1. No category

An Evaluation of Factors Affecting the Survival of Escherichia coli in
Sea Water'
III.
Antibiotics
A. F. CARLUCCI2
AND
DAVID PRAMER
Department of Agricultuiral Microbiology, Rutgers, The State University, New Brunswick, New Jersey
Received for publication December 7, 1959
biotics on the survival of cells of E. coli in sea water
were described previously (Carlucci and Pramer, 1960).
The following methods were employed to measure the
biological effects of antibiotics in sea water.
Growth of E. coli in sea water. A quantity of sea water
was supplemented with 0.5 per cent Bacto-peptone9
and freed of microorganisms by filtration (Millipore).
To each of a series of sterilized Bellco nephlo-flasks,'0
water was added aseptically and solutions of the antibiotics that were filter sterilized were added at appropriate levels to the flasks. All flasks were inoculated
equally with a suspension of cells of E. coli and incubated at 28 C on a rotary shaker. Growth was measured
turbidimetrically using a Klett-Summerson colorimeter" with filter no. 6.
Growth of the native micropopulation of sea water.
To each of a series of sterilized Bellco nephlo-flasks,
50 ml of sea water were added and each flask was
supplemented with 0.5 per cent Bacto-peptone that
was sterilized separately. The antibiotics were added,
and the flasks incubated and tested as described in the
preceding experiment with E. coli.
Growth of the bacterial population of sea water on agar
medium. Sea water was plated on the surface of sea
water nutrient agar prepared to contain the antibiotics
at various concentrations. Colony counts were made
after 7 days of incubation at 20 C.
Effect on ammonification. The procedure used was
identical to that employed to determine the effect of
antibiotics on growth of the- native micropopulation of
sea water. The production of ammonium from peptone
was determined using Nessler's reagent and a KlettSummerson colorimeter with filter no. 42.
Effect on nitrification. A nitrifying enrichment culture was obtained by adding 0.1 per cent (NH4)2SO4
to samples of sea water and incubating at 28 C on a
rotary shaker. When nitrite was produced, transfers
were made to fresh water samples containing (NH4)2SO4. By repetition of this procedure an enrichment
culture capable of oxidizing ammonium to nitrite but
not nitrite to nitrate was obtained. Samples of sea
It has been repeatedly suggested that sea water
contains heat labile toxic substances (ZoBell, 1936;
Krassilnikov, 1938; Ketchum et al., 1949, 1952; Nusbaum and Garver, 1955; Orlob, 1956). Although the
source and identity of these substances remain obscure, they are believed to be of microbial origin and
have been described as antibiotics (Rosenfeld and ZoBell, 1947; Vaccaro et al., 1950; Richou et al., 1955;
Greenberg, 1956). Therefore, the production, stability,
and some biological effects of antibiotics in sea water
were investigated.
MATERIALS AND METHODS
The antibiotics employed were chloramphenicol,3
chlortetracycline hydrochloride,4 neomycin sulfate,5
oxytetracycline hydrochloride,6 potassium penicillin
G,7 polymyxin sulfate,8 and streptomycin sulfate.7
The replica plate method (Lechevalier and Corke,
1953) was used to provide information regarding the
occurrence of antibiotic-producing bacteria in sea
water. The surfaces of plates containing nutrient agar
prepared with sea water were inoculated with 0.1 ml
of a freshly collected water sample, and the plates were
incubated 7 days at 28 C before replication. Plates
which contained approximately 50 discreet colonies
were replicated, incubated 5 days, and then flooded
with a suspension of Escherichia coli or Bacillus subtilis.
After storage at 28 C for 24 hr, the plates were examined for evidence of antibiotic production.
Procedures used to determine the influence of anti' Paper of the journal series, New Jersey Agricultural Experiment Station, Rutgers, The State University, Department
of Agricultural Microbiology, New Brunswick, New Jersey.
This investigation was supported in part by research grant
E1437 from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, U. S. Public Health
Service.
