Antibacterial agents and heavy metal resistance in

Gram-negative bacteria isolated from seawater, shrimp

and sediment in Iskenderun Bay, Turkey
Fatih Matyar
a,

, Aysenur Kaya
b
, Sadk Diner
b
a
ukurova University, Faculty of Education, Department of Science and Technology Education, 01330 Balcal, Adana, Turkey
b
ukurova University, Faculty of Science and Letters, Department of Biology, 01330 Balcal, Adana, Turkey
A R T I C L E D A T A A B S T R A C T
Article history:
Received 1 February 2008
Received in revised form 23 July 2008
Accepted 10 August 2008
Available online 19 September 2008
The aimof the present study was to determine the level of antibiotic resistance patterns and
distribution of heavy metal resistance of bacterial isolates from seawater, sediment and
shrimps, and to determine if there is a relationship between antibiotic and heavy metal
resistance. We undertook studies in 2007 in the industrially polluted Iskenderun Bay, on the
south coast of Turkey. The resistance of 236 Gram-negative bacterial isolates (49 from
seawater, 90 from sediment and 97 from shrimp) to 16 different antibiotics, and to 5 heavy
metals, was investigated by agar diffusion and agar dilution methods, respectively. Atotal of
31 species of bacteria were isolated: the most common strains isolated from all samples
were Escherichia coli (11.4%), Aeromonas hydrophila (9.7%) and Stenotrophomonas maltophilia
(9.3%). There was a high incidence of resistance to ampicillin (93.2%), streptomycin (90.2%)
and cefazolin (81.3%), and a low incidence of resistance to imipenem (16.5%), meropenem
(13.9%) and cefepime (8.0%). Some 56.8%of all bacteria isolated fromseawater, sediment and
shrimp were resistant to 7 or more antibiotics. Most isolates showed tolerance to different
concentrations of heavy metals, and minimal inhibition concentrations ranged from
12.5 g/ml to N3200 g/ml. The bacteria from seawater, sediment and shrimp showed high
resistance to cadmium of 69.4%, 88.9%, and 81.1% respectively, and low resistance to
manganese of 2%, 6.7% and 11.3% respectively. The seawater and sediment isolates which
were metal resistant also showed a high resistance to three antibiotics: streptomycin,
ampicillin and trimethoprim-sulphamethoxazole. In contrast, the shrimp isolates which
were metal resistant were resistant to four antibiotics: cefazolin, nitrofurantoin, cefuroxime
and ampicillin. Our results show that Iskenderun Bay has a significant proportion of
antibiotic and heavy metal resistant Gram-negative bacteria, and these bacteria constitute a
potential risk for public health.
2008 Elsevier B.V. All rights reserved.
Keywords:
Antibiotic resistance
Gram-negative bacteria
Heavy metal
Shrimp
1. Introduction
Bacterial resistance to antibiotics is an emerging public health
concern because of the wide availability of antibiotics and
their improper usage without proper prescription (Davis and
Amabile-Cuevas, 2003).
In aquatic environments, the presence of antibiotic resis-
tant pathogen bacteria belonging to the Enterobacteriaceae is
of particular concern. Many antibiotics persist in the sedi-
ment, and elsewhere in the aquatic environment, for several
months following administration (Pouliquen and Le, 1996;
Hirsch et al., 1999). Their residues may affect the microbial
S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 7 ( 2 0 0 8 ) 2 7 9 2 8 5
Corresponding author. Tel.: +90 322 338 60 84 2789; fax: +90 322 338 68 30.
E-mail address: fmatyar@cu.edu.tr (F. Matyar).
0048-9697/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2008.08.014
avai l abl e at www. sci encedi r ect . com
www. el sevi er. com/ l ocat e/ sci t ot env
community in the sediment and introduce antibiotic resis-
tance bacteria (Tendencia and De la Pena, 2002).
Among the Gram-negative bacteria, vibrios cause epidemic
and pandemic diarrhea outbreaks in many regions of the
world. They are free-living aquatic bacteria, common in
warmer waters, above 17 C and can tolerate a wide range of
salinities, depending on the species (Wright et al., 1996).
