm column.
The sample was eluted by gravity at 20C with
a 0. 2 M potassium phosphate buffer, pH 6. 2.
30 ml/hr.
The rate of elution was
The presence of peptides in the eluate, collected in 10 ml
fractions, was detected by the absorbance at 280 nm.
The different
fractions were pooled according to their reactions with ninhydrin and
tested for inhibitory activity.
20
Inhibitor Identification
The different fractions from the Bio-Rex 70 X column were
spotted on filter paper and tested for the presence of peptides and
lipids with ninhydrin spray (16) and Rhodamine 6G (7 5).
The ninhydrin and Rhodamine 6G solutions were prepared
according to the methods described by Johnston (63).
21
RESULTS
Inhibitor Production
Among the four strains of Achromobacter species tested, only
A43 appeared to produce an inhibitor(s) antagonistic toward the outgrowth of C_. botulinum type E spores, when the spent medium (SM)
was tested at l/10th strength (Table 1).
The outgrowth, but not the
germination of the spores, was inhibited by this compound (Figure 1).
The initial reduction cf optical density (O.D. ) due to germinating
spores was the same in media with or without SM but the subsequent
increase in O.D. due to outgrowth was reduced in SM containing
media.
Tylosin lactate (14) and NaNO? (104) have also been shown to
inhibit outgrowth rather than germination of C3. botulinum spores.
Table 1.
Production of anti-C^. botulinum
type E factor3- by Achromobacter
species.
Achromobacter
Strain
NCMB 131
NCMB 132
A43
A44
Inhibitory
Activity
+
C. botulinum type E Beluga spores were
suspended in spent media of Achromobacter,
and germination and outgrowth followed by
O.D. at 630 nm.
22
Figure 1.
Germination and outgrowth of C^. botulinum type E, Beluga,
in Achromobacter A43 spent medium (SM) of TPN (TPNSM), PM (PM-SM) and RCM (RCM-SM).
23
This inhibitor(s) was produced in various media (Figure 1).
The
lower level of inhibition production in TPN may be related to a longer
lag period and a reduced growth rate of Achromobacter A43 in this
medium (Figure 2).
At 48 hr, Achromobacter A43 had not yet
reached the maximum growth in TPN.
On the other hand, Achromo-
bacter A43 culture had reached or near the maximum growth in PM
and RCM at 48 hr when the SM had been obtained.
Figure 3 shows the degree of inhibitory effect of PM-SM collected during the 48 hr growth period of Achromobacter A 43 and
Achromobacter A44.
The pH was also determined to show whether the inhibitory effect
was due to change in pH.
Many antagonistic effects, especially those
observed with acid producing bacteria, have been attributed to the
acidity (101).
The pH of the Achromobacter A43-SM increased from
7. 0 to 7. 9 at the end of 48 hr (Figure 3).
If the effect was due to
pH alone, the increase would have been stimulatory rather than
inhibitory to C^ botulinum type E.
In any event, the inhibitory effect
was determined after the pH of SM had been adjusted to 7. 0.
Inhibitor Characterization
Table 2 summarizes the characteristics of the inhibitor produced by Achromobacter A43.
The inhibitor was dializable and no
inhibitory activity remained in the non-dializable fraction of SM.
The
24
jyJH>
1. 0
0.8
0.6 \
O.D
0.4
0. 2
6—&
16
Figure 2
24
32
Time in hours
40
48
Growth of Achromobacter A43 in perigo medium (PM),
reinforced clostridia medium (RCM) and tryptone-peptone
yeast extract-glucose-NaCl medium (TPN).
56
25
lA)
Figure 3.
Inhibitory effects of Achromobacter spent medium (PM-SM),
collected at various growth stages in PM, on JC. botulinum
type E, Beluga, spores.
26
activity was recoverable from the dialysate after lyophilization.
The
molecular weight of the inhibitor was less than 1, 000 Daltons as it
readily passed through both PSED and PSAC filters.
When the
lyophilized dialysate was vacuum distilled at 40° C. the inhibitory
activity remained in the residue and not in the distillate, indicating
that the inhibitory compound was not volatile.
To test the pH sensi-
tivity of the inhibitor, the lyophilized dialysate reconstituted in distilled water was used instead of SM to avoid cloudiness at low pH's.
The pH of the test solutions were readjusted to 7. 0 before dilution
with PM for the inhibition tests.
The inhibition was no longer
observed after exposure to acidf solutions of pH 2. Q or below.
Table 2.
Summary of physico-chemical characteristics of
Achromobacter A43 inhibitor.
1. Dialysable.
2. Passes through molecular weight cut off 1,000 filter.
3. Active fraction in m. w. 800 ~ 1, 000 eluate in Bio-Gel
P-2.
4. Not vacuum distillable at 40° C.
5. Inactivated at pH < 2. 0.
6. Inactivated at 65oC/10 min or above
7. Stable at 37° C or below for 12 days.
8. Ninhydrin reaction of active fraction--deep purple.
9. Rhodamine 6G reaction of active fraction--yellow with
purple hue.
27
The inhibitor was heat labile.
Autoclaving at 121° C for 15 min
or heating at 850C for 10 min destroyed its activity.
