EPIDEMIOLOGYAntibiotic Susceptibility of Non-Cholera Vibri

EPIDEMIOLOGY
Antibiotic Susceptibility of Non-Cholera Vibrios
Isolated from Farmed and Wild Marine Fish
(Argyrosomus japonicus), Implications for Public Health
Justine Fri,
1
Roland Ndip Ndip,
2
Henry Akum Njom,
1
and Anna Maria Clarke
1
This study aimed to evaluate the antibiogram and antibiotic
resistance genes (ARGs) of Vibrio isolates
recovered from a marine fish (Argyrosomus japonicus) and
water samples from two commercial dusky kob
aquaculture farms and the Kariega estuary, South Africa, and to
evaluate these findings for their public health
implications. A total of 277 molecularly confirmed Vibrio
isolates consisting of 126 Vibrio fluvialis, 45 Vibrio
vulnificus, 30 Vibrio Parahaemolyticus, and 76 vibrios
belonging to species of the genus other than Vibrio
cholerae were subjected to susceptibility testing to 15
antibiotics by the disc diffusion method. Multiple
antibiotic resistance index ( MARI) was used to determine the

antibiotic resistance-associated health risk,
while polymerase chain reaction was used to evaluate the
presence of 14 ARGs for nonsusceptible strains.
Highest resistances were recorded to amoxicillin (76.2%),
ampicillin (67.5%), erythromycin (38.3%), and
doxycycline (35.0%), while susceptibilities were highest to
gentamicin (100%), followed by norfloxacin
(97.8%), florfenicol (90.3%), tetracycline (87.7%), and
chloramphenicol (87.4%). We recorded a 58.5%
multidrug resistance (resistance to ‡2 antimicrobial classes).
MARI did not vary significantly between sites
( p > 0.05); however, values of >0.2 were recorded in 40%
(108/277) of all strains tested. ARG markers, ampC,
blaOXA, tetA, tetM, dfr1, sul1, sul2, ermB, nptII, strA, and
SXT integrase, were detected in one or more strains
with ermB (82.5%), sul2 (53.8%), strA (44%), dfr1 (42.3%),
and tetM (38.3%) being the most abundant.
Healthy marine finfish (dusky kob) and their environment can
serve as reservoirs for antibiotic resistant vibrios
and ARGs, which could be disseminated to humans and other
susceptible bacteria and this therefore becomes a
public health concern.
Keywords: Vibrio, marine fish, antibiotic drug resistance,
antibiotic resistant genes, public health
Introduction
Antimicrobials are widely used for the preventionand treatment
of bacterial diseases in food animals.1
They are among the most widely administered drugs ap-
proved for animal health and management. Global estimates
indicate higher volumes of antimicrobials used in food-
producing animals, exceeding those used in humans.
2

Most
animal feeds are supplemented with various concentrations
of antimicrobials ranging from subtherapeutic to full doses.
Moreover, almost all the classes of antibiotics used in the
treatment of human infections are also used in food animals,
including the newest classes of drugs such as third- and
fourth-generation cephalosporins, fluoroquinolones, glyco-
peptides, and streptogramins.
2
In aquaculture, the control of antibiotic usage varies widely
from country to country.
3
Most developed countries either have
registered antibiotics for bacterial disease control, with well -
defined regulations regarding their usage, or no longer permit
the registration of products for nontherapeutic purposes such as
growth promoters.
4,5
However, about 90% of aquaculture in-
dustries are located in developing countries with weaker con-
trol strategies. Although antibiotics play an important role in
the prevention and treatment of bacterial diseases both in hu-
mans and food animals, the indiscriminate use of these anti -
microbials has led to the emergence of antibiotic-resistant
bacteria and antibiotic resistance genes (ARGs) in the environ-
ment. Members of the genus Vibrio are abundant in the marine
environment and may contaminate edible fish. Vibrio cholerae,
Vibrio parahaemolyticus, Vibrio Vulnificus, and Vibrio
fluvialis