The introduction of heavy metals, in various forms in the
environment, can cause considerable modifications in the
structure and function of microbial communities (Doelman
et al., 1994). In last decade a number of studies have reported
that antibiotic resistant bacteria may arise in the environment
through co- or cross-resistance to metals or co-regulation of
resistance pathways (Wireman et al., 1997; McArthur and
Tuckfield, 2000; Berg et al., 2005; Akinbowale et al., 2007).
Plasmids are known to carry resistance to antibiotics and
heavy metals (Smith et al., 1993; Sobecky, 1999). Marine
bacteria adsorb, accumulate and transform heavy metals in
most food chains (Chan and Dean, 1988).
Heavy metals (Fe, Zn, Mn, Co, Cu, Ni, V, Mo) are essential
micronutrients for bacteria since they are incorporated into
enzymes and cofactors, however they are toxic in high
concentrations because of binding to enzymes and DNA, and
by production of oxygen radicals through the Fenton reaction
(Lopez-Maury et al., 2002). Trace metals are significant
contaminants in many aquatic systems, due in part to
anthropogenic sources such as industrial and mining inputs.
Coal-fired power plants generate large quantities of ash
through the combustion process, and this waste contains
residues of multiple trace metal such as arsenic and selenium.
The ashresidues are commonly mixedwiththe intake water of
the plant before being deposited in settling basins and this
flow-through water is then typically discharged into aquatic
systems (Rowe et al., 2002). The problem of antibiotic
resistance is global. An organism's expression of a novel gene
coding for drug resistance in remote communities has
implications for the developed world. Once a resistant organ-
ism is introduced into a population, it is rapidly disseminated
(Wenzel and Edmond, 2000).
In Turkey, Iskenderun Bay, on the south coast of the
country bordering the Mediterranean Sea (3638E, 3605N), is
a key location for the study of antibiotic resistant bacteria and
heavy metal contamination in the aquatic environment. It is
known that there are significant levels of accumulated heavy
metals in fish tissues (Trkmen et al., 2005, 2006; Firat et al., in
press). The Bay is of great economic importance for fishing and
aquaculture of numerous species of crustaceans and fish.
However, the Bay is also polluted by iron and steel processing
and production, a fertilizer factory, a refinery, a coal-fired
power plant, and the wreck of the M/V Ulla carrying coal-fired
power plant ash, all of which discharge a lot of amount of
processed or unprocessed wastes into the Bay. In addition,
domestic wastes, including hospital wastes are discharge into
the Bay.
To our knowledge, our present study is the first to
determine the prevalence and resistance to antimicrobial
agents and heavy metals of Gram-negative bacteria isolated
from Iskenderun Bay.
The specific purposes of this study were: (i) to identify the
Gram-negative bacterial strains recovered in Iskenderun Bay;
(ii) to determine the level of antibiotic resistance rates against
widely used antibiotics in Turkey; (iii) to determine the heavy
metal resistance of the bacteria; and (iv) to investigate if there
was a relationship between the antibiotic and heavy metal
resistance.
2. Materials and methods
2.1. Sample collection
Samples were collected fromFebruary to September 2007 from
Iskenderun Bay on the south coast of Turkey (Fig. 1). On each
sampling occasion, samples were collected from seawater,
sediment and shrimp. Water samples were collected 020 cm
below from the surface, using 250 ml sterile bottles (APHA,
1992). Sediment samples were collected from the upper layer
(05 cm depth), and stored in 250 ml sterile bottles. Penaeid
shrimp samples were obtained from fisherman and packed in
sterile bags. All samples were brought to the laboratory in an
ice chest, and processed within 4 h of collection.
2.2. Bacterial isolation
Bacteria fromthe seawater, sediment and shrimp were isolated
using the spread plate technique. One milliliter of the water
sample was serially diluted in sterile water and plated. One
gram of the sediment was homogenised in 9 ml sterile water,
and a serial 10-fold dilution up to 10
5
was prepared in sterile
water and plated. Intestinal contents of shrimps were taken
aseptically, and 1 g of this was homogenised in 9 ml sterile
water, the homogenised samples were then diluted using 10-
fold serial dilution up to 10
7
in sterile water.
