Jpn. J. Infect. Dig., 53, Ill‑115, 2000

Original Article

Inhibition ofAeromonas caviae and A. sobria by

Sodium Chloride, Citric Acid, Ascorbic Acid, Potassium Sorbate
and Extracts of Thymus vulgaris
Bayan M. Abu‑Ghazaleh*
Department ofBiology, Hashemite UniversiOJ, Zayqa, Jo71dan(Received April 3, 2000. Accepted June I 6, 2000)

SUMMARY: The respective and combined effects of sodium chloride, ascorbic acid, citric acid, potassium
sorbate, and Thymus vulgaris extract on the growth ofAeromonas caviae and Aenmonas sobria wereinvestlgated.

Sodiumchloride (3%) significantly reduced the growth and 4% NaCl inhibited growth of the tested strains.

AsCorbic acid (0. 1%), potassium sorbate (0.05%), and citric acid (0.03%) slightly inhibited growth. T. vulgaris
extract (0.3%) greatly reduced the growth. Various combinations of these compounds prevented growth of thetested strains. A combination'ofNaCl (3%) and ascorbic acid (0. 1%), citric acid (0.03%) and potassium sorbate(0.05%), or citric acid (0.03%) and ascorbic acid (0. 1%) inhibited growth ofA. caviae and A. sobria. In fishhomogenates, the addition of ascorbic acid (0. 1 %) and citric acid (0.03%) was the most effective combination
tested.

the wrapping material (9)･ To my knowledge, there.are no

INTRODUC TION

published data on the effect of preservatives on A. cavlae and

Aey10mOnaS SpP. have recently come to be considered an

A. sobria. Therefore, this study was conducted to investigate

important cause or fわod‑bone diseases in humans, and are

the respective effect of citric acid, ascorbic acid, potassium

implicated as a cause of gastroenteritis in both developed andsorbate, Sodium chloride, Thymus vulgaris extract, and combi‑

developlng COun廿ies. In studies of bacterial pathogens associ‑nations of these compounds on the two Aeromonas spp.

atedwith diarrhea, A. hydrophila and A. caviae were isolated
fTrom both adult and pediatric patients (1 ,2). In another study,

A4ATERIALS AND METHODS

Aeromonas spp. were recovered from 52% of children withacute diarrhea (3). Many types of food, particularly

Bacterial strains: Two A. sobria strainsand one A. caviae

contaminated seafわod, Were implicated as the cause or fわodstrain were used in this study. A. sobria 172 was isolated &om

poisoning (4). Aeromonas spp. were isolated from 34% of

local fish, and A. sobria 74 and A. caviae 166 were isolated

fin fish taken from retail outlets in New Zealand (5) and infrom imported fish. All strains had been previously isolated

the United Kingdom, 1 5 out of47 (approximately 32%) fishby the author, and were identified at the genus and species

purchased from retail outlets were found to be contaminatedlevels using the method described by Camahan et al. (10)with Aeromonas (6). In Jordan, 13% of sampled retail fish

and Kirov (ll). In brief; samples were enriched in 0.1%

(locally grown and imported) were contaminated with
Aey10mOnaS Spp., a large proportion of which were A. caviae

alkaline peptone water, and incubated at 34oC for 1 8‑24 h. A

The initial bacterial content in fresh fish is affected by theloopful of the above culture was streaked on MacConkey agar
and incubated ovemight at 34oC. Lactose fermenter andlactose non‑fermenter colonies appeanng on the MacConkey

method of harvestlng. Trawled fish carry bacterial loads thatagar plates were plated on non‑selective medium (nutrientor A. sobria (unpublished data).

are lOl 1 00 times greater than those of line‑caught fish. Thisagar) and an oxidase test was performed. Strains that wereis due to the fact that the trawling net drags along the sea

bottom, stirring up the mud and contaminating the fish (7).

oxidase positive were subjected to the followlng tests: Gram
stain, glucose fermentation using a triple sugar iron agar (TSI)