are the most significant human pathogens of the genus that may
cause mild to potentially fatal foodborne illnesses, especially
from consumption of contaminated seafood.
6–8
The acquisition
of ARGs by these pathogenic bacteria is of concern as these
may
1
Microbial Pathogenicity and Molecular Epidemiology Research
Group ( MPMERG), Department of Biochemistry and
Microbiology,
University of Fort Hare, Alice, South Africa.
2
Department of Microbiology and Parasitology, University of
Buea, Buea, Cameroon.
MICROBIAL DRUG RESISTANCE
Volume 24, Number 9, 2018
ª Mary Ann Liebert, Inc.
DOI: 10.1089/mdr.2017.0276
1296
cause treatment failure and difficulties in human disease man-
agement. Previous studies have reported antibiotic resistance or
resistance genes in vibrios isolated from shrimps
9,10
and other

seafood.
11,12
These pathogenic bacteria with acquired ARGs may serve
as reservoirs of resistance genes to susceptible bacteria. The
accumulation of antibiotic residues in edible fish tissues may
also alter human intestinal flora and cause food poisoning or
allergies.
13
Some of these residues may be detected from
several months
14
up to a few years posttreatment with an-
timicrobials.
15
The World Health Organization considers the
emergence of drug-resistant bacteria, particularly multidrug-
resistant strains, to have critical consequences for human
medicine.
16
Data on the actual supply, consumption con-
centrations, and volumes of antimicrobials utilized in South
African animal health and aquaculture are scanty. It is im-
perative to continuously monitor the drug resistance pattern
of potentially pathogenic bacteria species as to determine
the risk they may pose to human health. We therefore
evaluated the susceptibility patterns of Vibrio strains (iso-
lated from water and fish from two commercial dusky kob
aquaculture farms and the Kariega Estuary) to antibiotics

commonly used in the treatment of fish and human vibriosis,
as well as their associated ARG markers.
Materials and Methods
Bacterial isolates and growth
A total of 277 Vibrio isolates (126 V. fluvialis, 45 V. vulnifi -
cus, 30 V. parahaemolyticus, and 76 vibrios belonging to other
species of the genus other than V. cholerae) randomly selected
from a pool of 606 vibrios (193 V. fluvialis, 74 V. vulnificus,
33
V. parahaemolyticus, and 306 belonging to other species of the
genus), isolated from 80 water samples and 120 dusky kobs in a
previous study,
17
were used in this study. Forty-four isolates
were from Kariega estuary (33�41¢S, 24�44¢E), 147 were
from
Farm 1 (13.0670�N, 59.5712�W), and 86 were from Farm 2
(32.9638�S, 27.8789�E), all located in the Eastern Cape prov-
ince, South Africa. All strains were previously maintained in
Tryptic Soy Broth supplemented with 25% glycerol and stored
at -80�C following molecular identification of the isolates. The
frozen cultures were resuscitated on brain–heart infusion agar
plates, and the plates were incubated at 37�C for 18–24 hr
before
antibiotic susceptibility testing and antibiotic resistance gene
detection.
Antibiotic susceptibility test
Antimicrobial susceptibility of Vibrio isolates to 15 antibi -
otics belonging to nine classes was performed according to the

standard guidelines recommended by Clinical Laboratory
Standards Institute (CLSI).
18
An inoculum for each isolate was
prepared by emulsifying colonies from an 18-hr pure culture in
3 ml of sterile normal saline (0.85%), and the turbidity was
adjusted to 0.5 McFarland standard (0.5 ml of 1% w/v BaCl2
and 99.5 ml of 1% v/v H2SO4) equivalent to 1.0 · 10
8
cfu/ml.
The bacterial suspension was uniformly streaked on Mueller
Hinton agar plates using sterile swabs and allowed for 3–5 min
before introduction of antibiotic discs. A panel of 15 antibiotic -
impregnated discs ( Mast Diagnostics, United Kingdom),
namely tetracycline (30 mg), erythromycin (15 mg), amoxicillin
(15 mg), streptomycin (10 mg), gentamycin (10 mg), cipro-
floxacin (5 mg), Chloramphenicol (30 mg), trimethoprim-
sulfamethoxazole (1.25/23.75 mg), norfloxacin (10 mg), ampi-
cillin (10 mg), doxycycline (30 mg), nalidixic acid (30 mg),
florfenicol (30 mg), imipenem (15 mg), and cefoxitin (30 mg),
were placed on inoculated plates with a mast disc dispenser
(Mast Diagnostics). These antibiotics were chosen based on
their use in the treatment of Vibrio infections in humans and
animals, as well as their use in aquaculture. Plates were incu-
bated at 37�C for 24 hr, and the diameters of the zones of in-
hibition were measured and results interpreted as susceptible
(S), intermediate (I), or resistant (R) according to CLSI inter -
pretive criteria.
18,19
Resistance to florfenicol was interpreted as