Its activity was
also partially reduced upon mild heating at 65° C for 10 min (Figure
4).
Storage at room temperature or at 37° C for 12 days had no effect
on its activity.
Lyophilized inhibitor stored in a refrigerator for 2
months did not lose activity.
No visible precipitate was observed when the reconstituted
lyophilized dialysate was treated with 20% trichloroacetic acid and
centrifuged at 44 G.
The "supernatant" when assayed, did not show
any inhibitory activity.
The inhibitor was either destroyed in the
extraction process, possibly by the acidity, or in the precipitate in
undetectable amount.
The inhibitory activity of the eluates from Bio-Gel P-2 column
was found in fractions 9 to 14 (Table 3).
These fractions all carried a
light yellowish brown color and from the reactions with 0. 5 M AgNO.,
and ninhydrin spray, appeared to contain sulfur containing peptides.
It was estimated from the elution time that the molecular weight of
the compounds in the active fractions were from 800 to 1, 000 Daltons.
The inhibitory activities of various fractions eluted from Bio-Gel P-2
column are presented in Table 4
Fraction by the Bio-Rex 70 X column showed that the inhibitory
activity was associated with a yellowish brown eluate in fractions 17
28
1. 2 "
PM control fT
1. 1 -A43-SM, after
85oC/10 min
1. 0 -
0.9 •-
0.8 ■-
O.D.
0.7 f
A43-SM, after
0.6 ■■
0. 5 ■■
0.4-1-
0.3
0. 2
3
Figure 4.
4
5
Time in hours
Effects of heated Achromobacter A43 spent medium (SM)
on germination and outgrowth of C^. botulinum type E,
Beluga, spores.
29
to 20.
The purple color of ninhydrin reactions indicated that almost
all the fractions appeared to contain some peptides, but the inhibitory
fractions gave the strongest color reactions.
It must be noted how-
ever, that other compounds with free amino groups, such as an amino
acid-containing phosphatide, would also react with ninhydrin to give a
purple color (124).
The presence of peptides in fractions 17 to 40 was
also confirmed spectrophotometrically by absorbance at 280 nm.
Table 3.
Fractions
1-8
9-14
15-18
19-25
26-28
29-35
control
a
Anti-C. botulinum type E activities of fractions eluted from
B io-Gel P-2 column.
Reactions with
AgN03 (0. 5 M)
increasing yellow ppt
purple ppt
purple ppt
purple ppt
white ppt
dark purple ppt
Ninhydrin
Reactions
none
deep purple
brownish pur•pie
light purple
light pink
faint yellow
deep purple
Inhibitory
Activity
+
ppt = preci pitate.
Rhodamine sprayed spots of the eluate on filter paper examined
under UV light suggested that all the fractions contained some lipid
material but fractions 17 to 19 might contain some anionic phosphatides such as phosphatidic serine, or phosphatidic acids as the spots
carried a slightly purple fluorescence (75).
Phosphatidic serine,
having a free amino group, would give a positive ninhydrin reaction.
The inhibitory activities of the fractions eluated from the Bio-Rex
30
column are summarized in Table 4„
Table 4.
Fractions
1-16
17
18
19
20
21-23
24-28
29-40
3.
Anti-C^ botulinum type E activities of fractions eluted from
the Bio-Rex 70 X column.
Ninhydrin
Reaction
light pur•pie
purple
purple
purple
purple
deep purple
purple
light pui•pie
Viewed under UV while wet.
Rhodamine 6G
Reaction3yellow
yellow with purple hue
yellow with purple hue
yellow with purple hue
yellow
yellow
yellow
yellow
Inhibitory
Activity
-
+
+
+
+
±
-
31
DISCUSSION
The information gathered on the inhibitor thus far raises the
possibility that it might be an antibiotic.
Indeed many bacteria,
notably the Bacillus and the Streptomyces species, produce a variety
of antibiotics.
Most of these are small cyclic peptides with molecular
weights below 3, 000 Daltons (17, 18).
They are active toward vege-
tative cells and also inhibit spore outgrowth.
Antibiotics such as
nisin and tylosin have been shown to prevent spore outgrowth of some
cheese spoilage clostridia species but to have no effect on germination (14).
Antifciotics shown to be active against various species of
clostridia include azaserine (26), 6-diazo-5-oxo-L-norleucine (DON)
(37), chlortetracycline (35) and tylosin (76) produced by Streptomyces
species, tyrocidin (58) and subtilin (64) produced by Bacillus species,
and nisin synthesized by S. lactis (122).
Also, nisin, tylosin and
subtilin have been shown to be particularly effective against C. botulinum (2 3, 64, 108).
Antibiotics produced by Achromobacter species,
however, have not been reported.
On the basis of the elution time from the Bio-Gel P-2 column and
the results from the ultra-filtration experiment, the molecular weight
of the A43 inhibitor appears to be between 800-1, 000 Daltons.
How-
ever, it must be noted that unless the compound is pure, the exact
determination of the molecular weight of compounds with molecular
32
weight around 1,000 Daltons are often difficult and inaccurate (11).