Fig. 1 Map of Turkey showing Iskenderun Bay (inset):
1. coal-fired power plant, 2. oil refinery, 3. fertilizer factory,
4. ironsteel factory.
280 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 7 ( 2 0 0 8 ) 2 7 9 2 8 5
To determine total counts, plate counts of Gram-negative
bacteria were made using MacConkey Agar (McC) (Merck),
inoculated with appropriate dilutions from the sample
homogenates, and incubated for 2472 h at 35 C. To evaluate
the incidence of resistant bacteria, media supplemented with
ampicillin (50 g ml
1
), streptomycin (25 g ml
1
), tetracycline
(25 g ml
1
) and chloramphenicol (25 g ml
1
) were used. All
plate counts were performed in triplicate.
2.3. Bacterial Identification
A total of 236 Gram-negative bacterial isolates were randomly
selected from the plates containing antibiotics: 49 isolates
from seawater, 90 from sediment and 97 from shrimp. The
isolates were purified onto McC agar and then maintained in
nutrient agar (Oxoid).
All the isolates were characterised by phenotypical char-
acteristics, namely Gram staining, oxidase and catalase
reactions, motility, OF glucose and gelatin liquefaction tests
according to Cowan (1974) and Lemos et al. (1985). Isolates
were then identified using the Becton Dickinson Crystal E/NF
identification software (BBL, Md, USA). A number of isolates
(20%) were re-examined to check reproducibility of the test.
2.4. Antibacterial susceptibility testing
Susceptibility testing was performed by an agar diffusion
test (Bauer et al., 1966), using MuellerHinton agar (Difco)
and 16 antibiotic discs representing 9 classes of antibiotics:
amikacin (AN, 30 g), ampicillin (AM, 10 g), nalidixic acid
(NA, 30 g), chloramphenicol (C, 30 g), tetracycline (TE,
30 g), nitrofurantoin (F/M, 300 g), streptomycin (S, 10 g),
gentamicin (GM, 10 g), imipenem (IPM, 10 g), cefazolin (CZ,
30 g), ceftizoxime (ZOX, 30 g), meropenem (MEM, 10 g),
cefuroxime (CXM, 30 g), cefepime (FEP, 30 g), kanamycin (K,
30 g) and trimethoprim-sulphamethoxazole (SXT, 1.25 and
23.75 g). The isolates were determined to be sensitive
according to the information supplied by manufacturer (BBL,
Md, USA).
Reference strains of Escherichia coli ATCC 25922 and Pseu-
domonas aeruginosa ATCC 27853, as recommended by NCCLS
(1997), were used as control organisms for verification of the
antibacterial effect of the discs. All discs were purchased from
Becton Dickinson (BBL, Md, USA).
For all isolates, we calculated the MAR index values (a/b,
where a represents the number of antibiotics the isolate
was resistant to, and b represents the total number of
antibiotics the isolate was tested against). A MAR index
value N0.2 is observed when isolates are exposed to high
risk sources of human or animal contamination, where
antibiotics use is common; in contrast a MAR index value b
or =0.2 observed when antibiotics are seldom or never used
(Krumperman, 1985).
2.5. Determination of the MIC of heavy metals
The Minimal Inhibitory Concentration (MIC) for each bacterial
isolate for five heavy metals was determined using Mueller
Hinton agar (Difco) containing Cd
+2
, Cu
+2
, Cr
+3
, Pb
+2
and Mn
+2
at concentrations ranging from 12.5 g/ml to 3200 g/ml. The
metals were added as CdCl
2
2H
2
O, CuSO
4
5H
2
O, CrCl
3
, Pb
(NO
3
)
2
and MnCl
2
2H
2
O (Merck). The isolates were considered
resistant if the MIC values exceeded that of the E. coli K-12
strain which was used as the control (Akinbowale et al., 2007).