2 Present address: Research Department, United Fruit
Company, La Lima, Honduras, C. A.
3Parke, I)avis and Company, Detroit, Michigan.
4American Cyanamid Company, Pearl River, New York.
5 The Upjohn Company, Kalamazoo, Michigan.
6 Chas. Pfizer and Company, Brooklyn, New York.
7E. R. Squibb and Sons, New Brunswick, New Jersey.
8 American Cyanamid Company, Stamford, Connecticut.
I Difco Laboratories, Inc., Detroit, Michigan.
1
11
Bellco Glass Company, Vineland, New Jersey.
Klett Manufacturing Company, New York City, New
York.
251
A. F. CARLUCCI AND D. PRAMER
252
water containing (NH4)2S04 and different levels of the
inoculated equally with the nitrifying
antibiotics
amount of nitrite produced after 6
and
the
culture
determined
was
quantitatively, using Griess'
days
and
a
Coleman
Junior spectrophotometerl2
reagents
at 540 m,u.
Effect on oxidation of thiosulfate. A culture of Thiobacillus thioparus was added to sea water supplemented
with 1.0 per cent Na2S203 -5H20. When pH indicated
that the culture had developed, transfers were made to
flasks containing fresh sea water supplemented with
Na2S203 and the antibiotics. The reaction of the solution was pH 8.0 and the extent to which thiosulfate
oxidation by T. thioparus was influenced by antibiotics was determined by measuring pH electrometrically after 6 days of incubation at 28 C on a rotary
shaker.
RESULTS AND DIscUSSION
Production of antibiotics by marine bacteria. A total
of 200 bacterial colonies that developed on sea water
nutrient agar was examined by the replica plate procedure but in no case was evidence obtained for production of an antibiotic active against either E. coli or
B. subtilis. Although these results indicate that antibiotic-producing marine bacteria did not occur commonly, their presence in sea water has been demonstrated by other investigators. Rosenfeld and ZoBell
(1947) tested 58 species of marine microorganisms for
antibiotic production and found nine that were antagonistic to various nonmarine forms when cultiwere
12
[VOL. 8
vated on agar media. However, little or no antibiotic
was produced by these same organisms when grown
in nutrient broth prepared with sea water. Antibioticproducing actinomycetes were isolated from marine
sAdiments but it was doubtful that the organisms were
indigenous to the sea (Grein and Meyers, 1958).
Stability of antibiotics in sea water. The persistence
of six antibiotics in untreated and filter sterilized (Millipore) sea water was determined. The antibiotics
used were chloramphenicol, chlortetracycline, neomycin, oxytetracycline, penicillin, and streptomycin.
Each compound was added to provide a concentration
of 200 ppm in separate 500-ml quantities of untreated
and sterilized sea water in 2-L Erlenmeyer flasks, and
the flasks were stored at 28 C for 30 days. Samples
were withdrawn periodically and their antibiotic content determined by a streak-dilution assay (Waksman
and Reilly, 1945) using E. coli and B. subtilis as test
organisms. No loss of chloramphenicol, oxytetracycline,
TABLE 1
Influence of antibiotics on the survival of cells of
Escherichia coli in sea water
Survival after 48 Hr
Antibiotic*
None.16.2~~~~~~~~~~~~~N
16 .2
N one
Chloramphenicol ............ .........
Neomycin ...............................
Oxytetracycline .........................
Polymyxin ..............................
Streptomycin ...........................
Coleman Instruments, Inc., Maywood, Illinois.
*
<0.01
16.5
13.1
<0.01
<0.01
Added at a concentration of 200 ppm to sea water.