Many antibiotics that would inhibit gram positive bacteria including
clostridia had molecular weights of approximately 1,000 Daltons or
below (17), with the exceptions of nisin (7,000 Daltons) (60) and
subtilin (3,200 Daltons) (107).
Tylosin lactate, which prevented the
outgrowth of C^. botulinum type E spores, has a molecular weight of
904 Daltons (56).
Some of the antibiotics cited above, however, were more heat
resistant than the Achromobacter A43 inhibitor.
Nisin was not
affected by a heat treatment of 100oC for 5 min (61).
Azaserine lost
only 40% of its activity after heating for 4 hr at 60° C (43).
Tylosin
lactate, incorporated at 100 ppm in whitefish chub and heated at 5565° C for 30 min had retained 0. 5 to 5% of the activity which remained
stable for at least a month in the refrigerator temperature (108).
A
mild heat treatment of 65° C for 10 min of Achromobacter A43 inhibitor, on the other hand, rendered it ineffective in preventing the outgrowth of C_. botulinum type E spores (Figure 4).
It must be noted,
however, that the inhibitor was tested at l/10th of its initial strength
and the residual activity shown might have been comparable to socalled heat resistant antibiotics.
The acid sensitivity of the inhibitor appeared to be similar to
tylosin which had a pKa of 7. 1 (76).
In contrast, nisin and chlortetra-
cycline were acid stable and were extracted by precipitation at pH
33
1.4 (15,35).
Most of the antibiotics effective against clostridia,
however, are active near the neutral pH with an exception of nisin (23).
C^. botulinum type E spores germinated in the presence of the
inhibitor appeared darkly stained under the microscope with little or
no outgrowth and apparently very little lysis.
This was similar to the
observation by Ramseier (I960) (96) with C^. butyricum, when nisin
was added to the lag phase cells.
However, even antibiotics having
known surfactant properties such as tyrocidin ,
may not cause
immediate lysis in all types of cells (58) and any delayed lysis may be
confused with autolysis.
It is unlikely that the Achromobacter A43 inhibitor is an inorganic compound as shown by its heat lability.
Most of the antibiotics
against clostridia discussed above are peptides.
DON and chlortetra-
cycline however belong to the class of azaamine and tetracycline
respectively (37, 120).
The reactive fractions eluted from the Bio-Rex
70 X column were apparently rich in peptides and/or lipids.
The
Rhodamine reaction in particular was suggestive of the presence of
phosphatidic amino acids (75).
Evidence is still lacking whether such
reactions were indeed by the inhibitor or unrelated compound eluted
at the same rate.
in composition.
Small peptide antibiotics may be very complicated
They may be associated with sugars, amines, acids
(including fatty acids, hydroxy acids and heterocyclic carboxylic
acids), D- and L-amino acids and other related compounds (18).
34
The role of antibiotics in the organisms producing them is still
another mystery.
Some suggested that antibiotics were merely car-
riers for disposing undesirable metabolic by-products such as D-amino
acids or that they were products of metabolic pathways that were
abandoned during evolution.
The inhibitors such as those produced by
Achromobacter A43 could have been one of the factors in limiting the
growth of (3. botulinum in fresh uncooked fish.
Pseudomonas species and Achromobacter species were the
dominant microbial flora in fresh fish and the former gradually
displaced the latter microorganisms and became the major spoilage
organism (109).
The role played by the Achromobacter species is not
known and has been considered negligible.
In light of the present
study, some Achromobacter species that are always present in raw
foods and may become a dominant flora in irradiated seafood (27,70)
may contribute in preventing the growth of C^. botulinum.
This study
has shown that a high concentration of the inhibitor can be produced
in laboratory media in less than 40 hr.
Moreover, this inhibitor is
active within the pH range that C. botulinum would germinate and
grow.
It remains to be seen if this inhibitor can be demonstrated in
fish flesh or how significant a role it plays in foods.
The smoking process, somehow eliminates Achromobacter
species (69).
Hence any post-smoking contaminating spores or spores
35
that have survived the smoking process can germinate, grow and
produce toxin in foods where inhibitors of this type has been eliminated.
36
BIBLIOGRAPHY
1.
Ando, Y. , (1971). The germination requirements of spores of
Clostridium botulinum type E. Jap. J. Microbiol. 15(6):515.
Z. Ando, Y. and lida, H. , (1970). Factors affecting the germination
of spores of Clostridium botulinum type E. Jap. J. Microbiol.
14:361.
3.
Ando, Y. and Katsuhiro, I. , (1957). Studies on growth and toxin
production of Clostridium botulinum type E in fish products. I.
On the growth in relation to the oxidation-reduction potential in
fish flesh. Bull. Jap. Soc. Scientific Fisheries Z3:458.
4.
Angelotti, R. , (1970). The heat resistance of Clostridium
botulinum type E in food, p. 404-409. In M. Herzberg (ed. ),
Proc. 1st U.S. -Japan Conf. on Toxic Microorganisms. U.S.
Dept. of the Interior.
5.
Anonymous 1, (1963). Epidemic botulism related to smoked fish
ingestion. Morbidity and Mortality Weekly Reports 12,40:3Z9.
U.S. Dept. ofH.E.W., Public Health Service, Atlanta, Georgia.
6.