3. Results and discussion
3.1. Bacterial flora
A total of 49, 97 and 90 isolates were obtained from seawater,
sediment and intestinal content of shrimps, respectively; the
frequency of the isolates in the different sources is shown in
Table 1. Sevenspecies were foundat relativelyhighfrequencies:
E. coli (11.4%), Aeromonas hydrophila (9.8%), Stenotrophomonas
maltophilia (9.3%), Flavimonas oryzihabitans (8.5%), Acinetobacter
lwoffi (8.1%), Klebsiella pneumoniae ssp. pneumoniae (7.2%) and Ci-
trobacter freundii (5.5%). E. coli, a potential enteric pathogen was
Table 1 Number of species of Gram-negative bacteria
isolated from seawater, sediment and shrimp
Species Sources
Seawater Sediment Shrimp Total
Acinetobacter bzumannii 3 3
Acinetobacter lwoffi 2 3 14 19
Aeromonas caviae 2 6 8
Aeromonas hydrophila 14 9 23
Aeromonas veronii 1 1
Burkholderia cepacia 1 8 9
Chryseobacterium
indologenes
2 2
Chryseobacterium
meningosepticum
1 1
Citrobacter freundii 2 11 13
Edwardsiella tarda 2 2
Empedobacter brevis 1 1
Enterobacter cloacae 2 1 2 5
Enterobacter sakazakii 1 1
Escherichia coli 2 25 27
Flavimonas oryzihabitans 11 4 5 20
Klebsiella oxytoca 3 3
Klebsiella pneumoniae
ssp. pneumoniae
4 13 17
Pantoea agglomerans 2 1 1 4
Proteus penneri 8 8
Proteus vulgaris 9 9
Pseudomonas aeruginosa 2 1 7 10
Pseudomonas fluorescens 4 4
Pseudomonas putida 1 3 4
Pseudomonas stutzeri 3 4 4 11
Salmonella typhi 1 1
Shewanella putrefaciens 1 1
Shigella species 1 1
Sphingomonas
paucimobilis
2 2
Stenotrophomonas
maltophilia
1 2 19 22
Vibrio metschnikovii 1 2 3
Yersinia enterocolitica
group
1 1
Total 49 90 97 236
281 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 7 ( 2 0 0 8 ) 2 7 9 2 8 5
recovered intensively from the sediment (92.6%), and a
previous study (Goyal et al., 1979) has noted that marine
sediments under polluted seawater might be a reservoir of
enteric bacteria. A second prevalent species, A. hydrophila,
was isolated intensively from the seawater (60.9%), and a
third prevalent species, S. maltophilia, was commonly recov-
ered from the intestinal content of shrimp (86.4%). The high
recovery of enteric bacteria indicates that seawater is highly
contaminated. Only one species of Vibrionaceae (Vibrio
metschnikovii) was isolated from all the samples examined,
even though Vibrios are normally very common in marine
and estuarine water environments. Vibrio metschnikovii is
largely distributed in the aquatic environment; human
infections are rarely observed. Of the known Vibrio species,
twelve occur in human clinical specimens. Of particular note
is V. parahaemolyticus, which causes acute gastroenteritis. In
Japan, this pathogen is responsible for 50 to 70% of the cases
of foodborne gastroenteritis, mostly after the consumption of
raw or undercooked seafood (Farmer et al., 2003).
Among all the samples examined, only single isolates of
Aeromonas veronii, Salmonella typhi, Shewanella putrefaciens and
Yersinia enterocolitica group were recovered from the sediment,
and only single isolates of Chryseobacterium meningosepticum,
Empedobacter brevis, Enterobacter sakazakii and Shigella species
(from shrimp) were recovered. In our study, thirty-one
bacterial species were found in all the samples examined.
Bacteria in marine sediments are highly abundant to diverse
(Danovaro et al., 2000), and their community structure can be
affected by the deterioration of environmental conditions in
marine coastal ecosystems (Meyer-Reil and Kster, 2000).
Dang et al. (2008), identified thirty-two different species from
the 89 OTC
100
-resistant isolates at Jiaozhou Bay; the isolates
were dominated by Enterobacteriaceae, Pseudomonadaceae
and Vibrionaceae.