TABLE 2
Some biological effects of antibiotics in sea water
Concentra-
Antibiotic
tion (ppm)
None
Chlortetracycline
Chloramphenicol
Neomycin
Oxytetracycline
Penicillin
Streptomycin
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
1
10
100
Growth in Sea Water Plus 0.5
Per Cent Peptone
Colony Count on Ammonification
Total population ea Water Agar (% of Control)
coli
S
E. coli
Total population (% of Control)
(% of control)
(% of control)
100
104.8
83.5
26.1
87.8
1.1
0.5
96.3
72.3
0
97.9
92.6
20.7
42.5
4.8
0
96.3
83.5
1.6
100
83.7
73.1
11.9
98.0
87.8
8.8
89.1
93.9
85.0
93.9
87.8
81.0
111.6
80.0
62.9
89.1
93.2
102.7
100
32.3
1.6
0
16.4
8.2
1.4
109.6
12.3
16.4
23.3
4.1
1.4
35.6
12.3
1.4
39.7
20.6
5.5
100
84.0
73.1
23.1
90.9
77.3
15.9
67.1
62.5
61.4
59.1
78.4
67.1
80.7
64.8
42.1
72.7
75.0
51.1
Nitrification
S203- Oxidation
(% of Control)
(pH)
100
91.3
82.6
4.4
95.4
87.9
12.6
69.6
0.5
4.8
95.7
91.3
5.6
21.7
16.2
16.2
13.0
4.8
5.3
5.0
4.8
5.3
8.0
5.9
8.2
8.0
5.0
7.9
8.1
5.2
4.8
8.0
5.5
8.1
8.1
7.5
8.1
8.1
19(;0]
SURVIVAL OF E. COLI IN SEA WATER. III
ineomycin, or streptomycin activity was observed,
although all but neomycin have been demonstrated to
be unstable and susceptible to microbiological degradation in soil (Pramer, 1958). Chlortetracycline and
penicillin were rapidly inactivated in sea water. The
former was less stable (90 per cent loss in 3.4 days)
than the latter (90 per cent loss in 9 days). No significant differences were noted between the persistence of
the antibiotics in untreated and sterilized water, indicating that the inactivation of chlortetracycline and
penicillin resulted primarily from chemical rather than
biological factors.
Biological effects of antibiotics in sea water. The influence of 200 ppm of each of five antibiotics on the
survival of cells of E. coli in sea water is shown in table
1. Neomycin and oxytetracycline had little or no effect
but viability of the test organism was decreased
greatly by chloramphenicol, polymyxin, and streptomycin. When these same antibiotics were added to sea
water at a level of 50 ppm, no significant effects on
survival of E. coli were observed.
The results of studies of the biological effects of 1,
10, and 100 ppm of chloramphenicol, chlortetracycline,
neomycin, oxytetracycline, penicillin, and streptomycin on microorganisms in sea water are summarized in
table 2. In general, the antibiotics reduced the number
of colonies that developed on sea water agar to a greater
extent than growth of E. coli or the native micropopulation in sea water broth. Escherichia coli was more
sensitive to increased levels of chloramphenicol,
neomycin, oxytetracycline, and penicillin, than was
the indigenous marine microflora. Chloramphenicol
and penicillin had the greatest effect on the development of E. coli in sea water and growing cells were
more sensitive to antibiotics than resting cells (table
1). Although the antibiotics influenced ammonification, the effects were not great. It appeared that relatively high concentrations of antibiotics were required
to cause marked inhibition of growth and ammonificationi by microorganisms in the sea. Nitrification and
thiosulfate oxidation were more sensitive to antibiotics than was ammonification. The nitrifying bacteria and sulfur-oxidizing bacteria tolerated relatively
high concentrations of chlortetracycline and oxytetracycline. Their sensitivity to streptomycin was reported previously (Starkey and Pramer, 1953). The
only previous work describing the influence of antibiotics on marine microorganisms is that of Cviic
(1933). He studied 41 bacteria isolated from sea water
and found that 70 per cent were sensitive to penicillin
and 75 per cent were sensitive to streptomycin. The
growth of 97 per cent of the species tested was inhibited by a combination of penicillin and streptomycin.