Also, during periods of heavy fishing, trawling nets become

sl肌t,and resistance to 0/129 vibriostatic agent (150 LLg). The

full, and fish are bmised from compression, which results instrains that were Gram‑negative rods, glucose‑fermenting, andresistant to 0/1 29 vibriostatic agent were further characterizedpoor quahty and decreased shelf life. After harvesting, fish
should be cleaned and cooled down to OoC as quickly as

by using the APト20E system (Biomerieux, Marcy l'Etoile,

possible (8). However, small boats, which leave the docks in

France) and other complementary tests (e.g., production of

the momlng and retum after several hours to store fish, often

gas from glucose fermentation and growth in 6% NaCl).

store them without ice. This increasesthe number of spoilage

The organisms were maintained on nutrient agar slants atbacteria, and necessitates the application of compounds to

4oC. To prepare the inoculum, nutrient broth (20 ml) was

eliminate spoilage bacteria and extend the shelf life of fish.

inoculated wi仇叩prOpriate culhres and incubated at 34oC

Chemicals may be added in a varietyofways,including spray‑for 20 h.

mg onto fish; dipping fishinto a solution contalnmg Chemicals;

Media and chemicals: Nutrient broth was obtained fromdirect incorporation into fish; or application of chemicals to

ADSA micro (Scharlau Chemie, Barcelona, Spain). Sodium*Corresponding author: Mailing address: P.0.Box 926966,

obtained from Gainland Chemical Co. (Hampshire, UK).

chloride, citric acid, ascorbic acid,and potassium sorbate were
Stock solutions or citric acid, ascorbic acid, and potassium

Amman 11110, Jordan. Fax: +962 6 5676213

Illsorbate were keshly prepared before each use and sterilized

occurred before growth ofA. caviae 1 66 could be observed.

by filtration through filter membranes (0.45 LLm, Micron

Addition of ascorbic acid (0.1%) alone to the growth

Separation Inc., Philadelphia, Pa., USA). Crude extract of T.

medium slightly inhibited growth of the strains, whereas the

vu/garis (extracted by hydrodistillation) was obtained frompercentages of inhibition after 24 h incubation were 1 l%and 24% for A. sobria 172 and A. caviae 166, respectively.
However,the strains grew in nutrient brothcontaining ascorbic

Systema Co. Ltd. (Amman, Jor血n).

Growth conditions: (1) Growth in nutrient broth : Flasks
containing 30 ml of nutrient broth (pH 7.2), nutrient brothacid a触r a lag of about 2 h.

Interaction between sodium chloride or citric acid andplus NaCl (3% or 4% W/V), nutrient broth plus citric acid(0.03% W/V), nutrient broth plus ascorbic acid (0. 1% W/V),
nutrient brothplus crude extract of T. vulgaris (0.3% V/V), or

to NaCl (3%) and ascorbic acid (0.1%) together caused

nutrient broth plus various combinations of these compounds

enhanced inhibition of both strains; growth of cells was not

were inoculated with 1 ml of ovemight‑grown bacterial

detected after 24 h incubation (Tables 1 and 2). Addition ofcultures. For experiments containlng potassium sorbate

citric acid (0.03%) and ascorbic acid (0.1%) together to the(0.05% W/V), the pH of nutrient broth was adjusted to 5.8

growth medium inhibited growth ofA. caviae 166 and A.
sobria 172 (Tables 1 and 2).

ascorbic acid･. Exposure ofA. caviae 166 and A. sobria 172

before addition of the tested compounds, then the tested

compounds were added to flasks contalnlng 30 ml of nutrient

Effect of potassium sorbate: The presence of potassium

broth bH 5.8)and treated as mentioned above. Alter inocula‑sorbate (0.05%) in the growth medium slightly inhibited

tion, the flasks were incubated static at 34oC. Samples were

growth of the tested strains. The percentages of inhibition

withdrawn froヮeach mask at suitable intervals (〜l h) anda洗er 24 h incubation were 12%, 15%, and 34% forA. caviae

grow血was monltOred for 24 h by measuring optical densityat

166, A. sobria 74, andA. sobria 172, respectively. However,

560 nm.spectrophotomebically (Spectronic, Cheshire, UK)･
During Incubation, at least five readings were obtained &om
Table I. Inhibition of growth ofA. caviae 166 in nutrient broth byeach mask.

combination of various compounds(2) Growth in rISh homogenates: Fish muscle, fat tissue,
and skin (60 g) were aseptically obtained from fresh fish,

NaCl

and fish homogenates were prepared by blending the above

potassium

io7.T sore.a/誓(%)

samples with 400 ml distilled water in a sterile blender. The

pH of the homogenate was 6.6 as determined by pH meter.