In our study, E. coli was the most frequently detected
microorganism. This bacterium is an indicator of fecal contam-
ination in aquatic environment. Iskenderun Bay is occasionally
fedwithdomestic effluents containing hospital wastes fromthe
Iskenderundrainagesystem, andlarge quantities of wastewater
have been discharged into this region.
The second most prevalent microorganism was A. hydro-
phila, a ubiquitous inhabitant of aquatic environments. The
genus Aeromonas comprises important human pathogens
causing primary and secondary septicemia in immunocom-
promised persons, serious wound infections in healthy
individuals and in patients undergoing medicinal leech
therapy, and a number of less well described illnesses,
such as peritonitis, meningitis and infections of the eye
(Janda and Abbott, 1998). Sewage and effluents from
domestic water purification plants have been suspected as
vehicles for transmission of pathogenic aeromonads in a
number of clinical cases (Monfort and Baleux, 1991).
The third most prevalent microorganism S. maltophilia
(formerly Pseudomonas maltophilia) is a ubiquitous aerobic
Gram-negative bacillus. It can be found in aquatic environ-
ments, in soils, on vegetation and even on some animals. It is
widespread in the domestic and hospital environments,
including those of patients with cystic fibrosis, particularly
in sites related to water and plumbing.
3.2. Antimicrobial resistance patterns
The principal findings were (i) the viable counts of total and
resistant bacteria from shrimp were higher than those from
seawater or sediment, (ii) the frequency of streptomycin
resistant bacteria from seawater was higher than all other
resistant flora, and (iii) the frequency of tetracycline-resistant,
ampicillin-resistant and chloramphenicol-resistant bacteria
fromthe intestinal contents of shrimp was higher than that in
seawater and sediment (Table 2).
Among the seawater isolates, a high percentage of bacteria
were resistant to streptomycin (100%), cefazolin (89.8%),
ampicillin (83.7%) and trimethoprim-sulphamethoxazole
(69.4%), whereas a low percentage of bacteria were resistant
to cefepime (12.3%) and meropenem (14.3%), (Fig. 2). The high
degree of resistance to ampicillin in the present study was
similar to the findings of Vaseeharan et al. (2005).
Among the sediment isolates, a high percentage were
resistant to streptomycin (96.7%), ampicillin (94.4%) and tri-
methoprim-sulphamethoxazole (78.9%), and a low percentage
were resistant to imipenem (4.4%), meropenem (4.4%),
Table 2 Concentrations of antibiotic resistant bacteria in seawater, sediment and shrimp
Resistant viable count (%)
Total viable count
(CFU g
1
)
Tetracycline
(25 g ml
1
)
Ampicillin
(50 g ml
1
)
Streptomycin
(25 g ml
1
)
Chloramphenicol
(25 g ml
1
)
McConkey Agar Seawater 410
4
10.3 26.5 39.1 18.3
Sediment 3.210
5
20.4 28.3 37.0 14.6
Shrimp 6.110
5
20.8 32.3 31.2 20.3
Fig. 2 Antibacterial resistance of bacteria isolated from
water, sediment and shrimp.
282 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 7 ( 2 0 0 8 ) 2 7 9 2 8 5
ceftizoxime (6.7%) and cefepime (6.7%). Resistance to imipenem
was similar to the results of Schwartz et al. (2003).
Among the bacteria isolated from the intestine of shrimp,
all (100%) were resistant to cefazolin, and high percentages
were resistant to nitrofurantoin (97.9%), cefuroxime (97.9%),
and ampicillin (96.9%), only 7.2% were resistant to cefepime.
In Iskenderun Bay, a high proportion of bacteria were
resistant to different antibacterial agents, although resistance
to carpenemes, third and fourth-generation cephalosporins
was relatively infrequent in the water and sediment isolates.
Antibiotic residues entering this aquatic environment from
different sources may increase the distribution of potential
drug-resistant pathogen bacteria.
The story of antibiotics and antibiotic resistance is of recent
origin, since antibiotics have been used for the treatment of
infectious diseases in humans and also for a number of non-
human applications (agriculture, animal husbandry, and
aquaculture) during the last half of the century only. The
subsequent half-century of antibiotic use has undoubtedly
influenced all aspects of microbial genetic ecology through
mutation, selection, and the flux of genetic information
(horizontal gene transfer) between microbes; this has been a
period of strong evolutionary pressure and extensive selection
(Mazel and Davies, 1999).