earlier reported.
20
A multiple antibiotic-resistant phenotype
was defined as resistance to ‡2 antibiotic classes.
Determination of multiple antibiotic resistance
index/multiple antibiotic resistance phenotype
Multiple antibiotic resistance index ( MARI) is an im-
portant epidemiological tool used in assessing human health
risk in relation to antibiotic resistance. This tool aids in
differentiating high-risk (value of >0.2) from low-risk (value
of <0.2) contamination with bacteria of potential hazard to
humans in relation to antibiotic resistance.
21
MARI for an isolate was computed as:
MARI = a/b, where ‘‘a’’ represents the number of antibi-
otics to which that isolate was resistant and ‘‘b’’ the total
number of antibiotics to which the isolate was exposed,
21
while the MARI for a given sample of several isolates for
each site was also derived using the mathematical formula
MARI = a/b.c, where ‘‘a’’ is the aggregate antibiotic re-
sistance of all the isolates, ‘‘b’’ is the number of antibiotics
tested, and ‘‘c’’ is the number of isolates in the sample.
21

Detection of antibiotic resistance genes
For all cultures maintained at -80�C, genomic DNA was
isolated by suspending isolated colonies from an 18-hr pure
culture in 200 ml of sterile distilled water. Cells were lysed by
boiling for 15 min in a digital Accu dri-block (Lasec, South
Africa) and cell debris removed by centrifugation at 13,000 g
for 5 min. The supernatant was used directly as template in the
polymerase chain reaction (PCR) assay or stored in 50 ml ali -
quots at -20�C until use. Nonsusceptible strains to various
antibiotics were subjected to detection of ARGs by PCR using
specific primers in a C1000 Touch Thermal Cycler (Bio-Rad,
Singapore, Malaysia). Table 1 shows the primer sequences and
cycling conditions used for the detection of the ARGs. Each
amplification was performed in a 25 ml reaction, consisting of
5 ml template DNA, 0.5 ml of each of the oligonucleotide (In-
qaba Biotec, Pretoria, South Africa), and 12.5 ml of OneTaq
Quick-Load 2 · PCR Master mix (New England BioLabs,
Ipswich, United Kingdom) and an appropriate volume of
nuclease-free water (New England BioLabs). PCR amplified
products were separated in ethidium bromide (0.5 mg/ml)
stained with 1% agarose gels by electrophoresis at 100 V for
45 min. Gels were visualized using a UV transilluminator
(Alliance 4.7 XD-79; UVITEC, Cambridge, United Kingdom).
Statistical analysis
A chi-square test and Goodman and Kruskal’s tau test
were used to determine the relationships between resistances
ANTIBIOTIC-RESISTANT VIBRIOS FROM MARINE FISH
1297
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8
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.
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T
in
te
g
ra
se
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X
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F
:
A
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1
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3
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fo
r
1
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m
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ra
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fo
ll
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x
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7
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.
3
0
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G
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m
in
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ly
c
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si
d
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1
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4
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3
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a
a
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st
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4
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,
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7
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fo
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4
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c
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a
l
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si
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fo
r
7
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in