AsC.rbic

GrowthReducti.na:i/?, aci,d.ハ

aAer ahr a洗er inf.r/?tYth00000000000000. 0 0 0 0(%)

0000000000

0 つJ 4 0 3 0 0 0 つJ 0

Citric

(%)

2h

4h

tJ 1 5 /▲U 100000000000

24h

0 0 0 0 0 0 0 0 2 ｣ 0 0 0 5 つJ ∠U O ′000000000T(%)b0 3 0 ′0 7 上U
2 2 1 2 2 ′061

The homogenate was sterilized by heatlng at 70oC for I 0 min,then cooled. A specific volume (30 ml) of the homogenate
was dispensedinto screw cap test tubes,and sodium chloride,

99

24

111

citric acid, ascorbic acid, Or combinations of these compounds

1were added per tube, aseptically. One milliliter of ovemight‑

O00

10101 0 2 0 2

つJ つJ tJgrown bacterial cultures was added to each test tube, and thetubes were incubated static at 34oC. Samples were withdrawn

0 0 .0 0 5 5 5000

斤om each tube at suitable intervalsand grow血was monitored

000 0 ｣ 1

000 4

0

75

4 0 一0 0

5

for 24 h spectrophotometrically.

l

All experiments in this study were performed five times,

04

andthe optical densityreadings presented arethe meanvalues.

a

a

a

a: The values represent growth in nutrient broth (pH 5.8).

b: Reduction in growth(%)=

The standard error was ± 0.01 for all readings.

Growth in brothwithout chemicals‑growth in brothwith chemical(S)Growth in brothwithout chemicals

RESULTS
Efrect of sodium chloride, citric acid and ascorbic acid:
The OD56. readings ofA. caviae 166 and A. sobria 172

Table 2. Inhibition ofgrowthofA. sobrE'a 172 in nutrient brothby
combination of various compounds

subjected to sodium chloride (3%) alone are presented inTables I and 2. The presence of sodium chloride (3%) in theNaClgrowth medium reduced the growth rate, whereas the per‑potassium

CitriCAsCorbic(07.Y sore.a/fe af.i/ll aCi,d.ハ(%)

centage of inhibition after 24 h of incubation was 61% forbothstrains. Also, a lag was observed beforeincreaseinnumber

(%)

(%)

Growth

2h

4h

24h

/b′ 3 人古Ia

0 tJ 4 0 つJ 0 O つJ 000000000000000000000000

Reduction

aRer a洗er aRer in!or/oITth

00000000000

0 0 0 0 0 0 0 0 0 0 0 3 0 .1 ｣ 0 3

1 000 1T(%)b

1 o L o L o o L 14叫.Oto 0 7340 t

for either strain could be detected, and the lag was longer for

61

A. sobria 172. The effect ofNaCl (3%) onA. sobria 74 is

99

shown in Figure. The percentage of inhibition after 24 h of

つJ 8 2 8

1‑ll

incubation was 64%, and the lag was shorter than that for A.

ll
1 1 1 0 1 O つJ 上U O 一 0 2 0 4 つJ 0sobria 172 and more similar to that forA. cavL'ae 166.