Anonymous 2, (1967). Type E botulism- -Chicago, Illinois,
Morbidity and Mortality Weekly Reports 16, Z4:193. U.S. Dept.
of H. E. W. , Public Health Service, Atlanta, Georgia.
7.
Baird-Parker, A. C. , (1969). Medical and veterinary significance
of sporeforming bacteria and their spores, p. 517-548. In
G. W. Gould and A. Hurst (ed. ), The Bacterial Spore. Academic
Press Inc. , New York.
8.
Baird-Parker, A. C. , (1971). Factors affecting the production of
bacterial food poisoning toxins. J. Appl. Bact. 34(1):181.
9.
Baird-Parker, A.C. andFreame, B. , (1967). Combined effect
of water activity, pH and temperature on the growth of Clostridium botulinum from spore and vegetative cell inocula. J. Appl.
Bact. 30:420.
10.
Barnes, E. M. and Melton, W. , (1971). Extracellular enzymatic
activity of poultry spoilage bacteria. J. Appl. Bact. 34:599.
37
11. Battersby, A.R. and Craig, L.C. , (1967). The molecular weight
determination of polypeptides. J. Amer. Chem. Soc. 73:1887.
12. Baumann, P., Doudoroff, M. andStanier, R.Y., (1968). A
study of the Moraxella group. II. Oxidase-negative species
(genus Acinctobacter). J. Bact. 95:1520.
13. Benjamin, M. J. W. , Wheather, D. M. and Shepherd, P. A.,
(1956). Inhibition and stimulation of growth and gas production by
clostridia. J. Appl. Bact. 19:159.
14. Bergere, J.L. andHermier, J. , (1970). Spore properties of
clostridia occurring in cheese. J. Appl. Bact. 33:167.
15. Berridge, J. , (1949).
Biochem. J. 45:486.
Preparation of the antibiotic nisin.
16. Block, R.J., Durrum, E. L. andZweig, G., (1955). p. 75-83.
A manual of paper chromatography and paper electrophoresis.
New York. Academic Press.
17. Bodanszky, M. and Perlman, D. , (1964). Are peptide antibiotics
small proteins? Nature, 204 No. 4961:840.
18. Bodanszky, M. and Perlman, D. , (1969).
Science 163:352.
Peptide antibiotics.
19. Bonventre, P.F. and Kempe, L.L. , (I960). Physiology of toxin
production by Clostridium botulinum type A and B. I. Growth,
autolysis and toxin production. J. Bact. 78:18.
20. Bott, T.L. , Deffner, J.S., McCoy, E. and Foster, E.M. ,
(1966). Clostridium botulinum type E in fish fr.om the Great
Lakes. J. Bact. 91:919.
21. Bronner, F. and Freund, T.S., (1971). Calcium accumulation
during sporulation of Bacillus megaterium, p. 189-190. In H.O.
Halv.orson, R. Hansen and L. L. Campbell, (ed. ), Spores V.
Amer. Soc. Microbiol. Wash., D. C.
22. Bruch, M.K., Bohrer, C. W. and Denny, C.B., (1969). Adaptation of biphasic culture technique to the sporulation of Clostridium
botulinum type E. J. Food Science, 33:108.
38
23. Campbell, L. L. and Winiarski, W. , (1959). Isolation and
properties of a subtilin resistant strains of Clostridium botulinum.
Appl. Microbiol. 7:285.
24. Christiansen, L. N. , Deffner J. , Foster, E.M. andSuziyama, H.,
(1968). Survival and outgrowth of Clostridium botulinum type E
in certain vacuum packed fish. J. Appl. Bact. 28:431.
25. Church, B.D. and Halvorson, H.O., (1957). Intermediate
metabolism of aerobic spores. I. Activation of glucose oxidation
in spores of Bacillus cereus var. terminalis. J. Bact. 73:470.
26.
Coffey, G.L,., Hillagas, A. B., Knudsen, M. P., Koepsell, H. J. ,
Oyaas, J.E. and Ehrlich, J. , (1954). Azaserine: Microbiological studies. Antibiotics and Chemotherapy 4:775.
27. Corlett, D.A. , Jr., Lee, J.S. and Sinnhuber, R.O., (1965).
Application of replica plating and computer analysis for rapid
identification of bacteria in some foods. II. Analysis of microbial flora in irradiated Dover sole (Microstomus pacificus).
Appl. Microbiol. 13:818.
28. Cristley, F.D. andHelz, G.E., (1961). Some observations on
the effect of filtrates of several representative concomitant
bacteria on Clostridium botulinum type A. Can. J. Microbiol.
7:633.
29.
Curran, H. R. and Evans, F.R. , (1945). Heat activation inducing
germination in spores of thermotolerant and thermophilic aerobic
bacteria. J. Bact. 49:335.
30. Curran, H. R. andEvans, F.R., (1954). The influence of iron or
manganese upon the formation of spores by mesophilic aerobes in
fluid organic media. J. Bact. 67:488.
31. Day, L. E. and Costilow, R.N. , (1964a). Physiology of the
sporulation process in Clostridium botulinum. I. Correlation of
morphological changes with catabolic activities, synthesis of
dipicolinic acid, and development of heat resistance. J. Bact.