3.3. Multiple antibiotic resistance index
Similar percentages of seawater bacteria (16.3%) and sediment
bacteria (16.7%) were resistant to 4 antibiotics, and there was
similar resistance in sediment (21.1%) and shrimp bacteria
(23.7%) to6antibiotics. Of thebacteriaisolatedfromshrimp, only
1.03%wereresistant to4antibiotics and42.3%wereresistant to6
or 7 antibiotics. These percentages contrast with the higher
percentages reported by Ogbondeminu and Olayemi (1993) and
Matyar (2007) who reported 55.9% and 58.3% of enteric isolates
fromfish were resistant to 3 or 4 antibiotics. Some isolates from
our seawater and sediment samples showed resistance to as
many as 16 antibiotics (2.0%, 3.3%respectively), whereas among
the shrimp bacteria 12.4% were resistant to 13 antibiotics, and
none were resistant to 14 or more antibiotics (Fig. 3).
High multiresistance incidence, similar to or higher than
those found in this study, have been reported in aquatic
environments and fish isolates (Vivekanadhan et al., 2002;
Miranda and Zemelman, 2002; Matyar et al., 2004). The
presence of antimicrobial agents at lowconcentration through
leaching or continued usage may lead to the development of
drug-resistant isolates and multiple antibiotic resistance
(MAR) in bacteria, which may result in resistance transfer to
pathogenic bacteria and reduced efficacy of antibiotic treat-
ment for human and animal diseases (Tendencia and De la
Pena, 2001). Multiresistant bacteria are spreading within the
hospital environment and have become a significant cause of
nosocomial infections (Medeiros et al., 1985). A number of
Fig. 3 Antibacterial multiresistance of bacteria isolated from
water, sediment and shrimp.
Table 3 Heavy metal tolerance in bacteria from seawater, sediment and shrimp
Heavy metal/
environment
n Metal concentrations (g/ml) with number of tolerant isolates Resistant
isolates
12.5 25 50 100 200 400 800 1600 3200 N3200 n %
Cadmium *
Seawater 49 1 14 9 13 5 4 2 1 34 69.4
Sediment 90 4 6 19 22 11 22 5 1 80 88,9
Shrimp 97 1 23 23 16 21 13 73 81,1
Copper
Seawater 49 7 24 8 3 5 1 1 18 36.7
Sediment 90 1 14 13 34 11 4 13 75 83.3
Shrimp 97 2 1 2 24 38 22 2 3 1 2 30 30.9
Chromium
Seawater 49 3 6 21 17 2 2 4.1
Sediment 90 23 51 14 1 1 16 17.8
Shrimp 97 1 2 75 15 2 1 1 4 4.1
Lead
Seawater 49 1 1 10 17 16 2 2 4 8.2
Sediment 90 6 44 26 13 1 14 15.6
Shrimp 97 4 1 4 1 2 12 51 9 10 3 13 13.4
Manganese
Seawater 49 1 13 5 17 12 1 1 2.0
Sediment 90 1 10 1 9 52 11 4 2 6 6.7
Shrimp 97 1 4 11 4 31 19 16 9 2 11 11.3
Minimal inhibition concentration of standard strain Escherichia coli K12.
283 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 7 ( 2 0 0 8 ) 2 7 9 2 8 5
studies have shown that the incidence of antibiotic resistance
is often reduced when the use of antibiotics is restricted
(Gaynes and Monnet, 1997; Frimodt-Mller et al., 1997).
In present study, the MAR indexes of both seawater and
sediment bacteria (0.2501) were higher than that of the
shrimp bacteria (0.2500.875). De Vicente et al. (1990) reported
that the number of strains multiresistant to several antibiotics
was high in seawater samples containing low levels of fecal
indicators. The MAR index values ranged from 0.25 to 0.69 for
the most prevalent bacteria in our study, the potential enteric
pathogen E. coli. Jones et al. (1986) reported that E. coli isolated
from English lake waters had a greater incidence of antibiotic
resistance than did the native aquatic bacteria. At the same
time a high incidence of antibiotic resistance in Pseudomonas
spp. was reported by Jones et al. (1986) for lake waters.