a
t
7
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.
2
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C
1298
obtained for each antibiotic from different sampling sites. A
p-value <0.05 was considered statistically significant.
Results
Antimicrobial resistance
Generally, the antibiotic susceptibilities of Vibrio isolates to
various antibiotics varied considerably. Of the 277 isolates
analyzed, 262 (94.6%) were resistant to at least one antibiotic.
Overall, the antimicrobial susceptibility pattern revealed gen-
tamicin (100%), norfloxacin (97.8%), florfenicol (90.3%),
tetracycline (87.7%), chloramphenicol (87.4%), streptomycin
(78.7%), imipenem (76.5%), sulfamethoxazole–trimethoprim
(76.2%), and nalidixic acid (74.4%) as the most effective an-
tibiotics against Vibrio strains tested (Table 2). In contrast,
resistance to cefoxitin (28.9%), doxycycline (35.0%), and
erythromycin (38.3%) was higher, with the highest resistance
recorded to ampicillin (67.5%) and amoxicillin (76.2%). Al -
though the total percentage of resistance recorded to cipro-
floxacin was very low (1.8%), almost half (44.8%) of the
nonsusceptible isolates showed an intermediary resistance
pattern. Considering the three human pathogenic species, the
high total resistance to amoxicillin was mostly contributed by

V. vulnificus (84.4%) and V. parahaemolyticus (56.7%) with a
much lower resistance (8.7%) of V. fluvialis to the drug. V.
vulnificus resistance to erythromycin, streptomycin, trimetho-
prim–sulfamethoxazole, and cefoxitin was also significantly
higher ( p < 0.05) than those recorded for V. parahaemolyticus
and V. fluvialis. Similarly, high resistance (35.6% for V. vul-
nificus and 46.7% for V. parahaemolyticus) was detected to
doxycycline, with a lower resistance (13.5%) of V. fluvialis to
the antibiotic. Table 2 shows the percentage susceptibility
profiles of 277 Vibrio isolates to 15 antibiotics.
When we compared resistances among the isolates obtained
from the three sites, there were marked differences in the anti -
microbial resistance patterns observed from isolates of the dif-
ferent sites (Fig. 1). Vibrio isolates recovered from Farm 2
showed significantly higher ( p < 0.05) resistance to
tetracycline,
doxycycline, erythromycin, trimethoprim–sulfamethoxazole,
chloramphenicol, florfenicol, and imipenem with significantly
lower ( p < 0.05) resistances recorded to ampicillin, nalidixic
acid, florfenicol, and cefoxitin than those from Farm 1 and
Kariega estuary (Fig. 1). The only antimicrobial to which there
was a significantly higher ( p < 0.05) resistance in isolates from
Kariega estuary compared to other sites was cefoxitin. None of
the strains recovered from Kariega estuary were resistant to
ciprofloxacin, norfloxacin, nor tetracycline. The percent resis -
tances obtained for each antibiotic for Vibrio isolates recovered
from the three sites are shown in Figure 1.
Multiresistance/MARI/multiple antibiotic
resistance phenotypes
Simultaneous resistance to various antimicrobials was
high (84.5%; 234/277), with a 58.5% (162/277) multidrug
resistance (defined as resistance to at least two classes of
antimicrobials) recorded (Table 3).

As earlier described,
21
a MARI of >0.2 differentiates iso-
lates between antibiotic resistance from low-risk and high-
risk sources. In this study, the MARI ranged from 0.0 to 0.67.
Forty percent (108/277) of isolates fell into a MARI of >0.2,
while 42.2% (117/277) belonged to the <0.2 group. Fifty-two
isolates (18.8%) had MARI = 0.2. Assessment of the aggre-
gate individual resistances of isolates from each sampling
location revealed that Kariega Estuary had the highest per -
centage of vibrios; this could possibly indicate areas of high
antibiotic usage/contamination (MARI = 0.24), this was fol -
lowed by Farm 2 ( MARI = 0.23), and Farm 1 had the lowest
percentage ( MARI = 0.21). However, the differences in
MARI between sampling locations were not significant
( p > 0.05).
Considering multidrug-resistant isolates only, the com-
pilation of the multiple antibiotic resistance phenotypes re-
sulted in 23, 39, and 47 different patterns exhibited by
isolates from Kariega, Farm 1, and Farm 2, respectively.
One isolate from Farm 2 showed resistance to 10 of the 15
antimicrobials tested, exhibiting the phenotype AMP
R
-
AML
R
-TE
R