88:690.
32. Day, L. E. and Costilow, R.N. , (1964b). Phys iology of the
sporulation process in Clostridium botulinum. II. Maturation of
forespores. J. Bact. 88:695.
39
33. Dawes, I.W., Kay, D. and Mandelstam, J. , (1969). Sporulation
in Bacillus subtilis. Establishment of a time scale for the
morphological events. J. Gen. Microbiol. 56:171.
34. Dion, H.W., Fusari, S.A. , Jakaibowski, Z.L., Zora, J.G. and
Bartz, Q.R., (1956). 6-Diazo-5-oxo-norleucine, a new tumorinhibitory substance. II. Isolation and characterization. J.
Amer. Chem. Soc. 78:3075.
35. Duggar, B.M. , (1948). Aureomycin: a product of the continuing
search for new antibiotics. N. Y. Acad. Sci. 51(2):177.
36. Duncan, C.L. , (1970). Arrest of growth from spores in semipreserved foods. J. Appl. Bact. 33:60.
37. Ehrich, J. , Coffey, G.L. , Fisher, M. W. , Hillegas, A. B. ,
Kohberger, D. L. , Machamer, H.E., Rightsel, W.A. and
Roegner, F. K. , (1956). 6-Diazo-5-oxo-L-norleucine, anew
tumor inhibitory substance. Antibiotics and Chemotherapy
6:487.
38. Eisenstadt, E. and Silver, S. , (1972). Calcium transport during
sporulation in Bacillus subtilis, p. 180-186. In H. D. Halvorson,
R. Hansen, and L. L. Campbell, (ed. ), Spores V, Amer. Soc.
Microbiol. Wash., D. C.
39. Ellison, J. S. , Mattern, C.F.T. and Daniel, W.A. , (1971).
Structural changes in Clostridium botulinum type E after treatment with Bioticin S5
J. Bact. 108:526.
40. Emeruwa, A. C. and Hawirko, R. Z. , (1973). Butyric type of
fermentation during sporulation of Clostridium botulinum. p. 40,
Art. G86, Abstracts of Annual Meeting, A.S.M. , Miami, 1973.
41. Emodi, A.S. and Lechowich, R.V. , (1969). Low temperature
growth of type E Clostridium botulinum spores. I. Effects of
sodium chloride, sodium nitrite and pH. J. Fd. Sci. 34:78.
42. Foster J. W. and Wynne, E.S., (1948). Physiological studies on
spore germination with special references to Clostridium
botulinum. IV. Inhibition of germination by unsaturated C.fatty acids. J. Bact. 55:495.
40
43. Fusari, S.A. , Frohardt, R. P. , Ryder, A., Haskell, T.H. ,
Johannesen, D.W., Elder, C. C. andBartz, Q. K. , (1954).
Azaserine, a new tumor-inhibiting substance. Isolation and
characterization. J. Amer. Chem. Soc. 76:2878.
44. G.M.P. (Good Manufacturing Practice), Federal Register, 1970.
Part 128a. Fish and seafood products. Subpart A. Smoked and
smoke-flavored fish 35:17401-17402.
45. Gould, G.W. , (1970).
J. Appl. Bact. 33:34.
Germination and the problem of dormancy.
46. Gould, G.W. and Dring, G.J., (1972). Biochemical mechanism
of spore germination, p. 401-408. InH.O. Halvorson, R. Hansen
andL.L. Campbell, (ed. ), Spores V. Amer. Soc. Microbiol.
Wash. , D. C.
47. Gould, G.W. andHitchins, A.D., (1963). Sensitization of
bacterial spores to lyzozyme and hydrogen peroxide with reagents
which rupture disulfide bonds. J. Gen. Micro. 33:413.
48. Gordon, R.A. and Murrell, W.G., (1967). Botulism.
sent situation reviewed. Fd. Mgf. 42(8):34.
The pre-
49. Grecz, N. , (1964). Natural antibiotics in some varieties of surface ripened cheese. In N. Molin, (ed. ), Microbial Inhibitors in
Food, Proc, 4th Int. Sym. Fd. Microbiol. , Almquist and
Wiksell, Stockholm.
50. Grecz, N. , Wagenaar, R.O. and Dack, G.M. , (1959).
Relation of fatty acids to the inhibition of Clostridium botulinum
in aged surface ripened cheese. Appl. Microbiol 7:228.
51. Grelet, N. , (1957).
Bact. 20:315.
Growth limitation and sporulation.
J. Appl.
52. Gullmar, B. and Molin, N. , (1967). p. 185. In M. Ingram and
T.A. Roberts, (ed. ), Botulism 66, Proc. 5th Int. Symp. Fd.
Microbiol. , Moscow, July 66. Chapman and Hall, London.
53. Hall, I.C. and Peterson, E. , (1923). The effect of certain
bacteria upon the toxin production of Bacillus botulinus in vitro.
J. Bact. 8:319.
41
54. Halvorson, H.O., (1957).
J. Appl. Bact. 20(3):305.
Rapid and simultaneous sporulation.