SUMMARY
The production of antibiotics active against Escherichia coli or Bacillus subtilis was not demonstrated
253
in tests of 200 marine bacteria. Chlortetracycline and
penicillin were rapidly inactivated in both sterilized
and untreated sea water, whereas no loss of chloramphenicol, neomycin, oxytetracycline, or streptomycin
was observed in samples of water that contained the
antibiotics at a concentration of 200 ppm and were
incubated at 28 C for 30 days. The biological effects
of antibiotics in sea water varied with the nature and
concentration of the antibiotic tested as well as with
the process investigated. In general, antibiotics reduced the numbers of colonies developing on sea water
agar and the growth in sea water broth of E. coli
and the indigenous bacterial flora. Nitrification and
thiosulfate oxidation were more sensitive to antibiotics than was ammonification. Some antibiotics
added to sea water persisted for long enough periods
to exert a biological effect. Nevertheless, there is no
evidence that antibiotics are produced under natural
conditions by marine microorganisms and contribute
to the death of cells of E. coli that enter the sea.
REFEREN-CES
CARLUCCI, A. F. AND PRA-MER, 1). 1960 An evaluation of
factors affecting the survival of Escherichia coli in sea
water. I Experimental procedures. Appl. Microbiol.,
8, 243-247.
Cviic, V. 1953 The bactericidal and bacteriostatical action
of antibiotics oIn marine bacteria. Acta Adriat., 5, 1-32.
GREENBERG, A. E. 1956 Survival of enteric organisms in sea
water. Public Health Repts., 71, 77-86.
GREIN, A. AND MIEYERS, S. P. 1958 Growth characteristics
and antibiotic production of actinomycetes isolated from
littoral sediments and materials suspended in sea water.
J. Bacteriol., 76, 457-463.
KETCHUMI, B. H., CAREY, C. L., AND BRIGGS, M. 1949 Preliminary studies on the viability and dispersal of coliform
bacteria in the sea. In Limnological aspects of water
supply and waste disposal, pp. 64-73. Am. Assoc. Advance.
Sci., Washington, D. C.
KETCHUM, B. H., AYERS, J. C., AND VACCARO, R. F. 1952
Processes contributing to the decrease of coliform bacteria
in a tidal estuary. Ecology, 33, 247-258.
KRASSILNIKOV, N. A. 1938 The bactericidal action of sea
water. Mikrobiologiya, 7, 329-334 (English summary).
LECHEVALIER, H. A. AND CORKE, C. T. 1953 The replica
plate method for screening antibiotic producing organisms.
Appl. Microbiol., 1, 110-112.
NuJSBAUM, I. AND GARVER, R. M. 1955 Survival of coliform
organisms in Pacific Coastal wN-aters. Sewage and Ind.
Wastes, 27, 1383-1390.
ORLOB, G. T. 1956 Viability of sewage bacteria in sea water.
Sewage and Ind. Wastes, 28, 1147-1167.
PRAMER, D. 1958 The persistence and biological effects of
antibiotics in soil. Appl. Microbiol., 6, 221-224.
RICHOU, R., NEANT, M., AND RIcHou, H. 1955 Surle pouvoir
bact6ricide de l'eau de mer a l'egard du staphylocoque.
Rev. Immunol., 19, 64-68.
STARKEY, R. L. AND PRAMER, D. 1953 The significance of
streptomycin in soil. Intern. Congr. Microbiol., 6, 344345.
ROSENFELD, W. D. AND ZOBELL, C. E. 1947 Antibiotic
production by marine microorganisms. J. Bacteriol., 54,
393-398.
254
A. F. CARLUCCI AND D. PRAMER
VACCARO, R. F., BRIGGS, MI. P., CAREY, C. L., AND KETCHUM,
B. H. 1950 Viability of Escherichia coli in sea water.
Am. J. Public Health, 40, 1257-1266.