-E
R
-S
R
-C
R
-SXT
R
-DO
R
-NA
R
-IPM
R
.
Prevalence of antimicrobial resistance genes
PCR assays carried out to detect the presence of antibiotic-
resistant gene markers revealed that 11 (ampC, blaOXA, tetA,
tetM, dfr1, sul1, sul2, ermB, nptII, strA, and SXT) of the 14
genes tested occurred in one or more Vibrio strains, with
ermB (82.5%), sul2 (53.8%), strA (44%), dfr1 (42.3%), and
tetM (38.3%) being the most abundant. Table 3 shows the
prevalence of ARGs detected in the study.
Genes (dfr1, Sul1, and Sul2) coding for sulfonamide re-
sistance were detected in 58.3% (7/12), 25% (3/12), and 50%
(6/12) of the V. fluvialis isolates, respectively, while only dfr1
(33.3%; 4/12) and sul2 (66.7%; 8/12) genes were detected in

the V. vulnificus isolates. None of these resistance genes were
found in the nonsusceptible V. parahaemolyticus strains.
Among the genes that code for streptomycin (aminogly-
cosides) resistance, strA was the most commonly detected
gene as it occurred in the V. vulnificus, V. parahaemolyticus,
and V. fluvialis strains. In contrast, the nptII was detected
only in V. vulnificus and V. fluvialis, while aacC2 was not
found in any of the Vibrio strains. The gene coding for
integrase of the SXT element was only found in V. para-
haemolyticus and was detected in one out of the four re-
sistant isolates. Of all the three genes tested which code for
erythromycin (macrolide) resistance, namely, ermA, ermB,
and ermC, only ermB was detected in all three Vibrio spe-
cies at high frequencies. b-lactam resistance, encoded by the
ampC and blaZ AGRs, was detected in all three Vibrio
spp. For the genes coding for tetracycline resistance, tetM
was detected in all the Vibrio species, while tetA was only
detected in the resistant V. parahaemolyticus isolates. Fig-
ure 2 shows representative gels for some antibiotic resis-
tance genes detected (combined from different PCR assays).
Discussion
Antibiotic sensitivity patterns among Vibrio
isolates and MARI
It is necessary to continuously assess antibiotic resistance
patterns of pathogenic bacteria in various foods and food
environments as part of monitoring the spread and emer-
gence of bacterial resistance. This helps in determining the
health risk associated with subsequent infection when hu-
mans consume possibly contaminated food or water. Al-
though intrinsic antibiotic resistance may occur, it is less

1299
T
a
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(l
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re
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(%
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tr
a
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)
3
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(2
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7
(2
3
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1
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3
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(0
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(9
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(7
6
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1
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(8
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ry
th

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m
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c
in
(E
R
Y
)
1
5
R
1
3
(2
8
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3
(1
0
)
2
4
(1
9

)
5
6
(7
3
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)
1
0
6
(3
8
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I
1
4
(3
1
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1
8
(6
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2
9
(2
3
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1
3
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7
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6
4
(2
3
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1
8
(4
0
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9
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7
3
(5
8
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(9
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1
0
7
(3
8
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m
o
x
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li
n
(A

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1
0
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3
8
(8
4
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1
7
(5
6
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1
1
(8
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6
5
(8

5
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2
1
1
(7
6
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2
(4
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8
(2
6
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1
7
(1
3
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5
(6
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8
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3
2
(1
1
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)
S
5
(1
1
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)
5
(1
6
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)
1
8
(1

4
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6
(7
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3
4
(1
2
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tr
e
p
to
m
y
c
in
(S
)
1
0

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1
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(2
2
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3
(1
0
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1
2
(9
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1
3
(1
7
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3
8
(1
3
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)
I
0
(0
)
2
(6
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7
)
8
(6
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1
1
(1
4
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2
1
(7
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3
5
(7
7
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1
5
(5
0
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1
0
8
(8
5
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5
2
(6
8
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2

1
8
(7
8
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e
n
ta
m
ic
in
(G
N
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1
0
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0
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(0
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(0
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(0
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4
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1
2
6
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6
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2
7
7
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(C
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5
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(0
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(3