O 0 03 tJ tJ

SodiuT chloride (4%) nearly completely inhibited growthofA. cavlae 166 andA. sobria 172; Only a slight increase in

0 0 0 5 くJ 5

optical densitywas observed after 24 h incubation in nutrient
broth with 4% NaCl (Tables 1 and 2).
The presence of citric acid (0.03%) alone in the growth
medium only slightly inhibited growthofA. caviae 166 or A.

a: The values reprcscnt growthin nuthent broth bH 5.8).

sobria 1 72; OD560 readings after 24 h incubation in nutrient

b: Reduction in grow血(%)=

broth withandwithout citric acid were nearly the same (Tables

Growth in brothwithout chemicals ‑growth in brothwithchemicaI(S)land 2). However, whenthe brothcontained citric acid, a lag

Growth in broth without chemicals

112

Table 3. Eqect of 7卑ymus vulgan's extract on (A) A. cavt'ae 166and(B) A. sobrt'a 172

(A)
Thyme

NaCI

lol:)" (%)

GrowthReduction

after2h alkr4h a触24h

0 0 tJ

0.12

0.25

0

0

0

0

000

in

5LUW(Loll.)
00 0 0l エリ 29 9 3 00

Reduction ina鮎r2h after4h alter24h
000

000 ‑

0 0 つJ

000

tJ

Loo

5

つJ つJ

growtha

(%)

J0

4 9

8 97 2

8: Reduction in growth (%)=

Growth in broth without chemicals‑growthin brothwithchemicaI(S)Growth in bro也without chemicals

Table 4. Inhibition ofgrowthofA. caviae 166 in fish homogenates bycombination of various compounds
NaCI

Citric

AsCorbic

GrowthReductioninacid acid(%)(%)

0 0 tJ 0

0000

(%)

aRer2･5haRer24h′0 20000 0 0

0 0 0 0 4 4 つJー(%)

00 ′0 3

410

tJ 0
O
2

tJ

l

a: Reduction in growth(%F4Growth in broth without chemicals一growth in brothwithchemical(S)Time (h)Growth in broth without chemicals

Figure. Influence ofNaCl, potassium sorbate, and combination of various

chemicals onthe growth ofA. sobria 74. The strain was inoculated

into NB medium (pH 7.2) containing no chemicals (+) or 3% NaCl

(0.3%) to the growth medium significantly reducedthe growth( ▲ ); or into NB medium (pH 5.8) containing no chemicals ( ○ ),rate. The percentages of inhibition ofA. caviae 166 and A.

0.05% potassiumsorbate (A), 0.05% potassiumsorbate plus 3% NaCl

sobria 172 after 24 h of incubation were 93% and 87%,(ロ), or 0.05% pottassium sorbate plus0.03% citric acid (◇).

respectively (Table 3). Addition of NaCl (3%) and thyme
extract (0.3%) together to the growth mediumcaused enhanceda lag was observed before increase in number could be

inhibition of both strains (Table 3).

detected (Tables 1 and 2, Figure). This inhibitory activitywas

Efrects of ascorbic acid and combination of ascorbic aciddue to the presence of potassium sorbate and not due to thewith sodium chloride and citric acid in fish homogenates:

low pH of the growth medium. The OD56. readings of the

The growth ofA. caviae 166 in fish homogenates containing

strains grown in nutrient broth at pH 5.8 for 24 h were nearly
similar to OD56. readings of strains grown in nutrient broth at

the tested compounds is presented in Table 4. The presence

pH 7.2 (Tables 1 and 2, Figure).
Interaction between sodium chloride or citric acid andinhibit growth ofA. caviae 166; OD56. readings after 24 hpotassium sorbate: The results of exposure ofA. caviae 166,acid were nearly仇e same. Addition of ascorbic acid (0. 1 %)A. sobria 172, andA. sobria 74 to NaCl (3%) and potassium
sorbate (0.05%) together are presented in Tables I and 2 andFigure. Addition ofNaCl (3%) and potassium sorbate (0.05%)

tion of the growth rate of the tested straln; the percentage oftogether caused enhanced inhibition of the tested strains,

strain showed growth a洗er a lag ormore than 2.5 h. On theof ascorbic acid (0. 1%) alone in fish homogenates did notincubation in fish homogenates with and without ascorbic

and NaCl (3%) together to fish homogでnates caused reduc‑growth inhibition after 24 h incubation was 3 1%, and thewherethe percentages of inl1ibition after 24 hincubation wereother hand, addition of ascorbic acid (0. 1%) and citric acid

40%, 63%,and 70% forA. caviae 166,A. sobria 172, andA.