WAKSMAN, S. A. AND REILLY, H. C. 1945 Agar-streak
[VOL. 8
method for assaying antibiotic substances. Ind. Eng.
Chem., 17, 556-558.
ZoBELL, C. E. 1936 Bactericidal action of sea water. Proc.
Soc. Exptl. Biol. Med., 34, 113-116.
An Evaluation of Factors Affecting the Survival of Escherichia coli in
Sea 'Water'
IV. Bacteriophages
A. F. CARLUCCI2
AND
DAVID PRAMER
Department of Agricultutral Microbiology, Ruttgers, The State University, New Bruenswick, New Jersey
Received for publication December 7, 1959
d'HWrelle (1926) suggested that bacteriophages
contribute to the self-purification process in natural
waters, but ZoBell (1946) reported that they occurred
sporadically and only in the littoral zone and concluded
that there was insufficient evidence for bacteriophages
to be considered of importance in limiting the bacterial
population of the open ocean. Nevertheless, it was
repeatedly stated in the literature (Carlucci and Pramer, 1959) that bacteriophages contribute to the rapid
death and paucity of bacteria in sea water. Recent
studies (Kriss and Rukina, 1947; Spencer, 1955) have
shown that bacteriophages are not limited to the littoral zone but occur at points distant from land and
at depths as great as 2000 meters. The bacteriophage
isolated (1955) and studied (1957) by Spencer was
active against several strains of the luminous marine
bacterium Photobacterium phosphoreum. It caused lysis
of host cells on sea water agar but not on tap water
agar and appeared to be indigenous to the sea.
The present report describes the occurrence, persistence, and activity of some bacteriophages in sea
water.
MATERIALS AND METHODS
Various methods for the isolation of bacteriophages
from sea water were tested, but the one that proved
most successful was similar to that used in a study of the
occurrence of bacteriophages in soil (Carlucci and Star' Paper of the journal series, New Jersey Agricultural Experiment Station, Rutgers, The State University, Department
of Agricultural Microbiology, New Brunswick, New Jersey.
This investigation was supported in part by research grant
E1437 from the National Institute of Allergy and Infectious
Diseases, National Institutes of Health, U. S. Public Health
Service.
2 Present address: Research Department, United Fruit
Company, La Lima, Honduras, C. A.
key, 1956). Portions of freshly collected samples of
sea water were added to flasks that contained nutrient
broth, and separate flasks were inoculated with cells
of Escherichia coli, Aerobacter aerogenes, and Serratia
marinorubra. The flasks were incubated for 48 hr on a
rotary shaker3 at 28 C after which their contents were
filtered through Morton sintered glass discs of ultrafine grade (Morton, 1944). This primary treatment
caused multiplication of phages present in the sea
water sample, enriched the preparation, and increased
the probability of phage isolation.
Each filtrate (1 ml) was added to a tube containing
5 ml of nutrient broth. The tubes were inoculated with
appropriate host cells and incubated on a rotary shaker
at 28 C. The presence of phage was expressed by
clearing of the culture medium. When lysis was observed the contents of the tube were filtered through
sintered glass and an aliquot of the filtrate was tested
for its ability to produce plaques. To obtain plaque
formation, approximately 10 ml of nutrient agar was
added to a Petri dish and permitted to solidify. A
0.5-ml aliquot of the filtrate being tested was added
to the surface of this agar layer and thoroughly mixed
with an additional 10 ml of nutrient agar that were
seeded with cells of the host bacterium and poured at
approximately 45 C. When the second agar layer had
solidified, the plates were incubated at 28 C for 24 hr
and examined for plaques. Phage titers were measured
by the same layering technique using aliquots of appropriate dilutions of filtrates. Plaque counts were made
after 24 hr incubation and the number of phage units
present in the filtrate was calculated from the plaque
count, the volume of filtrate plated, and the dilution
used. Procedures employed to measure the survival of
3
New Brunswick Scientific Instrument Company, New
Brunswick, New Jersey.