55. Halvorson, H.O. , (1962). Physiology of sporulation. p. 223264. In.I.C. Gunsalus andR.Y. Stanier, (ed. ), The Bacteria
IV. Academic Pres s.
56. Hamill, R. L. , Haney, M.E., Jr., Stampes, M. and Wiley, P. F. ,
(1961). Tylosin, a new antibiotic: II. Isolation, properties, and
preparation of desmycosin, a microbiologically active degradation product. Antibiotics and Chemotherapy 11:328.
57. Hitzman, D.O., Halvorson, H.O. and Ukita, T. , (1957).
Requirements for production and germination of spores of
anaerobic bacteria.. J. Bact. 74:1.
58. Hotchkiss, R. D. , (1944). Gramicidin, tyrocidin and tyrothricin.
Adv. in Enzymology 4:153.
59. Ingram, M. and Dainty, R.H. , (1971). Changes caused by
microbes in spoilage of meat. J. Appl. Bact. 34(1):21.
60. Ingram, L. , Tombs, M. P. and Hurst, A., (1967). Mobility,
molecular weight relationships of small proteins and peptides in
acrylamide gel electrophoresis. Analyt. Biochem. 20:24.
61. Jarvis, B. , (1967). Resistance to nisin and production of nisin inactivating enzymes by several Bacillus species. J. Gen.
Microbiol. 47:33.
62. Johannsen, A., (1965). Clostridium botulinum type E in foods
and the environment generally. J. Appl. Bact. 28:90.
63. Johnston, P. V. , (1971). Basic lipid methodology, p. 46-47,
Spec. Pub. 19. U. of Illinois at Urbana. Champagne.
64. Kaufmann, O. W. , Ordal, Z.J. andElbisi, H.M. , (1954). The
effect of several antibiotics on certain sporeforming organisms
involved in food spoilage. Food. Res. 19:483.
65. Kautter, D.A., Harmon, S.M. , Lynt, R.K. , Jr., and Lilly, T. ,
Jr. , (1966). Antagonistic effect on Clostridium botulinum type E
by organisms resembling it. Appl. Microbiol. 14:616.
42
66. Kautter, D.A., Lynt, R.K. , Jr., Solomon, H. M. , Lilly, T. ,
Jr., and Harmon, S.M., (1970). The detection, identification,
and isolation of Clostridium botulinum, p. 236-246. In M.
Herzberg, (ed. ), 1st U.S. -Japan Conf. on Toxic Microorganisms,
U.S. Dept. of the Interior.
67. Keyman, A., Evenchik, Z., Halvorson, H.O. and Hastings,
J. W. , (1964). Activation of bacterial endospores. J. Bact
88:313.
68. Lee, J.S. and Harrison, J. M. , (1968). Microbial flora of
Pacific Lake (Merluccius productus). Appl. Microbiol. 16:1937.
69. Lee, J.S. andPfeifer, D. K. , (1973). Aerobic microbial flora of
smoked salmon. J. of Milk and Food Tech. 36:143.
70. Lee, J.S., Willett, L. , Robison, S.M. and Sinnhuber, R.O.,
(1967). Comparative effects of chlortetracycline, freezing, and
■y radiation on microbial population of ocean perch. Appl.
Microbiol. 15:368.
71. Lee, W.H. and Ordal, Z.J., (1963). Reversible activation for
germination and subsequent changes in bacterial spores. J. Bact.
85:207.
72. Leifson, E. , (1931).
Bacterial spores.
J. Bact. 21:331.
73. Levinson, H.S. and Hyatt, M. T. , (1969). Activities of Bacillus
megaterium spore germination, p. 262-272. In L.L. Campbell,
(ed.), Spores IV. A. S. M. , Bethesda, Md.
74. Lewis, K. H. and Hall, H.E., (1970). Botulism: Potential
hazards of food preservation, p. 384-387. In M. Herzberg,
(ed. ), Proc. of the 1 st U. S. -Japan Conf. on Toxic Microorganisms. U.S. Dept. of the Interior.
75. Marinetti, G.V. , Erbland, J. and Kochem, J. , (1957). Quantitative chromatography of phosphatides. Fed. Proc. 16:837.
76. McGuire, J. M. , Boniece, W.S., Higgins, C.E., Hoehn, M. M. ,
Stark, W.M. , Westhead, J. and Wolfe, R.N. , (1961). Tylosin,
a new antibiotic: I. Microbiological studies. Antibiotics and
Chemotherapy. 11:320.
43
77. Meyer, K. F. and Gunnison, J.B., (1929). Botulism due to home
canned Bartlett pears. J. Infect. Dis. 45:135.
78. Mol, J. H. H. , (1957). The temperature characteristics of spore
germination and growth of Bacillus cereus. J. Appl. Bact.
20(3):454.
79. Munsey, D.T., Boucher, B. and Silverman, G. J. , (1973).
Microbiol alteration of redox-potential in food and model systems, p. 19, Article El 10, Abstracts of the Annual Meeting.
Amer. Soc. Microbiol. , Miami, Florida. 1973.
80. Murrell, W.G. , (1967). The biochemistry of the bacterial endospore. Adv. Microbiol. Physiol. 1:133.
81. Murrell, W.G., (1969). Chemical composition, p. 215-273.
In G. W. Gould and A. Hurst, (ed.), The Bacterial Spore.