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3
(2
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1
(1
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5
(1
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2
4
(5
3
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1
7
(5
6
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)
6
0
(4
7
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2
3
(3
0
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1
2
4
(4
4
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2
1
(4
6
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)
1
2
(4
0
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6
3
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5
2
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1
5
8
(5
7
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ra

m
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(C
)
3
0
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1
(2
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5
(1
6
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)
5
(4
.0

)
8
(1
0
.5
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1
9
(6
.9
)
I
3
(6
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2
(6
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7
(5
.6
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4

(5
.3
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1
6
(5
7
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4
2
(9
3
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2
4
(8
0
)
1
1
5
(9
1

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6
5
(8
5
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2
4
2
(8
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ri
m
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th
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–
su

lf
a
m
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(S
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1
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5
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3
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5
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1
2
(2

6
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2
(6
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1
2
(9
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2
8
(3
6
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5
4
(1
9
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2

(4
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1
(3
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4
(3
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5
(6
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1
2
(4
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3
1
(6

8
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2
7
(9
0
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1
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(8
7
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3
(5
6
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1
1
(7
6
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o
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o
x
a
c
in
(N
O
R
)
1
0
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0
(0
)
1
(3
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)
1
(0

.8
)
0
(0
)
2
(0
.7
)
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0
(0
)
2
(6
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0
(0
)
2
(2
.6
)

4
(1
.4
)
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4
5
(1
0
0
)
2
7
(9
0
)
1
2
5
(9
9
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)
7
4
(9
7

.4
)
2
7
1
(9
7
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m
p
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il
li
n
(A
M
P
)
1
0
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2
9
(6

4
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)
1
9
(6
3
.3
)
9
2
(7
3
)
4
7
(6
1
.8
)
1
8
7
(6
7
.5
)

I
1
0
(2
2
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)
7
(2
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)
2
2
(1
7
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)
1
9
(2
5
)
5
8
(2
0

.9
)
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6
(1
3
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4
(1
3
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)
1
2
(9
.5
)
1
0
(1
3
.2
)
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2

(1
1
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)
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o
x
y
c
y
c
li
n
e
(D
O
)
3
0
R
1
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1300
likely to lead to high frequencies of resistance. An increase
in bacterial resistance to an antimicrobial generally points to
the widespread and indiscriminate use of these antimicro-
bials, while low frequency in bacterial resistance may be
related to low contamination with antibiotics in the area. In
this study, antimicrobial resistance results revealed that
most (84.5%) Vibrio isolates were resistant to more than one
antimicrobial, with over half of the total isolates (58.5%)
being multidrug resistant. The high resistances revealed by
Vibrio isolates and individual species to b-lactam antibiotics
(ampicillin and amoxicillin) are similar to those reported in
other studies.
12,31–34
However, our results of <50% resis-
tance of V. vulnificus to erythromycin and sulfamethoxazole,
the resistance by V. parahaemolyticus to erythromycin and
V. fluvialis to cotrimoxazole, erythromycin, and chloram-
phenicol are contrary to that reported by Okoh et al.
34
Al-

though resistance to streptomycin was considered to be low,
our findings revealed a higher percent resistance than has
been reported before.
12,35
The percentage of resistance re-
corded to ciprofloxacin might have been low (1.8%), but
almost half (44.8%) of the nonsusceptible isolates showed
an intermediary resistant pattern which possibly may be
completely resistant to the antibiotic overtime.
In this study, gentamicin was the most effective antibiotic
to vibrios as all isolates were susceptible to the drug. This
result is similar to those earlier reported.
12,32–34
The high
susceptibility to gentamicin, together with a >90% suscep-
tibility to norfloxacin and florfenicol, could be an indication
of fairly low contamination of the aquatic environment and
consequently its inhabitants by these antibiotics. In South
Africa, florfenicol (AQUAFLOR, NUFLOR) is the only
antibiotic currently registered for use in aquatic animals,
although other antibiotics such as oxytetracycline hydro-
chloride and oxytetracycline dehydrate have been approved
FIG. 1. Percentage of an-
tibiotic resistance of 277
Vibrio strains. AML,
amoxicillin; AMP, ampicil-
lin; C, chloramphenicol; CIP,
ciprofloxacin; DO, doxycy-
cline; E, erythromycin; FFC,