(0.03%) together to fish homogenates inhibited growth ofA.

sobria 74, respectively. There was a lag of about 4 h before

caviae 166; increase in optical densitywas not observed a洗er

growth of the tested strains could be detected.

24 h incubation. Similar results were obtained for A. sobria

Exposure ofA. caviae 166, A. sobria 172, andA. sobria 74(data not shown).

to citric acid (0.03%) and potassium sorbate (0.05%) together
completely inhibited growth of these strains. Increase in

DISCUSSION

optical densitywas not observed after 24 h incubation in

Aenomonas spp. are able to grow and produce exotoxins at

nutrient broth containing these compounds (Tables 1 and 2,

both low and moderate temperatures (C.g., 5oC and 37oC,

Figure).

respectively) ( I 2), which capacitysuggests that, in food stored

Effect of T. vutgaris extract: Addition of thyme extract113

at ambient or refrigeradon temperature, they could be ofpublic

Zhao et al. (17) showed that enterohemorrhagic E. colt

health significance. Therefore, addition of chemicals before

O157:H7 was inactivated in apple cider at higher rates in thestorage to control the growth ofAeylDmOnaS SPP. lS intended

presence of 0.1% potassium sorbate and 0.1% sodium

to extend the shelf life of food products. In this study, thebenzoate together. Other studies have reported that citricand

respecdve effects ofNaCl, cibic acid, ascorbic acid, potassium

lactic acids also potentiate theantimicrobial action of sorbate

sorbate, extracts of T. vulgaris, and combinations of these(23).

compounds on the growth ofA. caviae and A. sobria were

The effects of chemicals on A. caviae 166 and A. sobria

investigated. The tested strains showed marginally similar

172 in fish homogenates were examined in this study. Both

responses after exposure to NaCl, citric acid, or ascorbic acid.
Variationsinlagtime were generally observed. Upon exposurestrains were sensitive to a combination of ascorbic acid andto 3% NaCl, A. sobria 172 showed a lag time longer than thatthe other hand, the tested strains were more resistant to theofA. caviae 166 orA. sobria 74. A. sobria 172 was isolated
from freshwater fish, while the latter strains were isolated

effect ofascorbic acid (0. 1%) alone or to the combination of
ascorbic acid (0.1%) and NaCl (3%) when treated in fishfrom salt water fish and were therefore adapted to growth inhomogenates than in broth. Fish homogenates are proteina‑

citric acid when treated in fish homogenates and in broth. On

an environment of relatively high osmotic pressure. Similar

ceous products (I 6% proteins), and, therefわre, bacteria may

findings were reported by Abu‑Ghazaleh et al. (13), whobe able to survive by assumlng a Protective coatlng Of thesereported that genetic and environmental factors innuence theprotei.naceous materials (24)･ Further･ proteins and othertolerance of food poISOnlng bacteria to heat. Also, Cheroutre‑
Vialette et al･ (14) emphasized the need to take into ace?unt

organlC molecules in fish homogenates may react with thechemicals added, causlng a decrease in their antimicrobial

the previous history of a product, and therefore the previous

activity. Banwart (9) reported that the composition of food ishistory of a strain, which could impact the lag tlme Of theone of the factors affecting the activlty Of chemicals agalnSt

microorganism.

microorganisms in it.

AsCorbic acid (0. 1 %) showed a slight inhibitory effect on

In conclusion, this study showed that various combinations

the tested Aeromonas spp. Similar results were obtained byof the tested compounds inhibited growth ofAey10mOnaS Sp.

Fletcher et al. (15), who reported that ascorbic acid showedThis study also showed that some c?mpounds 〔e･g･, NaClsome inhibitory properties toward Campylobacterjejuni. Cibic(3%), a苧Corbic acid, citric acid, potass..um sorbate〕 increased

acid (0.03%) did not demonstrate an antimicrobial effect on
the Aeromonas spp. tested. Other studies have reported thatcitric acid is not as effective as other acids or preservatives.

the lag tlme OfAeromonがSP. ApplicatlOn Ofthese compounds

may Increase the shelf life of stored foods.

The growth ofEscherichia colt and Listeria monocytogenes

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