Academic Press Inc. , New York.
82.
Murrell, W.G. and Scott, J. , (1966). The heat resistance of
bacterial spores at various water activities. J. Gen. Microbiol.
43:411.
83.
Murrell, W.G. and Warth, A. D. , (1965). Composition and heat
resistant of bacterial spores, p. 1-24, In L. L. Campbell and
H.O. Halvorson, (ed.), Spores III. Amer. Soc. Microbiol.,
Ann Arbor, Mich.
84. Ohye, D. F. , Christian, J.H.B. and Scott, W. J. , (1966). Influence of temperature on the water relations of growth of Clostridium botulinum type E, p. 136-143. In M. Ingram and T.A.
Roberts, Botulism 66, Proc. 5th Int. Symp. Fd. Microbiol. ,
Moscow, July 66. Chapman and Hall, London.
85. Ohye, D.F. and Scott, W.J. , (1957). Studies in the physiology
of Clostridium botulinum type E. Australian!. Biol. Sci. 10:85.
86. Pace, P.J., Krumbiegel, E.R., (1973). Clostridium botulinum
and smoked fish production: 1963-1972. J. Milk and Food Tech.
36:42.
44
87. Pace, P. J. , Krumbiegel, E.R., Angelotti, R. and Wisniewski,
H. J. , (1967a). Demonstration and isolation of Clostridium
botulinum types from whitefish chubs collected at fish smoking
plants of the Milwaukee area. Appl. Microbiol. 15:877.
88. Pace, P.J., Krumbiegel, E.R. and Wisniewski, H. J. , (1972).
The interrelationship of heat and relative humidity in the destruction of Clostridium botulinum type E in whitefish chubs. Appl.
Microbiol. 23:750.
89. Pace, P.J. , Krumbiegel, E.R., Wisniewski, H. J. and Angelotti,
R. , (1967b). The distribution of Clostridium botulinum types fish
processed by smoking plants of the Milwaukee area, p. 40-48. In
M. Ingram and T. A. Roberts, (ed. ), Botulism 66, Proc. 5th
Intern. Symp. Food Microbiol. , Moscow, Chapman and Hall,
London.
90. Pace, P.J., Wisniewski, H. J. and Angelotti, R. , (1968).
Sensitivity of an enrichment culture procedure for detection of
Clostridium botulinum type E in raw and smoked whitefish chubs.
Appl. Microbiol. 16:673.
91. Perigo, J.A., Whiting, E. and Bashford, T.E., (1967). Observations on the inhibition of vegetative cells of Clostridium
sporagenes by nitrite which has been autoclaved in a laboratory
medium, discussed in the context of sub-lethally processed cured
meats. J. Fd. Tech. 2:377.
92. Pivnick, H. , Barnett, H. W. , Nordin, H. R. and Rubin, L.J.,
(1969). Factors affecting the safety of canned cured, shelf-stable
luncheon meat inoculated with Clostridium botulinum. Can. Inst.
Fd. Tech. 2:141.
93. Powell, J.F. , (1950). Factors affecting the germination of thick
suspensions of Bacillus subtilis spores in L-alanine solution.
J. Gen. Microbiol. 4:330.
94. Powell, H. M. and Strange, R.E., (1953). Biochemical changes
occurring during the germination of bacterial spores. Biochem.
J. 54:205.
95. Quortrup, E.R. and Sudheimer, R.L., (1943). Some ecological
relations of Pseudomonas aeruginosa to Clostridium botulinum
type C. J. Bact. 45:551.
45
96.
Ramseier, H.R., (I960). Die Wirkung von nisin auf Clostridium butyrieum praazm. Arkiv. Mikrobiol. 37:57.
97.
Rieman, H. , (1967). The effects of numbers of spores of
Clostridium botulinum type E on growth and toxin production in
inhibitory environments, p. 148-157. In M. Ingram and T. A.
Roberts, (ed.), Botulism 66, Proc. 5th Intern. Symp. Food
Microbiol. , Moscow, Chapman and Hall, London.
98.
Rode, L.J. and Foster, J. W. , (I960). Mechanical germination
of bacterial spores. Proc. Nat. Acad. Sci. , U.S.A., 46:118.
99. Royaud, M. , Turpin, A. , Mangalo, R. and Bizini, R. , (1955).
Croissance et toxigenese. Ann. Inst. Pasteur. 88:24.
100.
Sakaguchi, G. and Tohyama, Y. , (1955). Studies on the toxin
production of Clostridium botulinum type E. Jap. J. Med. Sci.
Biol. 8:247.
101.
Saleh, M.A. and Ordal, Z.J., (1955). Studies on the growth
and toxin production of Clostridium botulinum in a pre-cooked
frozen food. Fd. Res. 20:340.
102.
Schmidt, C.F., Lechowich, R.V. and Folinazzo, J. F. , (1961).
Growth and toxin production by type E Clostridium botulinum
below 40oF. J. Fd. Sci. 26:626.
103.
Schmidt, C. F. , Hank, W.K. and Lechowich, R.V. , (1962).