3.4. Heavy metal resistance
In the present study, resistance to five heavy metals (Cd
+2
, Cu
+2
,
Cr
+3
, Pb
+2
and Mn
+2
) was investigated for all isolates. Trends
in heavy metal resistance vary depending on the sample:
seawater, CdNCuNPbNCrNMn; sediment, CdNCuNCrNPbNMn;
andshrimpCdNCuNPbNMnNCr (Table3). Akinbowaleet al. (2007)
found that the resistance to 7 heavy metals was in the order
of CuNPb=CrNMnNZnNCdNCo for sediment isolates. Chandy
(1998) has studied the effects of 16 heavy metals on marine bac-
teria and observed the following order of toxicity: HgNAgN-
As=Cd=Cr=Co=Cu=Fe=PbNMo=SbNNi=V=ZnNMnNSr.
In present study in Iskenderun Bay, resistance to heavy
metals was as follows (Table 3) for the samples of seawater,
sediment and shrimp isolates: cadmium, 69.4%, 88.9% and
81.1%; copper, 36.7%, 83.3%, and 30.9%; chromium, 4.1%, 17.8%,
and 4.1%; lead, 8.2%, 15.6% and 13.4%; and manganese, 2.0%,
6.7% and 11.3%.
The MICs of the isolates ranged from 12.5 g/ml to
N3200 g/ml. Sediment isolates showed a high resistance to
cadmium, copper, chromium and lead, than did seawater and
shrimp isolates, whereas shrimp isolates showed a higher
resistance to manganese than did the seawater and sediment
isolates. Tolerance to maximum MIC (N3200 g/ml) for copper
was found in a high proportion of sediment isolates (14.4%),
whereas minimumMIC (12.5 g/ml) for lead was found among
the shrimp isolates (4.1%).
Heavy metal resistance detected in the present study could
be the result of heavy metal contamination from coal-fired
power plant, oil refinery and ironsteel factories.
3.5. Interaction of metal resistance and antibiotic resistance
The seawater and sediment isolates which were metal
resistant also showed a high resistance to three antibiotics:
streptomycin, ampicillin and trimethoprim-sulphamethoxa-
zole. In contrast the shrimp isolates, which were metal
resistant, were resistant to four antibiotics: cefazolin, nitro-
furantoin, cefuroxime and ampicillin. With all five of the
metals tested, there was a tendency towards a high frequency
of ampicillin resistance among all the isolates.
The present study is one of only a few that addresses the
incidence of antibiotic and heavy metal resistance in bacteria
from seawater, sediment and shrimp samples. Most previous
studies have focused on samples from freshwater (Miranda
and Castillo, 1998), metal-contaminated streams (Wright et al.,
2006) and clinical isolates (Ugur and Ceylan, 2003).
Increasing pressure from environmental pollution can be
accompanied by resistance of aquatic organisms to various
antimicrobials. The increase in heavy metal and antibiotics
resistance for some pathogens is a significant global problem,
complicating the battle against infectious diseases. Some of
these microorganisms are found in fishery products, which
have a very crucial role in food chain, and this may pose
important public health problems.
3.6. Recommendations
Antibiotic and heavy metal resistance in the environment
involves processes at multiple scales. The present study
focuses on a part of the eastern Mediterranean region, and
seawater, sediment and shrimp samples from a polluted
marine environment. Further studies are needed to under-
stand better the metal and antibiotic resistant bacteria, in fish,
aquatic environments and sediments. The geographic scope
of those studies should include other parts of the Turkish
coasts on the Mediterranean and Aegean Seas. Furthermore,
more studies should be designed at the molecular level to
evaluate the presence of plasmid-encoded resistant genes,
and to identify the mechanisms involved in the association
between antibiotic and metal resistance.
Acknowledgement
We would like to thank BAPKOM, (Cukurova University) for
providing the financial support of this work (Project number:
EF2007BAP3).
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