florfenicol; FOX, cefoxitin;
GN, gentamicin; IPM, imi-
penem; NA, nalidixic acid;
NOR, norfloxacin; TE, tetra-
cycline; S, streptomycin;
SXT, trimethoprim–
sulfamethoxazole.
Table 3. Antibiotic Resistant Genes Detected in Nonsusceptible
Vibrio Isolates
Group ARG tested
Frequency of occurrence
V. vulnificus (%) V. parahaemolyticus (%) V. fluvialis (%)
Total (%)
Sulfonamides dfr1 4/12 (33.3) 0/2 (0) 58.33 (7/12) 11/26 (42.3)
sul2 8/12 (66.7) 0/2 (0) 50 (6/12) 14/26 (53.8)
sul1 0/12 (0) 0/2 (0) 3/12 (25) 3/26 (11.54)
Aminoglycosides nptII 2/10 (20) 0/3 (0) 41.67 (5/12) 7/25
(28.0)
aacC2 0/10 (0) 0/3 (0) 0/12 (0) 0/25 (0)
strA 6/10 (60) 2/3 (66.67) 3/12 (25) 11/25 (44)
Integrase SXT 0/11 (0) 1/4 (25) 0/10 (0) 1/25 (4)
Macrolides ermB 11/13 (91.67) 2/3 (66.67) 20/24 (83.33) 33/40
(82.5)
ermA 0/13 (0) 0/3 (0) 0/24 (0) 0/40 (0)
ermC 0/13 (0) 0/3 (0) 0/24 (0) 0/40 (0)
b-lactams ampC 12/25 (48) 3/15 (20) 6/30 (20) 21/70 (30)
blaOXA 9/25 (36) 2/15 (13.33) 1/30 (3.33) 12/70 (17.1)

Tetracyclines tetA — 2/7 (28.57) 0/10 (0) 2/17 (11.76)
tetM 2/16 (12.50) 8/14 (57.14) 8/17 (47.06) 18/47 (38.3)
ARG, antibiotic resistant gene.
1301
for extra-label uses against fish diseases under veterinarian
supervision.
36
Low resistance (6.1%) to florfenicol is
therefore encouraging. However, frequent monitoring and
effective action to maintain the low levels of resistance to
this antibiotic, particularly in aquaculture settings, are nec-
essary to prevent further development and spread of resis-
tance to this antibiotic.
Doxycycline, ciprofloxacin, and norfloxacin are among first
line drugs administered against infections caused by vibrios.
Based on our results, while ciprofloxacin and norfloxacin ap-
pear to be effective against the isolates tested, it is concerning
to
record resistances of 35.6% (V. vulnificus) and 46.3% (V.
parahaemolyticus), respectively, to doxycycline. In addition,
despite the absence of antibiotics in fish meals (as testified by
the fish farmers of both Farm 1 and 2), the increased levels of
antibiotic resistance to cefoxitin, doxycycline, erythromycin,
ampicillin, and amoxicillin are an indication of antibiotic res -
idues in the environment. This could possibly be from human
and animal waste, runoffs from farmlands, or hospital waste

that finally ends up in the estuaries or the residues could be
from
larval growth stages of fish in aquaculture. Although the culture
of finfishes may not include antibiotics, most larval stages do
include antibiotic treatment to increase survival rates from
pathogenic bacteria.
37
The transfer of antibiotic-resistant bac-
teria’s DNA from fish hatcheries to other pathogenic bacteria
has been documented.
38
Subsequently, these are often associ-
ated with environmental and human health problems, including
bacterial resistance and persistence of disease in the aquatic
environment.
13
In turn, through the food chain or contact with
contaminated water, humans become infected with resistant
bacteria. Antibiotic resistance in food animals and the subse-
quent transfer of these resistant organisms to humans have been
reported.
1,3,39
Resistance to antimicrobials has also been re-
ported in aquaculture products or environments, as well as in
finfish,
40–42
with residues detected from several months