Radiation sterilization of food. II. Some aspects of the growth,
sporulation and radiation resistance of spores of Clostridium
botulinum type E. J. Fd. Sci. 27:77.
104.
Segner, W.P., Schmidt, C.F. andBotz,
(1966). Effect of
sodium chloride, potassium nitrate and sodium nitrite on the
recovery of heated bacterial spores. J. Fd. Tech. 1:147.
105.
Setlow, P. and Kornberg, A. , (1970). Biochemical studies of
bacterial sporulation and germination. XXIII. Nucleotide
metabolism during spore germination. J. Biol. Chem. 245:3645.
106.
Shaeffer, P. , (1969). Sporulation and the production of antibiotics exoenzymes and exotoxins. Bact. Rev. 33:48.
46
107.
Sheehan, J. C. , Drummond, P.E., Gardner, J. N , Maeda, K. ,
Mania, D. , Nakamura, S. , Sen, A.K. and Stock, J. A. , (1963).
The structure of teomycin. J. Amer. Chem. Soc. 85:2867.
108.
Sheneman, J.M. , (1965). Prevention of type E Clostridium
botulinum toxin formation in smoked whitefish chubs with
tylosin lactate. J. Fd. Sci. 30(2):337.
109.
Shewan, J. M. , (1971). The microbiology of fish and fishery
products--a progress report. J. Appl. Bact. 34(2):299.
110.
Silliker, J, H. , Greenberg, R.A. and Schack, W. R. , (1958).
Effect of individual curing ingredients on the shelf stability of
canned comminuted meats, Fd. Tech. 12:551.
111.
Slepecky, R.A., (1972). Ecology of bacterial sporeformers,
p. 297-313. InH.O. Halvorson, R. Hanson and L. L. Campbell,
(ed. ), Spores V. Amer. Soc. Microbiol. Wash., D. C.
112.
Spencer, R. , (1967). Factors in cured meat and fish products
affecting spore germination, growth and toxin production,
p. 123-135. In R. Ingram and T. A. Roberts, (ed. ), Botulism
66, Proc. 5th Int. Symp. Fd. Microbiol. , Moscow, July 66.
Chapman and Hall, London.
113.
Spencer, R. , (1971). Nitrite in curing--microbiological implications, presented at the 17th European Meeting of Meat
Research Workers.
114.
Stevens, CO. and Bergstrom, G.R., (1967). The multiple
nature of crystalling egg-white lysozyme. Proc. Soc. Exptl.
Biol. Med. 124:187.
115.
Strasdine, G.A., (1967).
botulinum type E spores.
116.
Strasdine, G.A. and Melville, J. , (1968). Growth and spore
production of Clostridium botulinum type E in chemically
defined media. J. Fish. Res. Bd. Can. 25(3):547.
117.
Thornley, M.J., (1967). A taxonomic study of Acinetobacter
and related genera. J. Gen. Microbiol. 49:211.
Rapid germination of Clostridium
J. Fish. Res. Bd. Can. 24(3):595.
47
118.
Torraini, A., Garrick, L. and Silber stein, Z. , (1969). Onset
of macromolecular synthesis at germination of bacterial spores,
p. 247-261. In L. L. Campbell, (ed. ), Spores IV. Amer. Soc.
Microbiol. Bethesda, Md.
119.
Treadwell, P.E., Jann, G.J. and Salle, A. J. , (1958). Studies
on factors affecting the rapid germination of Clostridium
botulinum. J. Bact. 76:549.
120.
Umezawa, H. , (ed. ), (1967). Index of antibiotics from Actinomycetes. p. 212, University Park Press, State College,
Pennsylvania.
121.
Ward.B.Q. and Carroll, B.J., (1966). Spore germination and
vegetative growth of Clostridium botulinum type E in synthetic
media. Can. J. Microbiol. 12:1145.
122.
Whitehead, H.R., (1933). A substance inhibiting bacterial
growth, produced by certain strains of lactic streptococci.
Biochem. J. 27:1793.
123.
Williams, O.B. and Blair, E., (1950). Spore formation in
synthetic media by Clostridium botulinum. p. 62. Bact. Proc.
1950.
124.
Williams, R. J. and Kirby, H. , (1948). Paper chromatography
using capillary ascent. Science 107:481.
125.
Woese, C.R. , (1959). Effect of withholding glutamic acid and
asparagine on the germination of spores of Bacillus subtilis.
J. Bact. 77:690.
126.
Wolf, J. and Mahmond, S.A.Z., (1957). The effect of L- and
D-alanine on the germination of some Bacillus spores. J. Appl.
Bact. 20(3):373.
127.
Wooley, B.C., and Collier, R.E., (1968). A synthetic medium
for germination, postgerminative development, and growth of
spores of Clostridium roseum. Can. J. Microbiol. 14:173.
128.
Wyne, E. S. and Foster, J. W. , (1948). Physiological studies on
spore germination with special reference to Clostricium botulinum. III. Carbon dioxide and germination, with a note on
carbon dioxide and aerobic spores. J. Bact. 55:331.
48
129.
Wynn, E.S., Mehl, D. A. and Schmieding, W.R., (1954).
Germination of Clostridium spores in buffered glucose. J.
Bact. 67:435.