14
up to
a few years posttreatment with antimicrobials.
15
Generally, the MARI for each site was slightly beyond
the threshold of low risk to humans. This result implies that
locations from which samples were collected have been
exposed to high antibiotic contamination. This conclusion
was unexpected in the recirculatory fish farm systems (Farm
1 and Farm 2), as both farms reported using an antibiotic-
free fish meals. These results might, therefore, indicate an
incoming water source to the fish farms, which is already
contaminated with antibiotic-resistant bacteria. The MARI
obtained from isolates from Kariega Estuary was higher
than that recorded in both farms, which we expected as the
estuary is exposed to contaminants from various sources.
Water bodies have been reported to be most contaminated,
with resistant bacteria from human and animal waste, in-
dustries and hospitals, farmland runoff, municipal sewage,
and poorly treated sewage.
43–46
These resistant bacteria
contaminate receiving water bodies and, therefore, aquatic
life, including fish.
47,48
This fact is reflected in many reports
that have revealed the presence of antibiotic-resistant Vibrio
in final effluents of wastewater treatment plants.

34,49–51
It is
unfortunate that most of these pathogens can eventually be
transferred through the food chain or skin abrasions to hu-
mans, causing illnesses that are difficult to treat with regular
antibiotics.
Prevalence of antibiotic resistance genes
The resistant genes ampC, blaZ, tetA, tetM, dfr1, sul1,
sul2, ermB, nptII, and strA were detected in two or more
isolates, while the SXT integrase was detected in one iso-
late. Other studies have reported resistant genes in Vibrio
species, which could be located in the bacterial chromosome
or plasmid mediated.
11,31
Yano et al.
10
also detected b-
lactamase and tetracycline-resistant genes in Vibrio species
isolated from cultured shrimps in Thailand. The inappro-
priate use of antimicrobials could lead to the emergence of
resistant bacteria and genes. Resistance genes are neither a
respecter of phylogeny nor geographical or ecological bor -
ders
52
; therefore, antibiotic-resistant bacteria or genes in the
incoming waters might have been sources of ARGs to the
farms. These resistant bacteria harboring resistant genes

may transfer these genes to susceptible bacteria, possibly
through plasmids.
Conclusion
Our findings represent the first report on antibiotic-
resistant vibrios with circulating ARGs from finfish aqua-
culture in South Africa. The findings of this study support
the hypothesis that marine fish and their environs may serve
as vehicles of antibiotic-resistant vibrios and resistance
genes to humans through the food chain or through contact
with water, and this is a public health concern. Continuous
monitoring of the antimicrobial-resistant patterns is there-
fore necessary to determine the emergence and spread of
resistance, particularly in controlled environments such as
aquaculture. This calls for better implementation of quality
FIG. 2. Representative gel
showing some antibiotic re-
sistant genes detected. (A)
Lanes 1 and 12 ( Molecular
weight markers; 1 kb plus),
Lanes 2 and 3 (tetM), Lanes
4 and 5 (ampC), Lanes 6 and
7 (tetA), Lane 8 (sul2), Lanes
9 and 10 (sul1), Lane 11
(ermA); (B) Lane 1 ( Mole-
cular weight marker; 1 kb
plus), Lanes 2 and 4 (nega-
tive), Lane 3 (SXT), Lanes 6–
7 (tetA), and Lanes 8 and 9
(blaOXA).
1302 FRI ET AL.

control strategies to prevent further transmission of resistant
bacteria to humans and ARGs to susceptible bacteria.
Acknowledgments
The authors are grateful to the South African Institute for
Aquatic Biodiversity (SAIAB) and NRF for their financial
support. They are also grateful to the directors of the fish
farms for provision of samples for the study.
Disclosure Statement
No competing financial interests exist.
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Address correspondence to:

Justine Fri, PhD
Microbial Pathogenicity and Molecular
Epidemiology Research Group ( MPMERG)
Department of Biochemistry and Microbiology
University of Fort Hare
Alice 5700
South Africa
E-mail: [email protected]
1304 FRI ET AL.
Reproduced with permission of copyright owner. Further
reproduction
prohibited without permission.

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